Patent Application
20220243164
Immunotherapy is currently at the cutting edge of both basic scientific research and pharmaceutically driven clinical application. This trend is in part due to the recent strides in targeted gene modification and the expanded use of CRISPR/Cas complex editing for therapeutic development. In order to identify genetic modifications of therapeutic interest, research organizations often have to screen thousands of genetic variants, which can include modification of an endogenous gene or insertion of an engineered gene. This drug discovery process is laborious, requiring significant manual labor within the laboratory, creating an industry-wide bottleneck due to the lack of adequate high-throughput technologies.
Biotech and pharmaceutical research and development activities have shifted to automating nearly all steps of the process. The workflows include liquid handling robots, powered by sophisticated laboratory management software, to enable high throughput discovery. However, transfection steps are limited to low throughput, poor efficiency technologies, and user-intensive systems that cannot be automated. Automated platforms for transfection not only have the potential to reduce process costs substantially, but also increase cell viability and the quantity of successfully engineered cells, all while reducing discovery time, which is critical in the competitive immunotherapy space.
A unique strength of transfection by way of electroporation is RNA delivery. Existing viral techniques to deliver DNA appear on par with transfection by way of electroporation, but there is a lack of GMP-quality non-retroviral RNA viruses. Therefore, companies with electroporation platforms have been the target of collaborations and acquisitions for the purpose of delivering mRNA into cells.
Current high-throughput gene transfer methods typically require the use of viral delivery (e.g., lentiviral vectors), in which viral particles infect a cell and transduce the genetic modification of interest. While a viral methodology can be applied to high-throughput automated systems, there are limitations in the production that extend timelines for research efforts: viral vectors have to be cloned, transfected into a viral production line, and then viral particles must be purified. This process can take research organizations months, significantly affecting their timelines for platform development while simultaneously increasing the cost of drug discovery. Additionally, the use of viral transduction for gene transfer is not amenable to genetic modification for all cell types, since some cells (such as specific immune cell subsets) are resistant to viral infection. Therefore, within the biotechnology industry there is an unmet need to have a high-throughput automated system for gene transfer that does not rely on viral delivery mechanisms.
In one aspect, the invention features a device for electro-mechanical delivery of a composition into a plurality of cells suspended in a liquid (e.g., a liquid flowing through the device), the device including a first and second electrode and an electroporation zone. The first electrode includes a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension, and the second electrode includes a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension. The electroporation zone is disposed between the first outlet and the second inlet and has a minimum cross-sectional dimension that is greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform transverse cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication.
In another aspect, the invention features a device for electroporating a composition into a plurality of cells suspended in a liquid (e.g., a liquid flowing through the device), the device including a first and second electrode and an electroporation zone. The first electrode includes a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension, and the second electrode includes a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension. The electroporation zone is disposed between the first outlet and the second inlet and has a minimum cross-sectional dimension that is greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform transverse cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication. Transfection of cells can occur within the electroporation zone via an electro-mechanical delivery mechanism that is distinct from the delivery mechanism in static electroporation systems.
In some embodiments of either of the preceding aspects, a transverse cross-section of the electroporation zone is a shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular shape (e.g., a shape having protrusions, such as protruding slots or grooves, irregular polygons, and/or curvilinear shapes). In some embodiments, the cross-section of the electroporation zone varies along the length (i.e., longitudinal axis or direction of flow) of the electroporation zone. In some embodiments, the shape is consistent along the length but varies in position relative to the central longitudinal axis along the length of the electroporation zone (e.g., the cross-sectional shape rotates about the central axis from one end of the electroporation zone to the other, such as a helix). In particular embodiments, the electroporation zone has a substantially circular transverse cross-section. In some embodiments, the electroporation zone has a transverse cross-sectional area of between about 7,850 μm2 and about 2,000 mm2 (e.g., between about 8,000 μm2 and about 1 mm2, between about 8,000 μm2 and about 10 mm2, between about 8,000 μm2 and about 100 mm2, between about 9,000 μm2 and 5 mm2, between about 1 mm2 and about 10 mm2, between about 1 mm2 and about 100 mm2, between about 3 mm2 and about 20 mm2, between about 10 mm2 and about 50 mm2, between about 25 mm2 and about 75 mm2, between about 50 mm2 and about 100 mm2, between about 75 mm2 and about 200 mm2, between about 100 mm2 and about 350 mm2, between about 150 mm2 and about 500 mm2, between about 300 mm2 and about 750 mm2, between about 500 mm2 and about 1,000 mm2, between about 750 mm2 and about 1,500 mm2, or between about 950 mm2 and about 2,000 mm2, e.g., about 8,000 μm2, about 9,000 μm2, about 1 mm2, about 5 mm2, about 10 mm2, about 15 mm2, about 20 mm2, about 25 mm2, about 50 mm2, about 60 mm2, about 75 mm2, about 80 mm2, about 100 mm2, about 150 mm2, about 200 mm2, about 250 mm2, about 300 mm2, about 350 mm2, about 400 mm2, about 450 mm2, about 500 mm2, about 600 mm2, about 700 mm2, about 800 mm2, about 900 mm2, about 1,000 mm2, about 1,100 mm2, about 1,200 mm2, about 1,300 mm2, about 1,400 mm2, about 1,500 mm2, about 1,600 mm2, about 1,700 mm2, about 1,800 mm2, about 1,900 mm2, or about 2,000 mm2).
In some embodiments, the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm). In some embodiments, the electroporation zone has a length of between 0.005 mm and 25 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 10 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 10 mm, between 7 mm and 15 mm, between 10 mm and 20 mm, or between 15 mm and 25 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, about 23 mm, or about 25 mm).
In some embodiments, a lumen of any of the first electrode and/or the second electrode has a minimum cross-sectional dimension of between 0.01 mm and 500 mm (e.g., between 0.01 mm and 0.1 mm, between 0.01 mm and 0.5 mm, between 0.01 mm and 10 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 50 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 30 mm and 300 mm, between 45 mm and 60 mm, between 50 mm and 100 mm, between 50 mm and 500 mm, between 75 mm and 150 mm, between 75 mm and 300 mm, between 100 mm and 200 mm, between 100 mm and 500 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm, about 450 mm, or about 500 mm).
In some embodiments, a ratio of the minimum cross-sectional dimension of a lumen of either of the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1).
In some embodiments, a ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 3:1 and 10:1, between 4:1 and 25:1, between 5:1 and 50:1, between 10:1 and 50:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1).
In some embodiments, a ratio of a transverse cross-sectional area of a lumen of any of the first electrode and/or the second electrode to the transverse cross-sectional area of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 3:1 and 10:1, between 4:1 and 25:1, between 5:1 and 50:1, between 10:1 and 50:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1).
In some embodiments, the device further includes a first reservoir (e.g., a sample bag) in fluidic communication with the first inlet and/or a second reservoir (e.g., a collection bag, e.g., a recovery bag) in fluidic communication with the second outlet. Additionally, the device may include a third reservoir in fluidic communication with the first lumen or the second lumen. The third reservoir may contain one or more reagents for transfection (e.g., reagents having a composition selected for transfection), e.g., a payload suspension, such as a genetic composition, to be delivered to the cells. In some embodiments, the third reservoir is configured to contain buffer, buffer additive, and/or payload suspension comprising the composition in a non-liquid phase (e.g., solid salt) that can be diluted before or during the electroporation or electro-mechanical process. In some embodiments, either of the first electrode or the second electrode has an additional inlet or outlet for fluidic communication with the third reservoir.
In some embodiments, either of the first electrode or the second electrode can be porous or a conductive fluid (e.g., conductive liquid).
A device of any of the preceding embodiments may include a delivery source in fluidic communication with the first inlet. The delivery source can be configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. A delivery source can also be configured to deliver other components, such as a composition (e.g., genetic composition) to be introduced to the cells (e.g., as a transfection reagent reservoir) or additional buffer or buffer additive, using a fluid displacement mechanism (e.g., a single syringe pump or multiple syringe pumps) in fluidic connection with the delivery source and the first lumen.
In some embodiments, the delivery source is configured to deliver components prior to (e.g. upstream of) or within the electroporation zone. In some embodiments, the delivery source is configured such that the fluid displacement mechanism (e.g., a single syringe pump or multiple syringe pumps) is dedicated to one buffer, buffer additive, and/or payload suspension and is in fluidic communication with the plurality of cells in suspension.
In some embodiments, the device further includes one or more additional electroporation zones (e.g., one, two, three, four, six, eight, ten, 11, 12, 24, 27, 36, 48, 64, 96, 384, 1536, or more) additional electroporation zones, which can be configured in parallel, in series, or a combination thereof. The one or more additional electroporation zones can each have a substantially uniform transverse cross-sectional area.
In some embodiments of any of the aforementioned embodiments, the device can further include a housing configured to encase the first electrode, second electrode, and the electroporation zone. The housing may include a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. In some embodiments, the housing further includes a thermal controller configured to increase the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device. The housing can be integral or releasably connected to the device.
In another aspect, the invention includes a device for electro-mechanical delivery of a composition into a plurality of cells suspended in a liquid, wherein the device includes a first electrode including a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension; a second electrode including a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension; a third inlet and a third outlet, wherein the third inlet and the third outlet are in fluidic communication with the first lumen, wherein the third inlet and the third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, wherein the fourth inlet and fourth outlet are in fluidic communication with the second lumen, wherein the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication. The transverse cross-section of the electroporation zone is a shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular shape (e.g., a shape having protrusions, e.g., protruding slots or grooves, irregular polygons, and/or curvilinear shapes). In some embodiments, the cross-section of the electroporation zone varies along the length (i.e., longitudinal axis or direction of flow) of the electroporation zone). In some embodiments, the shape is consistent along the length but varies in position relative to the central longitudinal axis along the length of the electroporation zone (e.g., the cross-sectional shape rotates about the central axis from one end of the electroporation zone to the other, such as a helix). In particular embodiments, the electroporation zone has a substantially circular transverse cross-section. In some embodiments, the electroporation zone has a transverse cross-sectional area of between about 7850 μm2 and about 2000 mm2 (e.g., between about 8,000 μm2 and about 1 mm2, between about 8,000 μm2 and about 10 mm2, between about 8,000 μm2 and about 100 mm2, between about 9,000 μm2 and 5 mm2, between about 1 mm2 and about 10 mm2, between about 1 mm2 and about 100 mm2, between about 3 mm2 and about 20 mm2, between about 10 mm2 and about 50 mm2, between about 25 mm2 and about 75 mm2, between about 50 mm2 and about 100 mm2, between about 75 mm2 and about 200 mm2, between about 100 mm2 and about 350 mm2, between about 150 mm2 and about 500 mm2, between about 300 mm2 and about 750 mm2, between about 500 mm2 and about 1,000 mm2, between about 750 mm2 and about 1,500 mm2, or between about 950 mm2 and about 2,000 mm2, e.g., about 8,000 μm2, about 9,000 μm2, about 1 mm2, about 5 mm2, about 10 mm2, about 15 mm2, about 20 mm2, about 25 mm2, about 50 mm2, about 60 mm2, about 75 mm2, about 80 mm2, about 100 mm2, about 150 mm2, about 200 mm2, about 250 mm2, about 300 mm2, about 350 mm2, about 400 mm2, about 450 mm2, about 500 mm2, about 600 mm2, about 700 mm2, about 800 mm2, about 900 mm2, about 1,000 mm2, about 1,100 mm2, about 1,200 mm2, about 1,300 mm2, about 1,400 mm2, about 1,500 mm2, about 1,600 mm2, about 1,700 mm2, about 1,800 mm2, about 1,900 mm2, or about 2,000 mm2).
In another aspect, the invention includes a device for transfecting a composition into a plurality of cells suspended in a liquid, wherein the device includes a first electrode including a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension; a second electrode including a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension; a third inlet and a third outlet, wherein the third inlet and the third outlet are in fluidic communication with the first lumen, wherein the third inlet and the third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, wherein the fourth inlet and fourth outlet are in fluidic communication with the second lumen, wherein the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication. The transverse cross-section of the electroporation zone is a shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular shape (e.g., a shape having protrusions, e.g., protruding slots or grooves, irregular polygons, and/or curvilinear shapes). In some embodiments, the cross-section of the electroporation zone varies along the length (i.e., longitudinal axis or direction of flow) of the electroporation zone). In some embodiments, the shape is consistent along the length but varies in position relative to the central longitudinal axis along the length of the electroporation zone (e.g., the cross-sectional shape rotates about the central axis from one end of the electroporation zone to the other, such as a helix). In particular embodiments, the electroporation zone has a substantially circular transverse cross-section. In some embodiments, the electroporation zone has a transverse cross-sectional area of between about 7850 μm2 and about 2000 mm2 (e.g., between about 8,000 μm2 and about 1 mm2, between about 8,000 μm2 and about 10 mm2, between about 8,000 μm2 and about 100 mm2, between about 9,000 μm2 and 5 mm2, between about 1 mm2 and about 10 mm2, between about 1 mm2 and about 100 mm2, between about 3 mm2 and about 20 mm2, between about 10 mm2 and about 50 mm2, between about 25 mm2 and about 75 mm2, between about 50 mm2 and about 100 mm2, between about 75 mm2 and about 200 mm2, between about 100 mm2 and about 350 mm2, between about 150 mm2 and about 500 mm2, between about 300 mm2 and about 750 mm2, between about 500 mm2 and about 1,000 mm2, between about 750 mm2 and about 1,500 mm2, or between about 950 mm2 and about 2,000 mm2, e.g., about 8,000 μm2, about 9,000 μm2, about 1 mm2, about 5 mm2, about 10 mm2, about 15 mm2, about 20 mm2, about 25 mm2, about 50 mm2, about 60 mm2, about 75 mm2, about 80 mm2, about 100 mm2, about 150 mm2, about 200 mm2, about 250 mm2, about 300 mm2, about 350 mm2, about 400 mm2, about 450 mm2, about 500 mm2, about 600 mm2, about 700 mm2, about 800 mm2, about 900 mm2, about 1,000 mm2, about 1,100 mm2, about 1,200 mm2, about 1,300 mm2, about 1,400 mm2, about 1,500 mm2, about 1,600 mm2, about 1,700 mm2, about 1,800 mm2, about 1,900 mm2, or about 2,000 mm2).
In some embodiments of any of the preceding aspects, the electroporation zone has a minimum cross-sectional dimension of between 0.1 mm and 50 mm (e.g., between 0.1 mm and 0.5 mm, between 0.1 mm and 1 mm, between 0.1 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 45 mm and 60 mm, between 50 mm and 100 mm, between 75 mm and 150 mm, between 100 mm and 200 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm).
In some embodiments of any of the preceding aspects, the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm). In some embodiments, the electroporation zone has a length of between 0.005 mm and 25 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 10 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 10 mm, between 7 mm and 15 mm, between 10 mm and 20 mm, or between 15 mm and 25 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, about 23 mm, or about 25 mm).
In some embodiments of any of the preceding aspects, a lumen of any of the first electrode and/or the second electrode has a minimum cross-sectional dimension of between 0.01 mm and 500 mm (e.g., between 0.01 mm and 0.1 mm, between 0.01 mm and 0.5 mm, between 0.01 mm and 10 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 50 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 30 mm and 300 mm, between 45 mm and 60 mm, between 50 mm and 100 mm, between 50 mm and 500 mm, between 75 mm and 150 mm, between 75 mm and 300 mm, between 100 mm and 200 mm, between 100 mm and 500 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm, about 450 mm, or about 500 mm). In some embodiments, a ratio of the minimum cross-sectional dimension of a lumen of any of the first electrode or the second electrode to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1).
In some embodiments of any of the preceding aspects, a ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 3:1 and 10:1, between 4:1 and 25:1, between 5:1 and 50:1, between 10:1 and 50:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1). In some embodiments, a ratio of a transverse cross-sectional area of a lumen of any of the first electrode and/or the second electrode to the transverse cross-sectional area of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 3:1 and 10:1, between 4:1 and 25:1, between 5:1 and 50:1, between 10:1 and 50:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1). Either the first or second electrode, or both, can be porous or a conductive fluid (e.g., liquid).
In some embodiments of any of the preceding aspects, the device further includes a first reservoir in fluidic communication with the first inlet. In some embodiments, the further includes a second reservoir in fluidic communication with the second outlet. In some embodiments, the device further includes a third reservoir in fluidic communication with the third inlet and the third outlet. In some embodiments, the device further includes a fourth reservoir in fluidic communication with the fourth inlet and the fourth outlet. In some embodiments, the device further includes a fifth reservoir in fluidic communication with a lumen of any of the first electrode or the second electrode, wherein any of the first electrode or the second electrode has at least one additional inlet for fluidic communication with the fifth reservoir. In some embodiments, the device further includes a fluid delivery source in fluidic communication with the first inlet, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, the device further includes a plurality of electroporation zones (e.g., arranged in series, in parallel, or a combination thereof). Each of the plurality of electroporation zones can have a substantially uniform transverse cross-sectional area.
In some embodiments of any of the preceding aspects, the device further includes a housing including a housing (e.g., a cartridge) configured to encase the first electrode, the second electrode, and the at least one electroporation zone of the device. The housing may include a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. In some embodiments, the housing further includes a thermal controller configured to increase the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device. In some embodiments, the housing is either integral to the device or releasably connected to the device. In some embodiments, the housing (e.g., cartridge) is configured for use with and/or insertion into an automated closed system that is used to deliver cell therapies to patients in a clinical or hospital setting.
In some embodiments, the housing further includes a cooling/heating area/enclosure for cell suspension and/or buffer storage during, before, and after electroporation of the specimen. In some embodiments, the system (e.g., device and housing) is externally powered.
In some embodiments, devices of the invention include a user interface or other alternative user interface that enables the user to select parameters such as flow rate, waveforms, applied potential, volume to transfect, time delay, cooling features, heating features, transfection status, progress and other parameters used to optimize the transfection protocol. In some embodiments, the user interface also contains pre-formulated parameter selections that enable the user to operate the system at standard conditions that have previously been validated by user or as recommended by the manufacturers. In some embodiments, the user interface may be connected to programming that allows for automated running of the system and/or running an algorithm to optimize transfection for a given sample of a known cell type. In some embodiments, the optimization algorithms have the ability to adjust transfection parameters independently or autonomously if the user selects this functionality. In some embodiments, the optimization algorithms allow for continuous adjustment of the parameters used in the transfection process that may depend on the cell type, conductivity of cell suspensions, volume of cell suspensions, viscosity, lifetime of the transfection cartridge(s), the physical state of the suspension, or the state of the transfection device(s).
In some embodiments, the optimization algorithms have the ability to perform predictive analysis based on known input cell-type parameters and to adjust transfection parameters accordingly. In some embodiments, the optimization algorithms adjust transfection parameters based on electrical signals within any of the devices of the invention. In some embodiments, the optimization algorithms adjust transfection parameters based on unique dimensionless input parameters. In some embodiments, the optimization algorithms have the ability to adjust transfection parameters based on unique multivariate combinations of parameters that are predictive of high viability results, high efficiency results, or matched viability and efficiency results. These unique multivariate combinations of parameters may be known relationships, such as Reynolds number, or can be new relationships, such as the relationship between shear rate and fluid velocity, channel diameter and fluid velocity, electric field and fluid velocity, etc. Generally speaking, there is a balance between the amount of electrical energy delivered via the applied electrical pulse and the amount mechanical energy delivered through fluid flow. Depending upon the desired outcome, e.g. high cell yield or high efficiency, an optimum combination of the electrical and mechanical effects exists. For a given electrical pulse condition, faster fluid flow decreases the amount of electrical energy exposed to the cell and tends to increase cell viability and cell yield. But this only occurs to a point, beyond which cell yield begins to decrease as the cells don't experience sufficient electrical energy for efficient payload delivery.
In another aspect, the invention includes a system for electro-mechanical delivery of a composition into a plurality of cells suspended in a liquid, wherein the system includes any of the aforementioned embodiments of the device.
In another aspect, the invention includes a system for electroporating a composition into a plurality of cells suspended in a liquid, wherein the system includes any of the aforementioned embodiments of the device (e.g., wherein the electroporating occurs via an electro-mechanical delivery mechanism).
In another aspect, the invention includes a system for electro-mechanical delivery of a composition into a plurality of cells suspended in a liquid, including a device and a source of electrical potential. The device includes a first electrode, a second electrode, and an electroporation zone. The first electrode includes a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension; and the second electrode includes a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension. The electroporation zone is disposed between the first outlet and the second inlet and has a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm). The electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication. The system further includes a source of electrical potential, wherein the first electrode and the second electrode of the device are releasably in operative contact with the source of electrical potential. In some embodiments, the device further includes a first reservoir in fluidic communication with the first inlet and/or a second reservoir in fluidic communication with the second outlet.
In another aspect, the invention includes a system for electroporating a composition into a plurality of cells suspended in a liquid, including a device and a source of electrical potential. The device includes a first electrode, a second electrode, and an electroporation zone. The first electrode includes a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension; and the second electrode includes a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension. The electroporation zone is disposed between the first outlet and the second inlet and has a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm). The electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluidic communication. The system further includes a source of electrical potential, wherein the first electrode and the second electrode of the device are releasably in operative contact with the source of electrical potential. In some embodiments, the device further includes a first reservoir in fluidic communication with the first inlet and/or a second reservoir in fluidic communication with the second outlet.
In some embodiments of either of the preceding aspects, the transverse cross-section of the electroporation zone is a shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular. In some embodiments, the electroporation zone has a substantially circular transverse cross-section. In some embodiments, the electroporation zone has a minimum cross-sectional dimension of between 0.1 mm and 50 mm (e.g., between 0.1 mm and 0.5 mm, between 0.1 mm and 1 mm, between 0.1 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 45 mm and 60 mm, between 50 mm and 100 mm, between 75 mm and 150 mm, between 100 mm and 200 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm).
In some embodiments of any of the preceding systems of the invention, the electroporation zone has a transverse cross-sectional area of between about 7,850 μm2 and about 2,000 mm2 (e.g., between about 8,000 μm2 and about 1 mm2, between about 8,000 μm2 and about 10 mm2, between about 8,000 μm2 and about 100 mm2, between about 9,000 μm2 and 5 mm2, between about 1 mm2 and about 10 mm2, between about 1 mm2 and about 100 mm2, between about 3 mm2 and about 20 mm2, between about 10 mm2 and about 50 mm2, between about 25 mm2 and about 75 mm2, between about 50 mm2 and about 100 mm2, between about 75 mm2 and about 200 mm2, between about 100 mm2 and about 350 mm2, between about 150 mm2 and about 500 mm2, between about 300 mm2 and about 750 mm2, between about 500 mm2 and about 1,000 mm2, between about 750 mm2 and about 1,500 mm2, or between about 950 mm2 and about 2,000 mm2, e.g., about 8,000 μm2, about 9,000 μm2, about 1 mm2, about 5 mm2, about 10 mm2, about 15 mm2, about 20 mm2, about 25 mm2, about 50 mm2, about 60 mm2, about 75 mm2, about 80 mm2, about 100 mm2, about 150 mm2, about 200 mm2, about 250 mm2, about 300 mm2, about 350 mm2, about 400 mm2, about 450 mm2, about 500 mm2, about 600 mm2, about 700 mm2, about 800 mm2, about 900 mm2, about 1,000 mm2, about 1,100 mm2, about 1,200 mm2, about 1,300 mm2, about 1,400 mm2, about 1,500 mm2, about 1,600 mm2, about 1,700 mm2, about 1,800 mm2, about 1,900 mm2, or about 2,000 mm2).
In some embodiments of any of the preceding systems, the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm). In some embodiments of the systems, the length of the electroporation zone is between 0.005 mm and 25 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 10 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 10 mm, between 7 mm and 15 mm, between 10 mm and 20 mm, or between 15 mm and 25 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, about 23 mm, or about 25 mm).
In some embodiments of any of the preceding systems, a lumen of any of the first electrode and/or the second electrode has a minimum cross-sectional dimension of between 0.01 mm and 500 mm (e.g., between 0.01 mm and 0.1 mm, between 0.01 mm and 0.5 mm, between 0.01 mm and 10 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 50 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 30 mm and 300 mm, between 45 mm and 60 mm, between 50 mm and 100 mm, between 50 mm and 500 mm, between 75 mm and 150 mm, between 75 mm and 300 mm, between 100 mm and 200 mm, between 100 mm and 500 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm, about 450 mm, or about 500 mm).
In some embodiments of the systems of the invention, a ratio of the minimum cross-sectional dimension of a lumen of any of the first electrode or the second electrode to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1). In some embodiments, a ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 3:1 and 10:1, between 4:1 and 25:1, between 5:1 and 50:1, between 10:1 and 50:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1).
In some embodiments, a ratio of a transverse cross-sectional area of a lumen of any of the first electrode and/or the second electrode to the transverse cross-sectional area of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:10 and 10:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 50:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 10:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 2:1 and 50:1, between 3:1 and 10:1, between 3:1 and 30:1, between 4:1 and 25:1, between 5:1 and 10:1, between 5:1 and 50:1, between 10:1 and 50:1, between 10:1 and 100:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1). Either of the first electrode or the second electrode, or both, can be porous or a conductive fluid (e.g., liquid).
In some embodiments of any of the preceding systems, the system includes a third reservoir in fluidic communication with a lumen of any of the first electrode or the second electrode, wherein any of the first electrode or the second electrode has an additional inlet for fluidic communication with the third reservoir. In some embodiments, the system further includes a fluid delivery source in fluidic communication with the first inlet, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet.
In some embodiments, the system of the invention further includes a controller operatively coupled to the source of electrical potential to deliver voltage pulses to the first electrode and the second electrode, wherein the voltage pulses generate an electrical potential difference between the first electrode and the second electrode, thus producing an electric field in the electroporation zone. In some embodiments, the system includes a plurality of electroporation zones (e.g., as part of a plurality of any embodiment(s) of the devices provided herein). Each of the plurality of electroporation zones can have a substantially uniform or non-uniform transverse cross-sectional area.
In some embodiments, the system further includes an outer structure including a housing configured to encase the first electrode, the second electrode, and the at least one electroporation zone of the device (e.g., wherein the outer structure further includes a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode). The housing may include a thermal controller configured to increase the temperature of the device and/or of the liquid in which the plurality of cells is suspended. The thermal controller can be a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. Additionally or alternatively, the thermal controller can be configured to decrease the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device.
In some embodiments of any of preceding the systems of the invention, the source of electrical potential is releasably connected to the first and second electrical inputs of the outer structure. The releasable connection between the first or second electrical inputs and the source of electrical potential can be selected from a group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof. The outer structure may be integral to, or releasably connected to, the device. In some embodiments, a housing (e.g., cartridge) is configured to energize a plurality of devices in parallel, in series, or offset in time, wherein the housing further includes a tray that accommodates a plurality of electroporation devices, wherein the tray is modified with two grid electrodes, wherein a first grid electrode is electrically isolated from a second grid electrode, wherein an exterior of the first electrode of each of the plurality of devices is releasably in operative contact with any of a first spring-loaded electrode, a first mechanically connected electrode, or a first inductively connected electrode, wherein an exterior of the second electrode of each of the plurality of devices is releasably in operative contact with any of a second spring-loaded electrode, a second mechanically connected electrode, or a second inductively coupled electrode, wherein each of the plurality of devices releasably enters the housing (e.g., cartridge) through an opening in the grid electrodes, wherein any of the first spring-loaded electrode, first mechanically connected electrode, or first inductively connected electrode of each device is in operative contact with the first grid electrode and any of the second spring-loaded electrode, second mechanically connected electrode, or second inductively connected electrode of each device is in operative contact with the second grid electrode, wherein the grid electrodes are connected to the source of electrical potential.
In some embodiments, the housing (e.g., cartridge) encapsulates one or more of the previously stated inventions or one or more electroporating devices used for continuous flow electroporation. In some embodiments, the housing (e.g., cartridge) is configured to allow use with and/or insertion into an automated closed system that delivers cell therapies to patients. In some embodiments, the housing further includes a cooling/heating area/enclosure for cell suspension and/or buffer storage during, before and after electroporation of the specimen. In some embodiments, the system (e.g., one or more devices and housing) is externally powered.
In some embodiments, the system also includes optimization algorithms that have the ability to adjust electroporation parameters independently or autonomously if the user selects this functionality.
These optimization algorithms allow for continuous adjustment of the parameters used in the electroporation process that may depend on the cell type, conductivity, volume of suspensions, viscosity, lifetime of the electroporating cartridge, the physical state of the suspension or the state of the electroporation device.
In some embodiments of any of the preceding aspects of the system, the source of electrical potential delivers voltage pulses to the grid electrodes, wherein the first grid electrode is energized at a particular applied voltage while the second grid electrode is energized at a particular applied voltage, wherein each of the plurality of devices is energized by the grid electrodes with an identical applied voltage pulse such that a magnitude of an electric field generated within each of the at least one electroporation zones of each device is substantially identical. In some embodiments, the source of electrical potential includes additional circuitry or programming configured to modulate the delivery of voltage pulses to the grid electrodes, wherein each of the plurality of devices may receive a different voltage from the grid electrodes, wherein a magnitude of an electric field generated within each of the at least one electroporation zones of each device is different.
In another aspect, the invention provides a system for electro-mechanical delivery of a composition into a plurality of cells suspended in a liquid, including: a device, including a first electrode including a first inlet, a first outlet, and a first lumen; a second electrode including a second inlet, a second outlet, and a second lumen; a third inlet and a third outlet, wherein the third inlet and the third outlet are in fluidic communication with the first lumen, wherein the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, wherein the fourth inlet and the fourth outlet are in fluidic communication with the second lumen, wherein the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm) and includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein a transverse cross-sectional area of the electroporation zone is substantially uniform; and wherein a ratio of a minimum cross-sectional dimension of the first lumen to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1), wherein a ratio of a minimum cross-sectional dimension of the second lumen to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1), and wherein the first outlet, the electroporation zone, and the second inlet are in fluidic communication; and a source of electrical potential, wherein the first and second electrodes of the device are releasably in operative contact with the source of electrical potential. The transverse cross-section of the electroporation zone is a closed shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular. The electroporation zone can have a substantially circular transverse cross-section.
In another aspect, the invention provides a system for electroporating a composition into a plurality of cells suspended in a liquid, including: a device, including a first electrode including a first inlet, a first outlet, and a first lumen; a second electrode including a second inlet, a second outlet, and a second lumen; a third inlet and a third outlet, wherein the third inlet and the third outlet are in fluidic communication with the first lumen, wherein the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, wherein the fourth inlet and the fourth outlet are in fluidic communication with the second lumen, wherein the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm) and includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein a transverse cross-sectional area of the electroporation zone is substantially uniform; and wherein a ratio of a minimum cross-sectional dimension of the first lumen to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1), wherein a ratio of a minimum cross-sectional dimension of the second lumen to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1), and wherein the first outlet, the electroporation zone, and the second inlet are in fluidic communication; and a source of electrical potential, wherein the first and second electrodes of the device are releasably in operative contact with the source of electrical potential. The transverse cross-section of the electroporation zone is a closed shape selected from a group consisting of circular, disk, elliptical, regular polygon, irregular polygon, curvilinear shape, star, parallelogram, trapezoidal, and irregular. The electroporation zone can have a substantially circular transverse cross-section.
In some embodiments of either of the preceding aspects, the electroporation zone has a minimum cross-sectional dimension of between 0.1 mm and 50 mm (e.g., between 0.1 mm and 0.5 mm, between 0.1 mm and 1 mm, between 0.1 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 45 mm and 60 mm, between 50 mm and 100 mm, between 75 mm and 150 mm, between 100 mm and 200 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm).
In some embodiments, the electroporation zone has a transverse cross-sectional area of between about 7,850 μm2 and about 2,000 mm2 (e.g., between about 8,000 μm2 and about 1 mm2, between about 8,000 μm2 and about 10 mm2, between about 8,000 μm2 and about 100 mm2, between about 9,000 μm2 and 5 mm2, between about 1 mm2 and about 10 mm2, between about 1 mm2 and about 100 mm2, between about 3 mm2 and about 20 mm2, between about 10 mm2 and about 50 mm2, between about 25 mm2 and about 75 mm2, between about 50 mm2 and about 100 mm2, between about 75 mm2 and about 200 mm2, between about 100 mm2 and about 350 mm2, between about 150 mm2 and about 500 mm2, between about 300 mm2 and about 750 mm2, between about 500 mm2 and about 1,000 mm2, between about 750 mm2 and about 1,500 mm2, or between about 950 mm2 and about 2,000 mm2, e.g., about 8,000 μm2, about 9,000 μm2, about 1 mm2, about 5 mm2, about 10 mm2, about 15 mm2, about 20 mm2, about 25 mm2, about 50 mm2, about 60 mm2, about 75 mm2, about 80 mm2, about 100 mm2, about 150 mm2, about 200 mm2, about 250 mm2, about 300 mm2, about 350 mm2, about 400 mm2, about 450 mm2, about 500 mm2, about 600 mm2, about 700 mm2, about 800 mm2, about 900 mm2, about 1,000 mm2, about 1,100 mm2, about 1,200 mm2, about 1,300 mm2, about 1,400 mm2, about 1,500 mm2, about 1,600 mm2, about 1,700 mm2, about 1,800 mm2, about 1,900 mm2, or about 2,000 mm2).
In some embodiments, the electroporation zone has a length of between 0.005 mm and 50 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.005 mm and 25 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.1 mm and 50 mm, between 0.5 mm and 5 mm, between 0.5 mm and 25 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 50 mm, between 15 mm and 25 mm, between 20 mm and 30 mm, between 25 mm and 40, or between 30 mm and 50 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm). In some embodiments of the system of the invention, the length of the electroporation zone is between 0.005 mm and 25 mm (e.g., between 0.005 mm and 0.05 mm, between 0.005 mm and 0.5 mm, between 0.01 mm and 1 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 10 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 10 mm, between 7 mm and 15 mm, between 10 mm and 20 mm, or between 15 mm and 25 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 12 mm, about 15 mm, about 18 mm, about 20 mm, about 23 mm, or about 25 mm).
In some embodiments, the device or system further includes a plurality of fluid constraining cavities for cell transfection that are in fluidic communication with the at least one electroporation zone and in which the plurality of cells experience similar or different electric fields and similar or different flow velocities (e.g., displacement velocities). In some embodiments, the electrodes may be in any configuration (e.g., manifold) with multiple inlets and outlets that lead to a constriction and support cell electroporation in multiple non-conductive fluid constraining cavities. In some embodiments, each of the plurality of fluid constraining cavities for cell electroporation includes an individual housing (e.g., cartridge) and can operate independently. In some embodiments, each individual housing (e.g., cartridge) is configured to be exposed to the same or different operating parameters that control flow velocities (e.g., displacement velocities), electric field, or a combination of other parameters. In some embodiments, each housing is configured to be integrated into one or more controller systems. In other embodiments, the one or more controller systems are configured to accept one or more individual housings.
In some embodiments, the device or system further includes a plurality of fluid constraining cavities for cell electroporation that are in fluidic communication with the at least one electroporation zone and in which the plurality of cells experience similar or different electric fields and similar or different flow velocities (e.g., displacement velocities). In some embodiments, the electrodes may be in any configuration (e.g., manifold) with multiple inlets and outlets that lead to a constriction and support cell electroporation in multiple non-conductive fluid constraining cavities. In some embodiments, each of the plurality of fluid constraining cavities for cell electroporation includes an individual housing (e.g., cartridge) and can operate independently. In some embodiments, each individual housing (e.g., cartridge) is configured to be exposed to the same or different operating parameters that control flow velocities (e.g., displacement velocities), electric field, or a combination of other parameters. In some embodiments, each housing is configured to be integrated into one or more controller systems. In other embodiments, the one or more controller systems are configured to accept one or more individual housings.
In some embodiments, a lumen of any of the first electrode and/or the second electrode has a minimum cross-sectional dimension of between 0.01 mm and 500 mm (e.g., between 0.01 mm and 0.1 mm, between 0.01 mm and 0.5 mm, between 0.01 mm and 10 mm, between 0.05 mm and 5 mm, between 0.1 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 50 mm, between 1 mm and 5 mm, between 1 mm and 10 mm, between 1 mm and 25 mm, between 3 mm and 15 mm, between 3 mm and 50 mm, between 10 mm and 20 mm, between 10 mm and 100 mm, between 15 mm and 30 mm, between 20 mm and 40 mm, between 20 mm and 200 mm, between 30 mm and 50, between 30 mm and 300 mm, between 45 mm and 60 mm, between 50 mm and 100 mm, between 50 mm and 500 mm, between 75 mm and 150 mm, between 75 mm and 300 mm, between 100 mm and 200 mm, between 100 mm and 500 mm, between 150 mm and 300 mm, between 200 mm and 400 mm, between 300 mm and 450 mm, or between 350 mm and 500 mm, e.g., about 0.005 mm, about 0.01 mm, about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 10 mm, about 15 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm, about 450 mm, or about 500 mm).
In some embodiments, a ratio of the minimum cross-sectional dimension of a lumen of any of the first electrode or the second electrode to the minimum cross-sectional dimension of the electroporation zone is between 1:10 and 10:1 (e.g., between 1:10 and 1:5, between 1:10 and 1:2, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 5:1, between 1:2 and 2:3, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 6:1, between 2:3 and 2:1, between 2:3 and 4:1, between 1:1 and 2:1, between 1:1 and 3:1, between 1:1 and 10:1, between 3:2 and 3:1, between 3:2 and 6:1, between 2:1 and 3:1, between 2:1 and 5:1, between 5:2 and 5:1, between 3:1 and 4:1, between 7:2 and 5:1, between 7:2 and 10:1, between 4:1 and 8:1, between 5:1 and 10:1, or between 7:1 and 10:1, e.g., about 1:10, about 1:5, about 1:2, about 2:3, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 9:2, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1). In some embodiments, a ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:10 and 10:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 50:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 10:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 2:1 and 50:1, between 3:1 and 10:1, between 3:1 and 30:1, between 4:1 and 25:1, between 5:1 and 10:1, between 5:1 and 50:1, between 10:1 and 50:1, between 10:1 and 100:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1). In some embodiments, a ratio of a transverse cross-sectional area of a lumen of any of the first electrode and/or the second electrode to the transverse cross-sectional area of the electroporation zone is between 1:100 and 100:1 (e.g., between 1:100 and 1:50, between 1:100 and 1:25, between 1:100 and 1:10, between 1:100 and 1:1, between 1:50 and 1:5, between 1:50 and 1:2, between 1:50 and 2:1, between 1:25 and 1:10, between 1:25 and 1:5, between 1:25 and 1:1, between 1:25 and 10:1, between 1:10 and 1:1, between 1:10 and 2:1, between 1:10 and 5:1, between 1:10 and 10:1, between 1:5 and 1:2, between 1:5 and 1:1, between 1:5 and 2:1, between 1:5 and 50:1, between 1:2 and 1:1, between 1:2 and 2:1, between 1:2 and 10:1, between 1:1 and 2:1, between 1:1 and 5:1, between 1:1 and 10:1, between 1:1 and 50:1, between 1:1 and 100:1, between 2:1 and 5:1, between 2:1 and 20:1, between 2:1 and 50:1, between 3:1 and 10:1, between 3:1 and 30:1, between 4:1 and 25:1, between 5:1 and 10:1, between 5:1 and 50:1, between 10:1 and 50:1, between 10:1 and 100:1, between 40:1 and 80:1, between 50:1 and 100:1, or between 75:1 and 90:1, e.g., about 1:100, about 1:75, about 1:50, about 1:25, about 1:10, about 1:5, about 1:2, about 1:1, about 3:2, about 2:1, about 5:2, about 3:1, about 7:2, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1).
In some embodiments, the system further includes a first reservoir in fluidic communication with the first inlet, a second reservoir in fluidic communication with the second outlet, a third reservoir in fluidic communication with the third inlet and the third outlet, a fourth reservoir in fluidic communication with the fourth inlet and the fourth outlet, and/or a fifth reservoir in fluidic communication with a lumen of any of the first electrode or the second electrode, e.g., wherein any of the first electrode or the second electrode has at least one additional inlet for fluidic communication with the fifth reservoir. In some embodiments, the system further includes a fluid delivery source in fluidic communication with the first inlet, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, the device further includes a plurality of electroporation zones, e.g., wherein each of the plurality of electroporation zones has a substantially uniform or non-uniform transverse cross-sectional area.
The system of any of the previous aspects can additionally include a controller operatively coupled to the source of electrical potential to deliver voltage pulses to the first and second electrodes to generate an electrical potential difference between the first and second electrodes, thus producing an electric field in the electroporation zone.
In some embodiments, the system further includes an outer structure including a housing configured to encase the first electrode, the second electrode, and the at least one electroporation zone of the device. The system can further include a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. The housing can further include a thermal controller configured to increase the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. Additionally or alternatively, the housing can further include a thermal controller configured to decrease the temperature of the device and/or of the liquid in which the plurality of cells is suspended, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device. In some embodiments, the source of electrical potential is releasably connected to the first and second electrical inputs of the outer structure, e.g., wherein the releasable connection between the first or second electrical inputs and the source of electrical potential is selected from a group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof. The outer structure and/or housing can be integral to, or releasably connected to, the device.
In some embodiments, the system further includes a plurality of devices, e.g., having a plurality of outer structures. In some embodiments, a housing is configured to energize a plurality of devices in parallel, in series, or offset in time, wherein the housing further includes a tray that accommodates a plurality of electroporation devices, wherein the tray is modified with two grid electrodes, wherein a first grid electrode is electrically isolated from a second grid electrode, wherein an exterior of the first electrode of each of the plurality of devices is releasably in operative contact with any of a first spring-loaded electrode, a first mechanically connected electrode, or a first inductively connected electrode, wherein an exterior of the second electrode of each of the plurality of devices is releasably in operative contact with any of a second spring-loaded electrode, a second mechanically connected electrode, or a second inductively coupled electrode, wherein each of the plurality of devices releasably enters the housing through an opening in the grid electrodes, wherein any of the first spring-loaded electrode, first mechanically connected electrode, or first inductively connected electrode of each device is in operative contact with the first grid electrode and any of the second spring-loaded electrode, second mechanically connected electrode, or second inductively connected electrode of each device is in operative contact with the second grid electrode, wherein the grid electrodes are connected to the source of electrical potential. In some embodiments, the source of electrical potential delivers voltage pulses to the grid electrodes, wherein the first grid electrode is energized at a particular applied voltage while the second grid electrode is energized at a particular applied voltage, wherein each of the plurality of devices is energized by the grid electrodes with an identical applied voltage pulse such that a magnitude of an electric field generated within each of the at least one electroporation zones of each device is substantially identical. In some embodiments, the source of electrical potential includes additional circuitry or programming configured to modulate the delivery of voltage pulses to the grid electrodes, wherein each of the plurality of devices may receive a different voltage from the grid electrodes, wherein a magnitude of an electric field generated within each of the at least one electroporation zones of each device may be different.
In another aspect, the invention provides a method of introducing a composition into a plurality of cells suspended in a flowing liquid using any of the devices or systems of the invention (e.g., by electro-mechanical delivery). In particular, methods of the invention include providing a device including a first electrode including a first outlet, a first inlet, and a first lumen including a minimum cross-sectional dimension; a second electrode including a second outlet, a second inlet, and a second lumen including a minimum cross-sectional dimension; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform cross sectional area; and wherein the first outlet, the electroporation zone, and the second inlet are in fluidic communication; applying an electrical potential difference between the first and second electrodes, thereby producing an electric field in the electroporation zone; and passing the plurality of cells and the composition through the electroporation zone, thereby enhancing permeability of the plurality of cells and introducing the composition into the plurality of cells. In some embodiments, the passing the plurality of the cells includes applying a fluid-driven positive pressure. In some embodiments, none of the first lumen, second lumen, or electroporation zone has a minimum cross-sectional dimension that causes a cross-sectional dimension of any of the plurality of cells suspended in the liquid to be compressed temporarily. The electroporation can be substantially non-thermal reversible electroporation, substantially non-thermal irreversible electroporation, or substantially thermal irreversible electroporation. In some embodiments, a flow rate of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 0.001 mL/min and 1,000 mL min (e.g., between 0.001 mL/min and 0.05 mL/min, between 0.001 mL/min and 0.1 mL/min, between 0.001 mL/min and 1 mL/min, between 0.05 mL/min and 0.5 mL/min, between 0.05 mL/min and 5 mL/min, between 0.1 mL/min and 1 mL/min, between 0.5 mL/min and 2 mL/min, between 1 mL/min and 5 mL/min, between 1 mL/min and 10 mL/min, between 1 mL/min and 100 mL/min, between 5 mL/min and 25 mL/min, between 5 mL/min and 150 mL/min, between 10 mL/min and 100 mL/min, between 15 mL/min and 150 mL/min, between 25 mL/min and 100 mL/min, between 25 mL/min and 200 mL/min, between 50 mL/min and 150 mL/min, between 50 mL/min and 250 mL/min, between 75 mL/min and 200 mL/min, between 75 mL/min and 350 mL/min, between 100 mL/min and 250 mL/min, between 100 mL/min and 400 mL/min, between 150 mL/min and 450 mL/min, between 200 mL/min and 500 mL/min, between 250 mL/min and 700 mL/min, between 300 mL/min and 1,000 mL/min, between 400 mL/min and 750 mL/min, between 500 mL/min and 1,000 mL/min, or between 750 mL/min and 1,000 mL/min, e.g., about 0.001 mL/min, about 0.01 mL/min, about 0.05 mL/min, about 0.1 mL/min, about 0.5 mL/min, about 1 mL/min, about 5 mL/min, about 10 mL/min, about 15 mL/min, about 20 mL/min, about 30 mL/min, about 40 mL/min, about 50 mL/min, about 60 mL/min, about 70 mL/min, about 80 mL/min, about 90 mL/min, about 100 mL/min, about 150 mL/min, about 200 mL/min, about 250 mL/min, about 300 mL/min, about 350 mL/min, about 400 mL/min, about 450 mL/min, about 500 mL/min, about 600 mL/min, about 700 mL/min, about 800 mL/min, about 900 mL/min, or about 1,000 mL/min), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet.
In some embodiments of any of the previous aspects, a Reynolds number of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 0.04 and 2.43×104 (e.g., between 10 and 2000, between 100 and 1600, between 100 and 1800, or between 183 and 1530), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, a maximum velocity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 5×10−5 m/s and 32.7 m/s (e.g., between 0.01 and 10 m/s, between 0.1 and 5 m/s, or between 0.26 and 2.08 m/s), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, shear rates of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone are between 0.1 1/s and 2×106 1/s (e.g., between 1/s and 100,000/s, between 10/s and 100,000/s, between 100/s and 100,000/s, between 1,000/s and 80,000/s, or between 2,600 and 54,000), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, a peak pressure of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1×10−3 Pa and 9.5×104 Pa (e.g., between 0.1 Pa and 10,000 Pa, between 1 Pa and 5,000 Pa, between 100 Pa and 3000 Pa, or between 136 Pa and 1600 Pa), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, an average velocity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1.5×10−5 m/s and 15.9 m/s (e.g., between 7.79×10−2 m/s and 7.81×10−1 m/s), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some embodiments, a kinematic viscosity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1×10−6 m2/s and 15×10−4 m2/s (e.g., between 1.3×10−6 m2/s and 5.6×10−4 m2/s), wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet.
In some embodiments of any of the previous aspects, a residence time in the electroporation zone of any of the plurality of cells suspended in the liquid is between 0.5 ms and 50 ms (e.g., between 0.5 ms and 5 ms, between 1 ms and 10 ms, between 1 ms and 15 ms, between 5 ms and 15 ms, between 10 ms and 20 ms, between 15 ms and 25 ms, between 20 ms and 30 ms, between 25 ms and 35 ms, between 30 ms and 40 ms, between 35 ms and 45 ms, or between 40 ms and 50 ms, e.g., about 0.5 ms, about 0.6 ms, about 0.7 ms, about 0.8 ms, about 0.9 ms, about 1 ms, about 1.5 ms, about 2 ms, about 2.5 ms, about 3 ms, about 3.5 ms, about 4 ms, about 4.5 ms, about 5 ms, about 5.5 ms, about 6 ms, about 6.5 ms, about 7 ms, about 7.5 ms, about 8 ms, about 8.5 ms, about 9 ms, about 9.5 ms, about 10 ms, about 10.5 ms, about 11 ms, about 11.5 ms, about 12 ms, about 12.5 ms, about 13 ms, about 13.5 ms, about 14 ms, about 14.5 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, or about 50 ms). In some embodiments, the residence time is from 5-20 ms (e.g., from 6-18 ms, 8-15 ms, or 10-14 ms).
In some embodiments of any of the preceding aspects, the plurality of cells has from 0% to about 25% phenotypic change (e.g., from about 0% to about 2.5%, from about 1% to about 5%, from about 1% to about 10%, from about 5% to about 15%, from about 10% to about 20%, from about 15% to about 25%, or from about 20% to about 25%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%) relative to a baseline measurement of cell phenotype upon exiting the second outlet of the device (e.g., within 48 hours after exiting the second outlet, e.g., within 24 hours after exiting the second outlet, e.g., between 1 minute and 24 hours, 5 minutes and 24 hours, 10 minutes and 24 hours, 30 minutes and 24 hours, 1 hour and 24 hours, or 2 hours and 24 hours after exiting the second outlet).
In some embodiments of any of the preceding aspects, the plurality of cells have no phenotypic change relative to a baseline measurement of cell phenotype upon exiting the second outlet of the device (e.g., within 48 hours after exiting the second outlet, e.g., within 24 hours after exiting the second outlet, e.g., between 1 minute and 24 hours, 5 minutes and 24 hours, 10 minutes and 24 hours, 30 minutes and 24 hours, 1 hour and 24 hours, or 2 hours and 24 hours after exiting the second outlet).
In some embodiments of any of the preceding aspects, the electric field is produced by voltage pulses, wherein the voltage pulses energize the first electrode at a particular applied voltage while the second electrode is energized at a particular applied voltage, thus applying an electrical potential difference between the first and second electrodes, wherein the voltage pulses each have an amplitude between −3 kV and 3 kV (e.g., between −3 kV and 1 kV, between −3 kV and −1.5 kV, between −2 kV and 2 kV, between −1.5 kV and 1.5 kV, between −1.5 kV and 2.5 kV, between −1 kV and 1 kV, between −1 kV and 2 kV, between −0.5 kV and 0.5 kV, between −0.5 kV and 1.5 kV, between −0.5 kV and 3 kV, between −0.01 kV and 2 kV, between 0 kV and 1 kV, between 0 kV and 2 kV, between 0 kV and 3 kV, between 0.01 kV and 0.1 kV, between 0.01 kV and 1 kV, between 0.02 kV and 0.2 kV, between 0.03 kV and 0.3 kV, between 0.04 kV and 0.4 kV, between 0.05 kV and 0.5 kV, between 0.05 kV and 1.5 kV, between 0.06 kV and 0.6 kV, between 0.07 kV and 0.7 kV, between 0.08 kV and 0.8 kV, between 0.09 kV and 0.9 kV, between 0.1 kV and 0.7 kV, between 0.1 kV and 1 kV, between 0.1 kV and 2 kV, between 0.1 kV and 3 kV, between 0.15 kV and 1.5 kV, between 0.2 and 0.6 kV, between 0.2 kV and 2 kV, between 0.25 kV and 2.5 kV, between 0.3 kV and 3 kV, between 0.5 kV and 1 kV, between 0.5 kV and 3 kV, between 0.6 kV and 1.5 kV, between 0.7 kV and 1.8 kV, between 0.8 kV and 2 kV, between 0.9 kV and 3 kV, between 1 kV and 2 kV, between 1.5 kV and 2.5 kV, or between 2 kV and 3 kV, e.g., about −3 kV, about −2.5 kV, about −2 kV, about −1.5 kV, about −1 kV, about −0.5 kV, about −0.01 kV, about 0 kV, about 0.01 kV, about 0.02 kV, about 0.03 kV, about 0.04 kV, about 0.05 kV, about 0.06 kV, about 0.07 kV, about 0.08 kV, about 0.09 kV, about 0.1 kV, about 0.2 kV, about 0.3 kV, about 0.4 kV, about 0.5 kV, about 0.6 kV, about 0.7 kV, about 0.8 kV, about 0.9 kV, about 1 kV, about 1.1 kV, about 1.2 kV, about 1.3 kV, about 1.4 kV, about 1.5 kV, about 1.6 kV, about 1.7 kV, about 1.8 kV, about 1.9 kV, about 2 kV, about 2.1 kV, about 2.2 kV, about 2.3 kV, about 2.4 kV, about 2.5 kV, about 2.6 kV, about 2.7 kV, about 2.8 kV, about 2.9 kV, or about 3 kV). In some embodiments, the first electrode is energized at a particular applied voltage while the second electrode is held at ground (e.g., 0 kV), thus applying an electrical potential difference between the first and second electrodes. In some embodiments, the voltage pulses have a duration of between 0.01 ms and 1,000 ms (e.g., between 0.01 ms and 0.1 ms, between 0.01 ms and 1 ms, between 0.01 ms and 10 ms, between 0.05 ms and 0.5 ms, between 0.05 ms and 1 ms, between 0.1 ms and 1 ms, between 0.1 ms and 5 ms, between 0.1 ms and 500 ms, between 0.5 ms and 2 ms, between 1 ms and 5 ms, between 1 ms and 10 ms, between 1 ms and 25 ms, between 1 ms and 100 ms, between 1 ms and 1,000 ms, between 5 ms and 25 ms, between 5 ms and 150 ms, between 10 ms and 100 ms, between 15 ms and 150 ms, between 25 ms and 100 ms, between 25 ms and 200 ms, between 50 ms and 150 ms, between 50 ms and 250 ms, between 75 ms and 200 ms, between 75 ms and 350 ms, between 100 ms and 250 ms, between 100 ms and 400 ms, between 150 ms and 450 ms, between 200 ms and 500 ms, between 250 ms and 700 ms, between 300 ms and 1,000 ms, between 400 ms and 750 ms, between 500 ms and 1,000 ms, or between 750 ms and 1,000 ms, e.g., about 0.01 ms, about 0.05 ms, about 0.1 ms, about 0.5 ms, about 1 ms, about 5 ms, about 10 ms, about 15 ms, about 20 ms, about 30 ms, about 40 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 150 ms, about 200 ms, about 250 ms, about 300 ms, about 350 ms, about 400 ms, about 450 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, or about 1,000 ms). In some embodiments, the voltage pulses are applied to the first and second electrodes at a frequency of between 1 Hz and 50,000 Hz (e.g., between 1 Hz and 10 Hz, between 1 Hz and 100 Hz, between 1 Hz and 1,000 Hz, between 5 Hz and 20 Hz, between 5 Hz and 2,000 Hz, between 10 Hz and 50 Hz, between 10 Hz and 100 Hz, between 10 Hz and 1,000 Hz, between 10 Hz and 10,000 Hz, between 20 Hz and 50 Hz, between 20 Hz and 100 Hz, between 20 Hz and 2,000 Hz, between 20 Hz and 20,000 Hz, between 50 Hz and 500 Hz, between 50 Hz and 1,000 Hz, between 50 Hz and 50,000 Hz, between 100 Hz and 200 Hz, between 100 Hz and 500 Hz, between 100 Hz and 1,000 Hz, between 100 Hz and 10,000 Hz, between 100 Hz and 50,000 Hz, between 200 Hz and 400 Hz, between 200 Hz and 750 Hz, between 200 Hz and 2,000 Hz, between 500 Hz and 1,000 Hz, between 750 Hz and 1,500 Hz, between 750 Hz and 10,000 Hz, between 1,000 Hz and 2,000 Hz, between 1,000 Hz and 5,000 Hz, between 1,000 Hz and 10,000 Hz, between 1,000 Hz and 50,000 Hz, between 5,000 Hz and 10,000 Hz, between 5,000 Hz and 20,000 Hz, between 5,000 Hz and 50,000 Hz, between 10,000 Hz and 15,000 Hz, between 10,000 Hz and 25,000 Hz, between 10,000 Hz and 50,000 Hz, between 20,000 Hz and 30,000 Hz, or between 20,000 and 50,000 Hz, e.g., about 1 Hz, about 5 Hz, about 10 Hz, about 20 Hz, about 50 Hz, about 75 Hz, about 100 Hz, about 150 Hz, about 200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900 Hz, about 1,000 Hz, about 2,000 Hz, about 5,000 Hz, about 10,000 Hz, about 15,000 Hz, about 20,000 Hz, about 30,000 Hz, about 40,000 Hz, or about 50,000 Hz).
In some embodiments, a waveform of the voltage pulse is selected from a group consisting of DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, arbitrary, and any superposition or combinations thereof. In some embodiments, the electric field generated from the voltage pulses has a magnitude of between 1 V/cm and 50,000 V/cm (e.g., between 1 V/cm and 50 V/cm, between 1 V/cm and 500 V/cm, between 1 V/cm and 1,000 V/cm, between 1 V/cm and 20,000 V/cm, between 5 V/cm and 10,000 V/cm, between 25 V/cm and 200 V/cm, between 50 V/cm and 250 V/cm, between 50 V/cm and 500 V/cm, between 50 V/cm and 15,000 V/cm, between 100 V/cm and 1,000 V/cm, between 300 V/cm and 500 V/cm, between 500 V/cm and 10,000 V/cm, between 1000 V/cm and 25,000 V/cm, between 5,000 V/cm and 25,000 V/cm, between 10,000 V/cm and 20,000 V/cm, between 10,000 V/cm and 50,000 V/cm, e.g., about 1 V/cm, about 2 V/cm, about 3 V/cm, about 4 V/cm, about 5 V/cm, about 6 V/cm, about 7 V/cm, about 8 V/cm, about 9 V/cm, about 10 V/cm, about 20 V/cm, about 30 V/cm, about 40 V/cm, about 50 V/cm, about 60 V/cm, about 70 V/cm, about 80 V/cm, about 90 V/cm, about 100 V/cm, about 150 V/cm, about 200 V/cm, about 250 V/cm, about 300 V/cm, about 350 V/cm, about 400 V/cm, about 450 V/cm, about 500 V/cm, about 550 V/cm, about 600 V/cm, about 650 V/cm, about 700 V/cm, about 750 V/cm, about 800 V/cm, about 900 V/cm, about 1,000 V/cm, about 2,000 V/cm, about 3,000 V/cm, about 4,000 V/cm, about 5,000 V/cm, about 6,000 V/cm, about 7,000 V/cm, about 8,000 V/cm, about 9,000 V/cm, about 10,000 V/cm, about 15,000 V/cm, about 20,000 V/cm, about 25,000 V/cm, about 30,000 V/cm, about 35,000 V/cm, about 40,000 V/cm, about 45,000 V/cm, or about 50,000 V/cm).
In some embodiments, a duty cycle of the voltage pulses is between 0.001% and 100% (e.g., between 0.001% and 0.1%, between 0.001% and 10%, between 0.01% and 1%, between 0.01% to 100%, between 0.1% and 5%, between 0.1% and 99%, between 1% and 10%, between 1% and 97%, between 2.5% and 20%, between 5% and 25%, between 5% and 40%, between 10% and 25%, between 10% and 50%, between 10% and 95%, between 15% and 60%, between 15% and 85%, between 20% and 40%, between 30% and 50%, between 40% and 60%, between 40% and 75%, between 50% and 85%, between 50% and 100%, between 75% and 100%, or between 90% and 100%, e.g., about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%).
In some embodiments, the liquid has a conductivity of between 0.001 mS/cm and 500 mS/cm (e.g., between 0.001 mS/cm and 0.05 mS/cm, between 0.001 mS/cm and 0.1 mS/cm, between 0.001 mS/cm and 1 mS/cm, between 0.05 mS/cm and 0.5 mS/cm, between 0.05 mS/cm and 5 mS/cm, between 0.1 mS/cm and 1 mS/cm, between 0.1 mS/cm and 100 mS/cm, between 0.5 mS/cm and 2 mS/cm, between 1 mS/cm and 5 mS/cm, between 1 mS/cm and 10 mS/cm, between 1 mS/cm and 100 mS/cm, between 1 mS/cm and 500 mS/cm, between 5 mS/cm and 25 mS/cm, between 5 mS/cm and 150 mS/cm, between 10 mS/cm and 100 mS/cm, between 10 mS/cm and 250 mS/cm, between 15 mS/cm and 150 mS/cm, between 25 mS/cm and 100 mS/cm, between 25 mS/cm and 200 mS/cm, between 50 mS/cm and 150 mS/cm, between 50 mS/cm and 250 mS/cm, between 50 mS/cm and 500 mS/cm, between 75 mS/cm and 200 mS/cm, between 75 mS/cm and 350 mS/cm, between 100 mS/cm and 250 mS/cm, between 100 mS/cm and 400 mS/cm, between 100 mS/cm and 500 mS/cm, between 150 mS/cm and 450 mS/cm, between 200 mS/cm and 500 mS/cm, between 300 mS/cm and 500 mS/cm, e.g., about 0.001 mS/cm, about 0.01 mS/cm, about 0.05 mS/cm, about 0.1 mS/cm, about 0.5 mS/cm, about 1 mS/cm, about 5 mS/cm, about 10 mS/cm, about 15 mS/cm, about 20 mS/cm, about 30 mS/cm, about 40 mS/cm, about 50 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, about 90 mS/cm, about 100 mS/cm, about 150 mS/cm, about 200 mS/cm, about 250 mS/cm, about 300 mS/cm, about 350 mS/cm, about 400 mS/cm, about 450 mS/cm, or about 500 mS/cm).
In some embodiments, a temperature of the plurality of cells suspended in the liquid is between 0° C. and 50° C. (between 0° C. and 5° C., between 2° C. and 15° C., between 3° C. and 30° C., between 4° C. and 10° C., between 4° C. and 25° C., between 5° C. and 30° C., between 7° C. and 35° C., between 10° C. and 25° C., between 10° C. and 40° C., between 15° C. and 50° C., between 20° C. and 40° C., between 25° and 50° C., or between 35° C. and 45° C., e.g., about 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C. about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C.).
In some embodiments, the method further includes storing the plurality of cells suspended in the liquid in a recovery buffer after poration. In some embodiments, the cells have a viability after introduction of the composition of between 0.1% and 99.9% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 99.9%, between 60% and 80%, between 75% and 99.9%, or between 85% and 99.9%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.9%).
In some embodiments, the composition is introduced into a plurality of the cells at an efficiency of between 0.1% and 99.9% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 99.9%, between 60% and 80%, between 75% and 99.9%, or between 85% and 99.9%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.9%).
In some embodiments, any of the methods of the invention produces a cell recovery number of between 104 cells and 1012 cells (e.g., between 104 cells and 105 cells, between 104 cells and 106 cells, between 104 cells and 107 cells, between 5×104 cells and 5×105 cells, between 105 cells and 106 cells, between 105 cells and 107 cells, between 105 cells and 1010 cells, between 2.5×105 cells and 106 cells, between 5×105 cells and 5×106 cells, between 106 cells and 107 cells, between 106 cells and 108 cells, between 106 cells and 1012 cells, between 5×106 cells and 5×107 cells, between 107 cells and 108 cells, between 107 cells and 109 cells, between 107 cells and 1012 cells, between 5×107 cells and 5×108 cells, between 108 cells and 109 cells, between 108 cells and 1010 cells, between 108 cells and 1012 cells, between 5×108 cells and 5×109 cells, between 109 cells and 1010 cells, between 109 cells and 1011 cells, between 1010 cells and 1011 cells, between 1010 cells and 1012 cells, or between 1011 cells and 1012 cells, e.g., about 104 cells, about 2.5×104 cells, about 5×104 cells, about 105 cells, about 2.5×105 cells, about 5×105 cells, about 106 cells, about 2.5×106 cells, about 5×106 cells, about 107 cells, about 2.5×107 cells, about 5×107 cells, about 108 cells, about 2.5×108 cells, about 5×108 cells, about 109 cells, about 2.5×109 cells, about 5×109 cells, about 1010 cells, about 5×1010 cells, about 1011 cells, or about 1012 cells).
In some embodiments, the method produces a cell recovery rate of between 0.1% and 100% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 100%, between 60% and 80%, between 75% and 100%, between 85% and 100%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%). In some embodiments, the method produces a live engineered cell yield (e.g., a recovery yield) of between 0.1% and 500% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 100%, between 60% and 80%, between 60% and 150%, between 75% and 100%, between 75% and 200%, between 85% and 150%, between 90% and 250%, between 100% and 200%, between 100% and 400%, between 150% and 300%, between 200% and 500%, or between 300% and 500%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, about 150%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%).
In some embodiments, the composition delivered to the plurality of cells (e.g., electro-mechanically delivered into the plurality of cells) includes at least one compound selected from the group consisting of therapeutic agents, vitamins, nanoparticles, charged molecules, uncharged molecules, engineered nucleases, DNA, RNA, CRISPR-Cas complex, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing nucleases, meganucleases (mns), megaTALs, enzymes, transposons, peptides, proteins, viruses, polymers, a ribonucleoprotein (RNP), and polysaccharides. In some embodiments, the composition has a concentration in the liquid of between 0.0001 μg/mL and 1,000 μg/mL (e.g., from about 0.0001 μg/mL to about 0.001 μg/mL, about 0.001 μg/mL to about 0.01 μg/mL, about 0.001 μg/mL to about 5 μg/mL, about 0.005 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 5 μg/mL, about 1 μg/mL to about 10 μg/mL, about 1 μg/mL to about 50 μg/mL, about 1 μg/mL to about 100 μg/mL, about 2.5 μg/mL to about 15 μg/mL, about 5 μg/mL to about 25 μg/mL, about 5 μg/mL to about 50 μg/mL, about 5 μg/mL to about 500 μg/mL, about 7.5 μg/mL to about 75 μg/mL, about 10 μg/mL to about 100 μg/mL, about 10 μg/mL to about 1,000 μg/mL, about 25 μg/mL to about 50 μg/mL, about 25 μg/mL to about 250 μg/mL, about 25 μg/mL to about 500 μg/mL, about 50 μg/mL to about 100 μg/mL, about 50 μg/mL to about 250 μg/mL, about 50 μg/mL to about 750 μg/mL, about 100 μg/mL to about 300 μg/mL, about 100 μg/mL to about 1,000 μg/mL, about 200 μg/mL to about 400 μg/mL, about 250 μg/mL to about 500 μg/mL, about 350 μg/mL to about 500 μg/mL, about 400 μg/mL to about 1,000 μg/mL, about 500 μg/mL to about 750 μg/mL, about 650 μg/mL to about 1,000 μg/mL, or about 800 μg/mL to about 1,000 μg/mL, e.g., about 0.0001 μg/mL, about 0.0005 μg/mL, about 0.001 μg/mL, about 0.005 μg/mL, about 0.01 μg/mL, about 0.02 μg/mL, about 0.03 μg/mL, about 0.04 μg/mL, about 0.05 μg/mL, about 0.06 μg/mL, about 0.07 μg/mL, about 0.08 μg/mL, about 0.09 μg/mL, about 0.1 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL, about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1 μg/mL, about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about 3 μg/mL, about 3.5 μg/mL, about 4 μg/mL, about 4.5 μg/mL, about 5 μg/mL, about 5.5 μg/mL, about 6 μg/mL, about 6.5 μg/mL, about 7 μg/mL, about 7.5 μg/mL, about 8 μg/mL, about 8.5 μg/mL, about 9 μg/mL, about 9.5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, about 100 μg/mL, about 200 μg/mL, about 250 μg/mL, about 300 μg/mL, about 350 μg/mL, about 400 μg/mL, about 450 μg/mL, about 500 μg/mL, about 550 μg/mL, about 600 μg/mL, about 650 μg/mL, about 700 μg/mL, about 750 μg/mL, about 800 μg/mL, about 850 μg/mL, about 900 μg/mL, about 950 μg/mL, or about 1,000 μg/mL).
In some embodiments of any of the preceding aspects, the plurality of cells suspended in the liquid includes eukaryotic cells (e.g., animal cells, e.g., human cells), prokaryotic cells (e.g., bacterial cells), plant cells, and/or synthetic cells. The cells can be primary cells (e.g., primary human cells), cells from a cell line (e.g., a human cell line), cells in suspension, adherent cells, stem cells, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), and/or immune cells (e.g., white blood cells (e.g., innate immune cells or adaptive immune cells)). In some embodiments, the cells (e.g., immune cells, e.g., T cells, B cell, natural killer cells, macrophages, monocytes, or antigen-presenting cells) are unstimulated cells, stimulated cells, or activated cells. In some embodiments, the cells are adaptive immune cells and/or innate immune cells. In some embodiments, the plurality of cells includes antigen presenting cells (APCs), monocytes, T-cells, B-cells, dendritic cells, macrophages, neutrophils, NK cells, Jurkat cells, THP-1 cells, human embryonic kidney (HEK-293) cells, Chinese hamster ovary (e.g., CHO-K1) cells, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). In some embodiments, the cells can be primary human T-cells, primary human macrophages, primary human monocytes, primary human NK cells, or primary human induced pluripotent stem cells (iPSCs). In some embodiments of any of the methods described herein, the method further includes storing the plurality of cells suspended in the liquid in a recovery buffer after poration.
In another aspect, the invention provides a kit including any of the devices or systems described herein. For example, in one aspect, the invention provides a kit for electro-mechanically delivering a composition into a plurality of cells suspended in a liquid, wherein the kit includes a plurality of devices, each of the plurality of devices including: a first electrode including a first outlet, a first inlet, and a first lumen including a minimum cross-sectional dimension; a second electrode including a second outlet, a second inlet, and a second lumen including a minimum cross-sectional dimension; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 7 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform cross-sectional area, wherein the application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone; and a plurality of outer structures configured to encase the plurality of devices, wherein each of the plurality of outer structures includes: a housing configured to encase the first electrode, second electrode, and the electroporation zone of the at least one device; a first electrical input operatively coupled to the first electrode; and a second electrical input operatively coupled to the second electrode. In some embodiments, the plurality of outer structures is integral to the plurality of devices. In some embodiments, the plurality of outer structures is releasably connected to the plurality of devices. In some embodiments, the housing further includes a thermal controller configured to increase a temperature of the at least one device, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease a temperature of the at least one device, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device.
In another aspect, the invention provides a kit for electroporating a composition into a plurality of cells suspended in a liquid, wherein the kit includes a plurality of devices, each of the plurality of devices including: a first electrode including a first outlet, a first inlet, and a first lumen including a minimum cross-sectional dimension; a second electrode including a second outlet, a second inlet, and a second lumen including a minimum cross-sectional dimension; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone includes a minimum cross-sectional dimension greater than about 100 μm (e.g., from 100 μm to 10 mm, from 150 μm to 15 mm, from 200 μm to 10 mm, from 250 μm to 5 mm, from 500 μm to 10 mm, from 1 mm to 10 mm, from 1 mm to 50 mm, from 5 mm to 25 mm, or from 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 7 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), wherein the electroporation zone has a substantially uniform cross-sectional area, wherein the application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone; and a plurality of outer structures configured to encase the plurality of devices, wherein each of the plurality of outer structures includes: a housing configured to encase the first electrode, second electrode, and the electroporation zone of the at least one device; a first electrical input operatively coupled to the first electrode; and a second electrical input operatively coupled to the second electrode. In some embodiments, the plurality of outer structures is integral to the plurality of devices. In some embodiments, the plurality of outer structures is releasably connected to the plurality of devices. In some embodiments, the housing further includes a thermal controller configured to increase a temperature of the at least one device, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease a temperature of the at least one device, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device.
In another aspect, the invention provides a kit for electro-mechanically delivering a composition into a plurality of cells suspended in a liquid, including: a plurality of devices, each of the plurality of devices including a device of the aforementioned embodiments; and a plurality of outer structures configured to encase the plurality of devices, wherein each of the plurality of outer structures includes: a housing configured to encase the first electrode, second electrode, and the electroporation zone of the at least one device; a first electrical input operatively coupled to the first electrode; and a second electrical input operatively coupled to the second electrode. In some embodiments, the plurality of outer structures is integral to the plurality of devices. In some embodiments, the plurality of outer structures is releasably connected to the plurality of devices. In some embodiments, the housing further includes a thermal controller configured to increase the temperature of the at least one device, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease the temperature of the at least one device, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device.
In another aspect, the invention provides a kit for electroporating a composition into plurality of cells suspended in a liquid, including: a plurality of devices, each of the plurality of devices including a device of the aforementioned embodiments; and a plurality of outer structures configured to encase the plurality of devices, wherein each of the plurality of outer structures includes: a housing configured to encase the first electrode, second electrode, and the electroporation zone of the at least one device; a first electrical input operatively coupled to the first electrode; and a second electrical input operatively coupled to the second electrode. In some embodiments, the plurality of outer structures is integral to the plurality of devices. In some embodiments, the plurality of outer structures is releasably connected to the plurality of devices. In some embodiments, the housing further includes a thermal controller configured to increase the temperature of the at least one device, wherein the thermal controller is a heating element selected from a group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to decrease the temperature of the at least one device, wherein the thermal controller is a cooling element selected from a group consisting of a liquid flow, an evaporative cooler, and a Peltier device.
In another aspect, the invention provides a device for electro-mechanically delivering a composition into plurality of cells suspended in a fluid, where the device includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are electroporated upon entering the electroporation zone.
In another aspect, the invention provides a device for electroporating a composition into plurality of cells suspended in a fluid, where the device includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are electroporated upon entering the electroporation zone.
In some embodiments of any of the preceding methods, the kit further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidically connected to a zone, e.g., the entry zone or recovery zone, of the device. For example, a first reservoir may be fluidically connected to the entry zone and a second reservoir may be fluidically connected to the recovery zone.
In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, ellipsoidal, polygonal, star, parallelogram, trapezoidal, and irregular.
In some cases, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone is between 0.01% to 100,000% of the cross-sectional dimension of the electroporation zone. For example, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 0.01% to about 1000% of the cross-sectional dimension of the electroporation zone, e.g., about 0.01% to about 1%, about 0.1% to about 10%, about 5% to about 25%, about 10% to about 50%, about 10% to about 1000%, about 25% to about 75%, about 25% to about 750%, or about 50% to about 1000% of the cross-sectional dimension of the electroporation zone. Alternatively, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 100% to about 100,000% of the of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000% of the cross-sectional dimension of the electroporation zone.
In some embodiments, the cross-sectional dimension of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 50 mm. In particular embodiments, the length of the electroporation zone is between 0.005 mm and 25 mm. In some embodiments, the cross-sectional dimension of any of the first electrode or the second electrode is between 0.1 mm to 500 mm. In particular embodiments, none of the entry zone, recovery zone, or electroporation zone reduce a cross-section dimension of any of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.
In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone. In some embodiments, the plurality of cells has no phenotypic change upon exiting the electroporation zone.
In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, second electrode, and the electroporation zone of the device. In some embodiments, the outer structure is integral to the device. In certain embodiments, the outer structure is releasably connected to the device.
In another aspect, the invention provides a device for electro-mechanical delivery of a composition into a plurality of cells suspended in a fluid, where the device includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; a third inlet and a third outlet, where the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, where the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are transfected upon entering the electroporation zone.
In another aspect, the invention provides a device for electroporating a composition into a plurality of cells suspended in a fluid, where the device includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; a third inlet and a third outlet, where the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, where the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are transfected upon entering the electroporation zone.
In some embodiments of either of the preceding aspects, the device further includes one or more reservoir, e.g., a first reservoir and a second reservoir, fluidically connected to a zone, e.g., the entry zone or recovery zone, of the device. For example, a first reservoir may be fluidically connected to the entry zone and a second reservoir may be fluidically connected to the recovery zone. In particular embodiments, the device includes a third reservoir fluidically connected to the third inlet and the third outlet and a fourth reservoir fluidically connected to the fourth inlet and the fourth outlet.
In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, ellipsoidal, polygonal, star, parallelogram, trapezoidal, and irregular.
In some embodiments, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone is between 0.01% to 100,000% of the cross-sectional dimension of the electroporation zone. For example, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 0.01% to about 1,000% of the cross-sectional dimension of the electroporation zone, e.g., about 0.01% to about 1%, about 0.1% to about 10%, about 5% to about 25%, about 10% to about 50%, about 10% to about 1,000%, about 25% to about 75%, about 25% to about 750%, or about 50% to about 100% of the cross-sectional dimension of the electroporation zone. Alternatively, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 100% to about 100,000% of the of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000% of the cross-sectional dimension of the electroporation zone.
In some embodiments, the cross-sectional dimension of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 50 mm. In particular embodiments, the length of the electroporation zone is between 0.005 mm and 25 mm. In some embodiments, the cross-sectional dimension of any of the first electrode or the second electrode is between 0.1 mm to 500 mm. In particular embodiments, none of the entry zone, recovery zone, or electroporation zone reduce a cross-section dimension of any of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.
In particular embodiments, the first and/or second electrodes is porous or a conductive fluid (e.g., liquid).
In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone. In some embodiments, the plurality of cells has no phenotypic change upon exiting the electroporation zone.
In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, second electrode, and the electroporation zone of the device. In some embodiments, the outer structure is integral to the device. In certain embodiments, the outer structure is releasably connected to the device.
In another aspect, the invention provides a system for electro-mechanical delivery of a composition into a plurality of cells suspended in a fluid, the system including a device that includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. The system further includes source of electrical potential, where the first and second electrodes of the device are releasably connected to the source of electrical potential. In the system, the plurality of cells suspended in the fluid are electroporated upon entering the electroporation zone.
In another aspect, the invention provides a system for electroporating a composition into a plurality of cells suspended in a fluid, the system including a device that includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. The system further includes source of electrical potential, where the first and second electrodes of the device are releasably connected to the source of electrical potential. In the system, the plurality of cells suspended in the fluid are electroporated upon entering the electroporation zone.
In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone. In some embodiments, the plurality of cells has no phenotypic change upon exiting the electroporation zone.
In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, second electrode, and the electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. In some embodiments, the releasable connection between the first or second electrical inputs and the source of electrical potential is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof.
In some embodiments, the outer structure is integral to the device. In certain embodiments, the outer structure is releasably connected to the device.
In some embodiments, any of the devices, systems, or methods of any of the previous aspects induces reversible or irreversible electroporation. In particular embodiments, the electroporation is substantially non-thermal reversible electroporation, substantially non-thermal irreversible electroporation, or substantially thermal irreversible electroporation.
In some embodiments, the releasable connection between the device and the source of electrical potential is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof. In particular embodiments, the releasable connection between the device and the source of electrical potential is a spring.
In some embodiments, the device further includes one or more reservoir, e.g., a first reservoir and a second reservoir, fluidically connected to a zone, e.g., the entry zone or recovery zone, of a device. For example, a first reservoir may be fluidically connected to the entry zone and a second reservoir may be fluidically connected to the recovery zone.
In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, ellipsoidal, polygonal, star, parallelogram, trapezoidal, and irregular.
In some embodiments, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone is between 0.01% and 100,000% of the cross-sectional dimension of the electroporation zone. For example, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 0.01% to about 1000% of the cross-sectional dimension of the electroporation zone, e.g., about 0.01% to about 1%, about 0.1% to about 10%, about 5% to about 25%, about 10% to about 50%, about 10% to about 1,000%, about 25% to about 75%, about 25% to about 750%, or about 50% to about 100% of the cross-sectional dimension of the electroporation zone. Alternatively, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 100% to about 100,000% of the of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000% of the cross-sectional dimension of the electroporation zone.
In some embodiments, the cross-sectional dimension of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 50 mm. In particular embodiments, the length of the electroporation zone is between 0.005 mm and 25 mm. In some embodiments, the cross-sectional dimension of any of the first electrode or the second electrode is between 0.1 mm to 500 mm. In particular embodiments, none of the entry zone, recovery zone, or electroporation zone reduce a cross-section dimension of any of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.
In further embodiments, the system includes a fluid delivery source fluidically connected to the entry zone, wherein the fluid delivery source is configured to deliver the plurality of cells suspended in the fluid through the entry zone to the recovery zone. In some embodiments, the delivery rate from the fluid delivery source is between 0.001 mL/min to 1,000 mL/min, e.g., 25 mL/min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is between 0.5 ms to 50 ms. In some embodiments, the conductivity of the fluid is between 0.001 mS/cm to 500 mS/cm, e.g., 1-20 mS/cm.
In further embodiments, the system includes a controller operatively coupled to the source of electrical potential to deliver voltage pulses to the first electrode and second electrodes to generate an electrical potential difference between the first and second electrodes. In some embodiments, the voltage pulses have an amplitude of −3 kV to 3 kV, e.g., 0.01 kV to 3 kV, e.g., 0.2-0.6 kV. In some cases, the duty cycle of the electroporation is between 0.001% to 100%, e.g., 10-95%. In some embodiments, the voltage pulses have a duration of between 0.01 ms to 1,000 ms, e.g., 1-10 ms. In certain embodiments, the voltage pulses are applied the first and second electrodes at a frequency between 1 Hz to 50,000 Hz, e.g., 100-500 Hz. The waveform of the voltage pulse may be DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, arbitrary, or any superposition or combination thereof. In particular embodiments, the electric field generated from the voltage pulses has a magnitude of between 1 V/cm to 50,000 V/cm, e.g., 100-1,000 V/cm.
In further embodiments, the system includes a housing (e.g., a housing structure) configured to house the electroporation device described herein. In further instances, the housing (e.g., housing structure) includes a thermal controller configured to increase or decrease the temperature of the housing or any component of the system thereof. In some embodiments, the thermal controller is a heating element, e.g., a heating block, liquid flow, battery powered heater, or a thin-film heater. In other embodiments, the thermal controller is a cooling element, e.g., liquid flow, evaporative cooler, or a thermoelectric, e.g., a Peltier, device.
In further embodiments, the system includes a plurality of cell porating devices, e.g., in series or in parallel. In particular embodiments, the system includes a plurality of outer structures for the plurality of cell porating devices.
In another aspect, the invention provides a system for electro-mechanical delivery of a composition into a plurality of cells suspended in a fluid, the system including a device that includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; a third inlet and a third outlet, where the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, where the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are transfected upon entering the electroporation zone. In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone. In some embodiments, the plurality of cells has no phenotypic change upon exiting the electroporation zone.
In another aspect, the invention provides a system for electroporating a composition into a plurality of cells suspended in a fluid, the system including a device that includes: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; a third inlet and a third outlet, where the third inlet and third outlet intersect the first electrode between the first inlet and the first outlet; a fourth inlet and a fourth outlet, where the fourth inlet and fourth outlet intersect the second electrode between the second inlet and the second outlet; and an electroporation zone, where the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, where the electroporation zone has a substantially uniform cross-section dimension, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone. In the device, the plurality of cells suspended in the fluid are electroporated upon entering the electroporation zone.
In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone. In some embodiments, the plurality of cells has no phenotypic change upon exiting the electroporation zone.
In further embodiments of any of the previous aspects, the device includes an outer structure having a housing (e.g., a housing structure) configured to encase the first electrode, second electrode, and the electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. In some embodiments, the releasable connection between the first or second electrical inputs and the source of electrical potential is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof.
In some embodiments, the outer structure is integral to the device. In certain embodiments, the outer structure is releasably connected to the device.
In some cases, the system induces reversible or irreversible electroporation. In particular embodiments, the electroporation is substantially non-thermal reversible electroporation, substantially non-thermal irreversible electroporation, or substantially thermal irreversible electroporation.
In some embodiments, the releasable connection between the device and the source of electrical potential is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, mechanical connection, inductive connection, or a combination thereof. In particular embodiments, the releasable connection between the device and the source of electrical potential is a spring.
In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidically connected to a zone, e.g., the entry zone or recovery zone, of a device. For example, a first reservoir may be fluidically connected to the entry zone and a second reservoir may be fluidically connected to the recovery zone.
In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, ellipsoidal, polygonal, star, parallelogram, trapezoidal, and irregular.
In some embodiments, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone is between 0.01% to 100,000% of the cross-sectional dimension of the electroporation zone. For example, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 0.01% to about 1000% of the cross-sectional dimension of the electroporation zone, e.g., about 0.01% to about 1%, about 0.1% to about 10%, about 5% to about 25%, about 10% to about 50%, about 10% to about 1,000%, about 25% to about 75%, about 25% to about 750%, or about 50% to about 100% of the cross-sectional dimension of the electroporation zone. Alternatively, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 100% to about 100,000% of the of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000% of the cross-sectional dimension of the electroporation zone.
In some embodiments, the cross-sectional dimension of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 50 mm. In particular embodiments, the length of the electroporation zone is between 0.005 mm and 25 mm. In some embodiments, the cross-sectional dimension of any of the first electrode or the second electrode is between 0.01 mm and 500 mm. In particular embodiments, none of the entry zone, recovery zone, or electroporation zone reduce a cross-section dimension of any of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.
In further embodiments, the system includes a fluid delivery source fluidically connected to the entry zone, wherein the fluid delivery source is configured to deliver the plurality of cells suspended in the fluid through the entry zone to the recovery zone. In some embodiments, the delivery rate from the fluid delivery source is between 0.001 mL/min and 1,000 mL/min, e.g., 25 mL/min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is between 0.5 ms and 50 ms. In some embodiments, the conductivity of the fluid is between 0.001 mS/cm and 500 mS/cm, e.g., between 1 mS/cm and 20 mS/cm.
In further embodiments, the system includes a controller operatively coupled to the source of electrical potential to deliver voltage pulses to the first electrode and second electrodes to generate an electrical potential difference between the first and second electrodes. In some embodiments, the voltage pulses have an amplitude of −3 kV to 3 kV, e.g., 0.01 kV to 3 kV, e.g., 0.2-0.6 kV. In some cases, the duty cycle of the electroporation is between 0.001% to 100%, e.g., 10-95%. In some embodiments, the voltage pulses have a duration of between 0.01 ms to 1,000 ms, e.g., 1-10 ms. In certain embodiments, the voltage pulses are applied the first and second electrodes at a frequency between 1 Hz to 50,000 Hz, e.g., 100-500 Hz. The waveform of the voltage pulse may be DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, arbitrary, or any superposition or combination thereof. In particular embodiments, the electric field generated from the voltage pulses has a magnitude of between 1 V/cm and 50,000 V/cm, e.g., between 100 V/cm and 1,000 V/cm.
In further embodiments, the system includes a housing (e.g., a housing structure) configured to house the electroporation device described herein. In further instances, the housing structure includes a thermal controller configured to increase or decrease the temperature of the housing structure or any component of the system thereof. In some embodiments, the thermal controller is a heating element, e.g., a heating block, liquid flow, battery powered heater, or a thin-film heater. In other embodiments, the thermal controller is a cooling element, e.g., liquid flow, evaporative cooler, or a thermoelectric, e.g., a Peltier, device.
In further embodiments, the system includes a plurality of cell porating devices, e.g., in series or in parallel. In particular embodiments, the system includes a plurality of outer structures for the plurality of cell porating devices.
In another aspect, the invention provides methods of introducing a composition into at least a portion of a plurality of cells suspended in a fluid, the method including the steps of: a. providing a device including: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and an electroporation zone, wherein the electroporation zone is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode, and where application of an electrical potential difference to the first and second electrodes produces an electric field in the electroporation zone; b. energizing the first and second electrodes to produce an electrical potential difference between the first and second electrodes, thereby producing an electric field in the electroporation zone; and c. passing the plurality of cells suspended in the fluid with the composition through the electric field in the electroporation zone of the device. In the method, flow of the plurality of cells suspended in fluid with the composition through the electric field in the electroporation zone enhances temporary permeability of the plurality of cells, thereby introducing the composition into at least a portion of the plurality of cells.
In further embodiments, the method includes assessing the health of a portion of the plurality of cells suspended in the fluid. In certain embodiments, the assessing includes measuring the viability of the portion of the plurality of cells suspended in the fluid. In some embodiments, the assessing includes measuring the transfection efficiency of the portion of the plurality of cells suspended in the fluid. In some embodiments, the assessing includes measuring the cell recovery rate of the portion of the plurality of cells suspended in the fluid. In certain embodiments, the assessing includes flow cytometry analysis of cell surface marker expression.
In some embodiments, the plurality of cells has from 0% to about 25% phenotypic change relative to a baseline measurement of cell phenotype upon exiting the electroporation zone of the device. In some cases, the plurality of cells has no phenotypic change upon exiting the electroporation zone of the device.
In some embodiments, the method induces reversible or irreversible electroporation. In particular embodiments, the electroporation is substantially non-thermal reversible electroporation, substantially non-thermal irreversible electroporation, or substantially thermal irreversible electroporation.
In some embodiments, cells suspended in the fluid with the composition are passed through the electric field in the electroporation zone of the device by the application of a positive pressure, e.g., a pump, e.g., a syringe pump or peristaltic pump.
In certain embodiments, cells in the plurality of cells in the sample may be mammalian cells, eukaryotes, human cells, animal cells, plant cells, synthetic cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, activated cells, immune cells, stem cells, blood cells, red blood cells, T cells, B cells, neutrophils, dendritic cells, antigen presenting cells (APCs), natural killer (NK) cells, monocytes, macrophages, or peripheral blood mononuclear cells (PBMCs), human embryonic kidney cells, e.g., HEK-293 cells, or Chinese hamster ovary (CHO) cells. In particular embodiments, the plurality of cells includes Jurkat cells. In particular embodiments, the plurality of cells includes primary human T-cells. In particular embodiments, the plurality of cells includes THP-1 cells. In particular embodiments, the plurality of cells includes primary human macrophages. In particular embodiments, the plurality of cells includes primary human monocytes. In particular embodiments, the plurality of cells includes natural killer (NK) cells. In particular embodiments, the plurality of cells includes Chinese hamster ovary cells. In particular embodiments, the plurality of cells includes human embryonic kidney cells. In particular embodiments, the plurality of cells includes B-cells. In particular embodiments, the plurality of cells includes primary human T-cells. In particular embodiments, the plurality of cells includes primary human monocytes. In particular embodiments, the plurality of cells includes primary human macrophages. In particular embodiments, the plurality of cells includes embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). In particular embodiments, the plurality of cells includes primary human induced pluripotent stem cells (iPSCs).
In some embodiments, the composition includes at least one compound selected from the group consisting of therapeutic agents, vitamins, nanoparticles, charged therapeutic agents, nanoparticles, charged molecules, e.g., ions in solution, uncharged molecules, nucleic acids, e.g., DNA or RNA, CRISPR-Cas complexes, proteins, polymers, ribonucleoproteins (RNPs), engineered nucleases, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing nucleases, meganucleases (MNs), megaTALs, enzymes, peptides, transposons, or polysaccharides, e.g., dextran, e.g., dextran sulfate. Compositions that can be delivered to cells in a suspension include nucleic acids (e.g., oligonucleotides, mRNA, or DNA), antibodies (or an antibody fragment, e.g., a bispecific fragment, a trispecific fragment, Fab, F(ab′)2, or a single-chain variable fragment (scFv)), amino acids, polypeptides (e.g., peptides or proteins), cells, bacteria, gene therapeutics, genome engineering therapeutics, epigenome engineering therapeutics, carbohydrates, chemical drugs, contrast agents, magnetic particles, polymer beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotic agents, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, anti-inflammatory agents, anti-microbial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacteriophages, adjuvants, vitamins, minerals, organelles, and combinations thereof. In certain embodiments, the composition is a nucleic acid (e.g., an oligonucleotide, mRNA, or DNA). In certain embodiments, the composition is an antibody. In certain embodiments, the composition is a polypeptide (e.g., a peptide or a protein).
In certain embodiments, the composition has a concentration in the fluid of between 0.0001 μg/mL and 1,000 μg/mL (e.g., from about 0.0001 μg/mL to about 0.001 μg/mL, about 0.001 μg/mL to about 0.01 μg/mL, about 0.001 μg/mL to about 5 μg/mL, about 0.005 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 5 μg/mL, about 1 μg/mL to about 10 μg/mL, about 1 μg/mL to about 50 μg/mL, about 1 μg/mL to about 100 μg/mL, about 2.5 μg/mL to about 15 μg/mL, about 5 μg/mL to about 25 μg/mL, about 5 μg/mL to about 50 μg/mL, about 5 μg/mL to about 500 μg/mL, about 7.5 μg/mL to about 75 μg/mL, about 10 μg/mL to about 100 μg/mL, about 10 μg/mL to about 1,000 μg/mL, about 25 μg/mL to about 50 μg/mL, about 25 μg/mL to about 250 μg/mL, about 25 μg/mL to about 500 μg/mL, about 50 μg/mL to about 100 μg/mL, about 50 μg/mL to about 250 μg/mL, about 50 μg/mL to about 750 μg/mL, about 100 μg/mL to about 300 μg/mL, about 100 μg/mL to about 1,000 μg/mL, about 200 μg/mL to about 400 μg/mL, about 250 μg/mL to about 500 μg/mL, about 350 μg/mL to about 500 μg/mL, about 400 μg/mL to about 1,000 μg/mL, about 500 μg/mL to about 750 μg/mL, about 650 μg/mL to about 1,000 μg/mL, or about 800 μg/mL to about 1,000 μg/mL, e.g., about 0.0001 μg/mL, about 0.0005 μg/mL, about 0.001 μg/mL, about 0.005 μg/mL, about 0.01 μg/mL, about 0.02 μg/mL, about 0.03 μg/mL, about 0.04 μg/mL, about 0.05 μg/mL, about 0.06 μg/mL, about 0.07 μg/mL, about 0.08 μg/mL, about 0.09 μg/mL, about 0.1 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL, about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1 μg/mL, about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about 3 μg/mL, about 3.5 μg/mL, about 4 μg/mL, about 4.5 μg/mL, about 5 μg/mL, about 5.5 μg/mL, about 6 μg/mL, about 6.5 μg/mL, about 7 μg/mL, about 7.5 μg/mL, about 8 μg/mL, about 8.5 μg/mL, about 9 μg/mL, about 9.5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, about 100 μg/mL, about 200 μg/mL, about 250 μg/mL, about 300 μg/mL, about 350 μg/mL, about 400 μg/mL, about 450 μg/mL, about 500 μg/mL, about 550 μg/mL, about 600 μg/mL, about 650 μg/mL, about 700 μg/mL, about 750 μg/mL, about 800 μg/mL, about 850 μg/mL, about 900 μg/mL, about 950 μg/mL, or about 1,000 μg/mL).
In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidically connected to a zone, e.g., the entry zone or recovery zone, of a device. For example, a first reservoir may be fluidically connected to the entry zone and a second reservoir may be fluidically connected to the recovery zone.
In some embodiments, the electroporation zone of the device has a uniform cross-sectional dimension. In other embodiments, the electroporation zone of the device has a non-uniform cross-sectional dimension. In further embodiments, the device further comprises a plurality of electroporation zones, where each of the plurality of electroporating zones, e.g., electroporation zone, may have a uniform cross-section or a non-uniform cross-section. In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, ellipsoidal, polygonal, star, parallelogram, trapezoidal, and irregular.
In some embodiments, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone is between 0.01% to 100,000% of the cross-sectional dimension of the electroporation zone. For example, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 0.01% to about 100% of the cross-sectional dimension of the electroporation zone, e.g., about 0.01% to about 1%, about 0.1% to about 10%, about 5% to about 25%, about 10% to about 50%, about 25% to about 75%, or about 50% to about 100% of the cross-sectional dimension of the electroporation zone. Alternatively, the cross-sectional dimension of the entry zone or the cross-sectional dimension of the recovery zone may be about 100% to about 100,000% of the of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000% of the cross-sectional dimension of the electroporation zone.
In some embodiments, the cross-sectional dimension of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 50 mm. In some embodiments, the length of the electroporation zone is between 0.005 mm and 25 mm. In some embodiments, the cross-sectional dimension of any of the first electrode or the second electrode is between 0.1 mm to 500 mm. In particular embodiments, none of the entry zone, recovery zone, or electroporation zone reduces a cross-section dimension of any of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.
In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, second electrode, and the electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operatively coupled to the first electrode and a second electrical input operatively coupled to the second electrode. In some embodiments, the outer structure is integral to the device. In certain embodiments, the outer structure is releasably connected to the device.
In some embodiments, the delivery rate from the fluid delivery source is between 0.001 mL/min to 1,000 mL/min, e.g., 20-30 mL/min, e.g., 25 mL/min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is between 0.5 ms and 50 ms. In some embodiments, the conductivity of the fluid is between 0.001 mS/cm to 500 mS/cm, e.g., 1-20 mS/cm.
In further embodiments, the method includes a controller operatively coupled to the source of electrical potential to deliver voltage pulses to the first electrode and second electrodes to generate an electrical potential difference between the first and second electrodes. In some embodiments, the voltage pulses have an amplitude of −3 kV to 3 kV, e.g., 0.2-0.6 kV. In some cases, the duty cycle of the electroporation is between 0.001% and 100%, e.g., between 10% and 95%. In some embodiments, the voltage pulses have a duration of between 0.01 ms and 1,000 ms, e.g., between 1 ms and 10 ms. In certain embodiments, the voltage pulses are applied the first and second electrodes at a frequency between 1 Hz to 50,000 Hz, e.g., 100-500 Hz. The waveform of the voltage pulse may be DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, arbitrary, or any superposition or combination thereof. In particular embodiments, the electric field generated from the voltage pulses has a magnitude of between 1 V/cm and 50,000 V/cm, e.g., between 100 V/cm and 1,000 V/cm.
In further embodiments, the method includes a housing structure configured to house the electroporation device described herein. In further instances, the housing structure includes a thermal controller configured to increase or decrease the temperature of the housing or any component of the system thereof. In some embodiments, the thermal controller is a heating element, e.g., a heating block, liquid flow, battery powered heater, or a thin-film heater. In other embodiments, the thermal controller is a cooling element, e.g., liquid flow, evaporative cooler, or thermoelectric, e.g., Peltier device. In certain embodiments, the temperature of the plurality of cells suspended in the fluid is between 0° C. and 50° C.
In further embodiments, the device includes a plurality of cell porating devices, e.g., in series or in parallel. In particular embodiments, the device includes a plurality of outer structures for the plurality of devices.
In some embodiments, the method further includes storing the plurality of cells suspended in the fluid in a recovery buffer after poration. In certain embodiments, the electroporated cells have a viability after introduction of the composition between 0.1% and 99.9%, e.g., 25% and 85%. In other embodiments, the efficiency of the introduction of the composition into the cells is between 0.1 and 99.9%, e.g., between 25% and 85%. In certain embodiments, the cell recovery rate is between 0.1% and 100%. In particular embodiments, the cell recovery yield is between 0.1% and 500%. In some embodiments, the number of recovered cells (e.g., live cells) is between 104 and 1012.
In another aspect, the invention provides a kit for electro-mechanical delivery of a composition into a plurality of cells suspended in a fluid, the kit including a plurality of devices as described herein, a plurality of outer structures as described herein, and a transfection buffer.
In another aspect, the invention provides a kit for electroporation of a composition into a plurality of cells suspended in a fluid, the kit including a plurality of devices as described herein, a plurality of outer structures as described herein, and a transfection buffer.
In some embodiments of any of the preceding aspects, the outer structures are integral to the plurality of cell devices. In certain embodiments, the outer structures are releasably connected to the plurality of devices.
The application file contains at least one drawing executed in color. Copies of this patent application with color drawings will be provided by the Office upon request of the payment of the necessary fee.
Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
The term “about,” as used herein, refers to +/−10% of a recited value.
The term “plurality,” as used herein, refers to more than one.
The term “substantially uniform,” as used herein, refers to +/−5% variance.
The term “minimum cross-sectional dimension,” as used herein, refers to a minimum length of a straight line that passes through the geometric center of a transverse cross-section of a lumen and intersects an inner wall of the lumen twice on the same plane of the transverse cross-section. The term “cross-sectional area,” unless otherwise specified, refers to the transverse cross-sectional area (e.g., along the plane perpendicular to the longitudinal axis or direction of flow).
The term “fluidically connected,” as used herein, refers to a direct connection between at least two device elements, e.g., an electroporation device, a reservoir, etc., that allows for fluid to move between such device elements without passing through an intervening element.
The term “fluidic communication,” as used herein, refers to an indirect connection between at least two device elements, e.g., an electroporation zone, a reservoir, etc., that allows for fluid to move between such device elements, e.g., through an intervening element, (e.g., through intervening tubing, an intervening channel, etc.). For example, in embodiments in which a fluid flows from a lumen of first electrode, through an electroporation zone, into a lumen of a second electrode, the first electrode is in fluidic communication with the second electrode.
The term “lumen,” as used herein, refers to an interior cavity of an electrode of the devices of the invention that allows for fluid to pass through. Part or all of a lumen of an electrode may be conductive or non-conductive. For example, a lumen of an electrode may encase a C-shaped conductive element that does not completely surround the perimeter of the lumen. In other embodiments, the electrode is substantially entirely composed of the conductive material that transmits current. When an electric potential difference is applied to a first and second electrode of the devices of the invention, an electric field that may be generated in a lumen of any one of the first or second electrodes is not high enough to cause cell electroporation to occur within the lumen.
The term “entry zone,” as used herein, comprises a lumen of a first electrode of the devices of the invention through which a fluid and a plurality of cells suspended in the fluid may pass prior to electroporation. An entry zone may further comprise an additional reservoir in fluidic communication with a lumen of a first electrode of the devices of the invention. When an electric potential difference is applied to a first and second electrode of the devices of the invention, the electric field that may be generated within an entry zone of the devices of the invention is not high enough to cause cell electroporation to occur.
The term “recovery zone,” as used herein, comprises a lumen of a second electrode of the devices of the invention through which a fluid and a plurality of cells suspended in the fluid may pass after electroporation. A recovery zone may further comprise an additional reservoir in fluidic communication with a lumen of a second electrode of the devices of the invention. When an electric potential difference is applied to a first and second electrode of the devices of the invention, the electric field that may be generated within a recovery zone of the devices of the invention is not high enough to cause cell electroporation to occur.
The term “electroporation zone,” as used herein, refers to a portion of a device that is disposed between, and in fluidic communication with, an outlet of an upstream electrode (e.g., a first outlet) and an inlet of a downstream electrode (e.g., a second inlet). The electric field is delivered to the fluid in the electroporation zone.
The term “transfection,” as used herein, refers to a process by which payloads can be introduced into cells utilizing means other than viral delivery methods, such as biological, chemical, electrical, mechanical, or physical methods.
The term “electroporation,” as used herein, refers to a process utilizing applied electric fields to create small pores in cell membranes through which payloads can be introduced into cells (e.g. as a method of transfection).
The term “electro-mechanical delivery,” as used herein, is a transfection process by which payloads can be introduced to cells utilizing any combination of applied electric fields and/or mechanical poration mechanisms. This delivery method has the potential to decrease and/or stabilize the overall electric field exposure of the cells in the electroporation zone, thereby enhancing cell viability and/or transfection efficiency, or both. The devices of the invention are configured to transfect cells via electro-mechanical delivery rather than by electroporation alone.
The present invention provides devices, systems, and methods for the transfection of cells, e.g., primary T cells, by electroporation at larger volumes, higher transfection efficiencies, higher throughputs, higher recovery rates, higher yields, and higher cell viabilities as compared with traditional cuvette based electroporation approaches or commercially available electroporation instruments. In particular, systems and methods are provided that can perform electroporation in a flow-through manner, a continuous manner, or using a plurality of electroporation devices of the invention to enhance throughput and cell numbers.
In general, devices of the present invention are configured to be flow through devices that may interface with existing liquid handling, pumps, or fluid transport apparatuses, such as conventional pipette tip robots or large-scale liquid handling systems, to provide continuous electroporation of cells suspended in a fluid. A device of the invention is configured for transfection of cells to occur within the electroporation zone via an electro-mechanical delivery mechanism that is distinct from the delivery mechanism in static electroporation systems. Devices of the invention typically feature three distinct regions: a first electrode having a first inlet and a first outlet, where a lumen of the first electrode defines an entry zone; a second electrode having a second inlet and a second outlet, where a lumen of the second electrode defines a recovery zone; and electroporation zone that is fluidically connected to the first outlet of the first electrode and the second inlet of the second electrode. An example of an embodiment of the device of the invention is shown in
In some cases, the first electrode and the second electrode may be electrically conductive wires, hollow cylinders, electrically conductive thin films, metal foams, mesh electrodes, liquid diffusible membranes, conductive liquids, or any combination thereof can be included in the device. The electrodes may be either aligned parallel with the axis of fluid flow of the device or may be aligned orthogonal to the axis of fluid flow of the device. For example, the first and second electrodes may be hollow cylindrical electrodes arranged in parallel with the axis of fluid flow within the device, such as the in the device of
When configured to be hollow cylindrical electrodes, the diameter of the electrode may be from about 0.1 mm to about 5 mm, e.g., from about 0.1 mm to about 1 mm, from about 0.5 mm to about 1.5 mm, from about 1 mm to about 2 mm, from about 1.5 mm to about 2.5 mm, about 2 mm to about 3 mm, from about 2.5 mm to about 3.5 mm, about 3 mm to about 4 mm, from about 3.5 mm to about 4.5 mm, or about 4 mm to about 5 mm, e.g., about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm, about 2.1 mm, about 2.2 mm, about 2.3 mm, about 2.4 mm, about 2.5 mm, about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm, about 3 mm, about 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, about 3.5 mm, about 3.6 mm, about 3.7 mm, about 3.8 mm, about 3.9 mm, about 4 mm, about 4.1 mm, about 4.2 mm, about 4.3 mm, about 4.4 mm, about 4.5 mm, about 4.6 mm, about 4.7 mm, about 4.8 mm, about 4.9 mm, or about 5 mm. An exemplary electrode outer diameter is 1.3 mm, corresponding to a 16 gauge electrode.
In some embodiments, when a device of the invention is configured to include hollow cylindrical electrodes, a lumen of an electrode, e.g., the first or second electrode, may include a zone, e.g., an entry zone or a recovery zone, that is not subject to the electric field of the electroporation zone. As is shown in
The electroporation zone fluidically connects the first and second electrodes of devices of the invention, and when the electrodes are energized, experiences a localized electric field therebetween. The cross-sectional shape of the electroporation zone may be of any suitable shape that allows cells to pass through the electroporation zone and the electric field within the electroporation zone. The cross-sectional shape may be, e.g., circular, ellipsoidal, or polygonal, e.g., square, rectangular, triangular, n-gon (e.g., a regular or irregular polygon having 4, 5, 6, 7, 8, 9, 10, or more sides), star, parallelogram, trapezoidal, or irregular, e.g., oval, or curvilinear shape. In some cases, the electroporation zone is a channel that has a substantially uniform cross-section dimension along its length, e.g., the electroporation zone may have a circular cross-section, where the diameter is constant from the fluidic connection to the entry zone to the fluidic connection of the recovery zone. In this configuration, the resulting electric field is more uniform, thus allowing for a more predictable electric field exposure of cells suspended in a fluid. Alternatively, the cross-sectional dimension of the electroporation zone may be varied along is length. For example, the cross-sectional dimension of the electroporation zone may either increase or decrease along its length, or may have more than one dimension change along its length, e.g., the cross-sectional dimension, e.g., the diameter, may increase or decrease by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at most 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In this configuration, the electroporation zone may have a truncated conical cross-section, with the diameter increasing from the top aperture to the bottom aperture or decreasing from the top aperture to the bottom aperture. In some cases, devices of the invention may include a plurality of electroporation zones fluidically connected in series, with each electroporation zone having either a uniform or non-uniform cross-section and each may have a different cross-section shape. As a non-limiting example, a device of the invention may include a plurality of serially-connected electroporation zones, each of the plurality of electroporation zones having a cylindrical cross-section of a different cross-sectional dimension, e.g., each has a different diameter.
In some embodiments, the cross-sectional dimension of the electroporation zone may be from about 0.005 mm to about 50 mm, e.g., about 0.005 mm to about 0.05 mm, about 0.01 mm to about 0.1 mm, about 0.05 mm to about 0.5 mm, about 0.1 mm to about 1 mm, about 0.5 mm to about 1 mm, from about 0.5 mm to about 2 mm, about 0.7 mm to about 1.5 mm, about 1 mm to about 5 mm, about 3 mm to about 7 mm, about 5 mm to about 10 mm, about 7 mm to about 12 mm, about 10 mm to about 15 mm, about 13 mm to about 18 mm, about 15 mm to about 20 mm, about 22 mm to about 30 mm about 25 mm to about 35 mm, about 30 mm to about 40 mm, about 35 mm to about 45 mm, or about 40 mm to about 50 mm, e.g., about 0.005 mm, about 0.006, about 0.007 mm, about 0.008 mm, about 0.009 mm, about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm, about 26 mm, about 27 mm, about 28 mm, about 29 mm, about 30 mm, about 31 mm, about 32 mm, about 33 mm, about 34 mm, about 35 mm, about 36 mm, about 37 mm, about 38 mm, about 39 mm, about 40 mm, about 41 mm, about 42 mm, about 43 mm, about 44 mm, about 45 mm, about 46 mm, about 47 mm, about 48 mm, about 49 mm, or about 50 mm. In general, the diameter of the electroporation zone is sized such that it does not have a constriction that contacts the cells to deform the cell membranes with the channel walls, e.g., poration of the cells is not induced by mechanical deformation due to cell squeezing—e.g., the cells can freely pass through the electroporation zone.
In some cases, the length of the electroporation zone may be from about 0.005 mm to about 50 mm, e.g., about 0.005 mm to about 0.05 mm, about 0.01 mm to about 0.1 mm, about 0.05 mm to about 0.5 mm, about 0.1 mm to about 1 mm, from about 0.5 mm to about 2 mm, about 1 mm to about 5 mm, about 3 mm to about 7 mm, about 4 mm to about 8 mm, about 5 mm to about 10 mm, about 7 mm to about 12 mm, about 10 mm to about 15 mm, about 13 mm to about 18 mm, about 15 mm to about 20 mm, about 22 mm to about 30 mm about 25 mm to about 35 mm, about 30 mm to about 40 mm, about 35 mm to about 45 mm, or about 40 mm to about 50 mm, e.g., about 0.005 mm, about 0.006, about 0.007 mm, about 0.008 mm, about 0.009 mm, about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm, about 26 mm, about 27 mm, about 28 mm, about 29 mm, about 30 mm, about 31 mm, about 32 mm, about 33 mm, about 34 mm, about 35 mm, about 36 mm, about 37 mm, about 38 mm, about 39 mm, about 40 mm, about 41 mm, about 42 mm, about 43 mm, about 44 mm, about 45 mm, about 46 mm, about 47 mm, about 48 mm, about 49 mm, or about 50 mm.
The cross-sectional dimension of the entry zone and/or the recovery zone may be independently substantially the same as the cross-sectional dimension of the electroporation zone. Alternatively, the entry zone and/or the recovery zone may be independently smaller or larger than the cross-sectional dimension of the electroporation zone. For example, when the cross-sectional dimension of the entry zone and/or the recovery zone is independently configured to be smaller than the cross-sectional dimension of the electroporation zone, the cross-sectional dimension of the entry zone and/or the recovery zone may be from about 0.01% to about 100% of the cross-sectional dimension of the electroporation zone, about 0.01% to about 1%, about 0.1% to about 10%, about 1% to about 5%, about 1% to about 10%, about 5% to about 25%, about 5% to about 10%, about 10% to about 25%, about 10% to about 50%, about 25% to about 75%, or about 50% to about 100%, e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
Alternatively, when the cross-sectional dimension of the entry zone and/or the recovery zone is independently configured to be larger than the cross-sectional dimension of the electroporation zone, the cross-sectional dimension of the entry zone and/or the recovery zone may be from about 100% to about 100,000% of the cross-sectional dimension of the electroporation zone, e.g., about 100% to about 1000%, about 100% to about 250%, about 100% to about 500%, about 250% to about 750%, about 500% to about 1,000%, about 500% to about 5,000%, about 1,000% to about 10,000%, about 5,000% to about 25,000%, about 10,000% to about 50,000%, about 25,000% to about 75,000%, or about 50,000% to about 100,000%, e.g., about 100%, about 150%, about 175%, about 200%, about 225%, about 250%, about 300%, about 250%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1,000%, about 2,000%, about 3,000%, about 4,000%, about 5,000%, about 6,000%, about 7,000%, about 8,000%, about 9,000%, about 10,000%, about 15,000%, about 20,000%, about 25,000%, about 30,000%, about 35,000%, about 40,000%, about 45,000%, about 50,000%, about 55,000%, about 60,000%, about 65,000%, about 70,000%, about 75,000%, about 80,000%, about 85,000%, about 90,000%, about 95,000%, or about 100,000%.
Devices of the invention may also include one or more reservoirs for fluid reagents, e.g., a buffer solution, or samples, e.g., a suspension of cells and a composition to be introduced to the cells. For example, devices of the invention may include a reservoir for the cells suspended in the fluid to flow in the first electrode into the electroporation zone and/or a reservoir for holding the cells that have been electroporated. Similarly, a reservoir for liquids to flow in additional components of a device, such as additional inlets that intersect the first or second electrodes, may be present. A single reservoir may also be connected to multiple devices of the invention, e.g., when the same liquid is to be introduced at two or more individual device of the invention configured to electroporate cells in parallel or in series. Alternatively, devices of the invention may be configured to mate with sources of the liquids, which may be external reservoirs such as vials, tubes, or pouches. Similarly, the device may be configured to mate with a separate component that houses the reservoirs. Reservoirs may be of any appropriate size, e.g., to hold 10 mL to 5000 mL, e.g., 10 mL to 3000 mL, 25 mL to 100 mL, 100 mL to 1000 mL, 40 mL to 300 mL, 1 mL to 100 mL, 10 mL to 500 mL, 250 mL to 750 mL, 250 mL to 1000 mL, or 1000 mL to 5000 mL. When multiple reservoirs are present, each reservoir may have the same or a different size.
In addition to the components discussed above, devices of the invention may include additional components. For example, the first and second electrodes of the devices of the invention may include one or more additional fluid inlets to allow for the introduction of non-sample fluids, e.g., buffer solutions, into the appropriate region of the device. For example, a recovery zone of a device of the invention may include an additional inlet and outlet to circulate a recovery buffer to aid in the closing of the pores opened in the cell membranes from the electroporation process.
One or more electroporation devices of the invention may be combined with various external components, e.g., power supplies, pumps, reservoirs (e.g., bags), controllers, reagents, liquids, and/or samples in the form of a system. In some embodiments, a system of the invention includes a plurality of devices of the invention and a source of electrical potential that is releasably connected to the first and second electrodes of the device(s) of the invention. In this configuration, the device(s) of the invention are connected to the source of electrical potential, and the first electrode is energized and the second electrode is held at ground. This creates a localized electric field in the electroporation zone, thus electroporating the cells that pass through the device(s). Electroporation systems incorporating devices of the invention may induce either reversible or irreversible electroporation to the cells that pass through the device and system of the invention. For example, devices and systems of the invention may induce substantially non-thermal reversible electroporation, substantially non-thermal irreversible electroporation, or substantially thermal irreversible electroporation on the cells suspended in the fluid.
In some cases, the releasable connection to the first and second electrodes may include any practical electro-mechanical connection that can maintain consistent electrical contact between the source of electrical potential and the first and second electrodes. Example electrical connections include, but are not limited to clamps, clips, e.g., alligator clips, springs, e.g., a leaf spring, an external sheath or sleeve, wire brushes, flexible conductors, pogo pins, mechanical connections, inductive connections, or a combination thereof. Other types of electrical connections are known in the art. For example, a spring-type electrode can be integrated into a conductive platform such as that shown in
The source of electrical potential is configured to deliver an applied voltage to one or more electrode in order to provide an electrical potential difference between the electrodes and thus establish a uniform electric field in the electroporation zone. In some cases, such as in a two-electrode electroporation circuit, the applied voltage is delivered to a first electrode and the second electrode is held at ground. Without wishing to be bound by any particular theory, an applied voltage delivered to the electrode is delivered at a particular amplitude, a particular frequency, a particular pulse shape, a particular duration, a particular number of pulses applied, and a particular duty cycle. These parameters, coupled to the geometry of the electroporation zone, will deliver a particular electric field within the electroporation zone that will be experienced by the cells suspended in a fluid. The electrical parameters described herein may be optimized for a particular cell line and/or composition being delivered to a particular cell line. The application of the electrical potential to the electrodes of devices(s) of the invention may be initiated and/or controlled by a controller, e.g., a computer with programming, operatively coupled to the source of electrical potential.
Along with the electrical potential parameters described herein, the geometry of devices of the invention, e.g., the shape and dimensions of the cross-section of the electroporation zone, control the shape and intensity of the resulting electric field within the electroporation zone. Typically, a device with an electroporation zone that has a uniform cross section will exhibit a uniform electric field along its length. In order to modulate the resulting electric field in the electroporation zone, the electroporation zone may include a plurality of different cross-sectional dimensions and/or different cross-section shapes along its length. As a non-limiting example, a device of the invention may include a plurality of serially-connected electroporation zones, each of the plurality of electroporation zones having a circular cross-section of a different cross-sectional dimension, e.g., each has a different diameter. In this configuration, the different diameter circular cross-sections of the electroporation zone each act as an independent electroporation zone, and each will induce a different electric field at every change in dimension with an identical applied voltage, e.g., a constant DC voltage.
In some cases, devices of the invention may include a plurality of electroporation zones fluidically connected in series, with each electroporation zone having either a uniform or non-uniform cross-section and each may have a different cross-section shape. Alternatively, a system of the invention may include a plurality of devices of the invention in a parallel configuration, with each device operating independently of each other to increase the overall throughput of the electroporation.
In some cases, the amplitude of the applied voltage is from about −3 kV to 3 kV, e.g., about −3 kV to about −0.1 kV, about −2 kV to about −0.1 kV, about −1 kV to about −0.1 kV, about −0.1 kV to about −0.01 kV, 0.01 kV to about 3 kV, e.g., about 0.01 kV to about 0.1 kV, about 0.02 kV to about 0.2 kV, about 0.03 kV to about 0.3 kV, about 0.04 kV to about 0.4 kV, about 0.05 kV to about 0.5 kV, about 0.06 kV to about 0.6 kV, about 0.07 kV to about 0.7 kV, about 0.08 kV to about 0.8 kV, about 0.09 kV to about 0.9 kV, about 0.1 kV to about 1 kV, about 0.1 kV to about 2.0 kV, about 0.1 kV to about 3 kV, about 0.15 kV to about 1.5 kV, about 0.2 kV to about 2 kV, about 0.25 kV to about 2.5 kV, or about 0.3 kV to about 3 kV, e.g., about 0.01 to about 1 kV, about 0.1 kV to about 0.7 kV, or about 0.2 to about 0.6 kV, e.g., about 0.01 kV, about 0.02 kV, about 0.03 kV, about 0.04 kV, about 0.05 kV, about 0.06 kV, about 0.07 kV, about 0.08 kV, about 0.09 kV, about 0.1 kV, about 0.2 kV, about 0.3 kV, about 0.4 kV, about 0.5 kV, about 0.6 kV, about 0.7 kV, about 0.8 kV, about 0.9 kV, about 1 kV, about 1.1 kV, about 1.2 kV, about 1.3 kV, about 1.4 kV, about 1.5 kV, about 1.6 kV, about 1.7 kV, about 1.8 kV, about 1.9 kV, about 2 kV, about 2.1 kV, about 2.2 kV, about 2.3 kV, about 2.4 kV, about 2.5 kV, about 2.6 kV, about 2.7 kV, about 2.8 kV, about 2.9 kV, or about 3 kV.
In some cases, the frequency of the applied voltage is from about 1 Hz to about 50,000 Hz, e.g., from about 1 Hz to about 1,000 Hz, about 1 Hz to about 500 Hz, about 100 Hz to about 500 Hz, about 100 Hz to about 5,000 Hz, about 500 Hz to about 10,000 Hz, about 1000 Hz to about 25,000 Hz, or from about 5,000 Hz to about 50,000 Hz, e.g., from about 10 Hz to about 1000 Hz, about 10 Hz to about 500 Hz, about 500 Hz to about 750 Hz, or about 100 Hz to about 500 Hz, e.g., from about 1 Hz, about 2 Hz, about 3 Hz, about 4 Hz, about 5 Hz, about 6 Hz, about 7 Hz, about 8 Hz, about 9 Hz, about 10 Hz, about 20 Hz, about 30 Hz, about 40 Hz, about 50 Hz, about 60 Hz, about 70 Hz, about 80 Hz, about 90 Hz, about 100 Hz, about 110 Hz, about 120 Hz, about 130 Hz, about 140 Hz, about 150 Hz, about 160 Hz, about 170 Hz, about 180 Hz, about 190 Hz, about 200 Hz, about 210 Hz, about 220 Hz, about 230 Hz, about 240 Hz, about 250 Hz, about 260 Hz, about 270 Hz, about 270 Hz, about 280 Hz, about 290 Hz about 300 Hz, about 310 Hz, about 320 Hz, about 330 Hz, about 340 Hz, about 350 Hz, about 360 Hz, about 370 Hz, about 380 Hz, about 390 Hz, about 400 Hz, about 410 Hz, about 420 Hz, about 430 Hz, about 440 Hz, about 450 Hz, about 460 Hz, about 470 Hz, about 480 Hz, about 490 Hz, about 500 Hz, about 510 Hz, about 520 Hz, about 530 Hz, about 540 Hz, about 550 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900 Hz, about 1,000 Hz, about 2,000 Hz, about 3,000 Hz, about 4,000 Hz, about 5,000 Hz, about 6,000 Hz, about 7,000 Hz, about 8,000 Hz, about 9,000 Hz, about 10,000 Hz, about 15,000 Hz, about 20,000 Hz, about 25,000 Hz, about 30,000 Hz, about 35,000 Hz, about 40,000 Hz, about 45,000 Hz, or about 50,000 Hz.
In some embodiments, the shape of the applied pulse, e.g., waveform, can be a square wave, pulse, a bipolar wave, a sine wave, a ramp, an asymmetric bipolar wave, or arbitrary. Other voltage waveforms are known in the art. The chosen waveform can be applied at any practical voltage pattern including, but not limited to, high voltage-low voltage, low voltage-high voltage, direct current (DC), alternating current (AC), unipolar, positive (+) polarity only, negative (−) polarity only, (+)/(−) polarity, (−)/(+) polarity, or any superposition or combination thereof. A skilled artisan can appreciate that these pulse parameters will depend on the cell line any electrical characteristics of the composition being delivered to the cell.
Applied voltage pulses can be delivered to the electroporation zone with durations from about 0.01 ms to about 1,000 ms, e.g., from about 0.01 ms to about 1 ms, about 0.1 ms to about 10 ms, about 0.1 ms to about 15 ms, about 1 ms to about 10 ms, about 1 ms to about 50 ms, about 10 ms to about 100 ms, about 25 ms to about 200 ms, about 50 ms to about 400 ms, about 100 ms to about 600 ms, about 300 ms to about 800 ms, or about 500 ms to about 1,000 ms, e.g., about 0.01 ms to 100 ms, about 0.1 ms to about 50 ms, or about 1 ms to about 10 ms, e.g., about 0.01 ms, about 0.02 ms, about 0.03 ms, about 0.04 ms, about 0.05 ms, about 0.06 ms, about 0.07 ms, about 0.08 ms, about 0.09 ms, about 0.1 ms, about 0.2 ms, about 0.3 ms, about 0.4 ms, about 0.5 ms, about 0.6 ms, about 0.7 ms, about 0.8 ms, about 0.9 ms, about 1 ms, about 2 ms, about 3 ms, about 4 ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 11 ms, about 12 ms, about 13 ms, about 14 ms, about 15 ms, about 20 ms, about 30 ms, about 40 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 150 ms, about 200 ms, about 250 ms, about 300 ms, about 350 ms, about 400 ms, about 450 ms, about 500 ms, about 550 ms, about 600 ms, about 650 ms, about 700 ms, about 750 ms, about 800 ms, about 850 ms, about 900 ms, about 950 ms, or about 1,000 ms.
In some cases, the number of applied voltage pulses delivered can be from 0 to about 1000, or more, e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 100 or more, e.g., 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 7-12, or 9-13, e.g., about 0.01 to about 1,000, e.g., from about 1 to about 10, about 1 to about 50, about 5 to about 10, about 5 to about 15, about 10 to about 100, about 25 to about 200, about 50 to about 400, about 100 to about 600, about 300 to about 800, or about 500 to about 1,000, e.g., about 1 to 100, about 1 to about 50, or about 1 to about 10, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1,000.
In some instances, the number of applied voltage pulses delivered can be from 1 to about 1,000,000. For example, in some instances, the number of applied voltage pulses delivered is from 1,000 to 1,000,000, e.g., from 1,000 to 10,000 (e.g., from 1,000 to 2,000, from 2,000 to 3,000, from 3,000 to 4,000, from 4,000 to 5,000, from 5,000 to 6,000, from 6,000 to 7,000, from 7,000 to 8,000, from 8,000 to 9,000, or from 9,000 to 10,000, e.g., about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, or about 10,000), from 10,000 to 100,000 (e.g., from 10,000 to 20,000, from 20,000 to 30,000, from 30,000 to 40,000, from 40,000 to 50,000, from 50,000 to 60,000, from 60,000 to 70,000, from 70,000 to 80,000, from 80,000 to 90,000, or from 90,000 to 100,000, e.g., about 10,000, about 25,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 75,000, about 80,000, about 90,000, or about 100,000), or from 100,000 to 1,000,000 (e.g., from 100,000 to 200,000, from 200,000 to 300,000, from 300,000 to 400,000, from 400,000 to 500,000, from 500,000 to 600,000, from 600,000 to 700,000, from 700,000 to 800,000, from 800,000 to 900,000, or from 900,000 to 1,000,000, e.g., about 100,000, about 200,000, about 250,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 750,000, about 800,000, about 900,000, or about 1,000,000).
The pulses of applied voltage can, in some instances, be delivered at a duty cycle of about 0.001% to about 100%, e.g., from about 0.001% to about 0.1%, about 0.01% to about 1%, about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, or about 50% to about 100%, e.g., about 0.01% to 100%, about 0.1% to about 99%, about 1% to about 97%, or about 10% to about 95%, e.g., about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
Device(s) of the invention, when the electrodes are connected to the source of electrical potential and energized, generate a localized electric field in the electroporation zone that electroporate cells that pass through. In some cases, the electric field generated in the electroporation zone has a magnitude from about 2 V/cm to about 50,000 V/cm, e.g., about 2 V/cm to about 1,000 V/cm, about 100 V/cm to about 1,000 V/cm, about 100 V/cm to about 5,000 V/cm, about 500 V/cm to about 10,000 V/cm, about 1000 V/cm to about 25,000 V/cm, or from about 5,000 V/cm to about 50,000 V/cm, e.g., from about 2 V/cm to about 20,000 V/cm, about 5 V/cm to about 10,000 V/cm, or about 100 V/cm to about 1,000 V/cm, e.g., from about 2 V/cm, about 3 V/cm, about 4 V/cm, about 5 V/cm, about 6 V/cm, about 7 V/cm, about 8 V/cm, about 9 V/cm, about 10 V/cm, about 20 V/cm, about 30 V/cm, about 40 V/cm, about 50 V/cm, about 60 V/cm, about 70 V/cm, about 80 V/cm, about 90 V/cm, about 100 V/cm, about 200 V/cm, about 300 V/cm, about 400 V/cm, about 500 V/cm, about 600 V/cm, about 700 V/cm, about 800 V/cm, about 900 V/cm, about 1,000 V/cm, about 2,000 V/cm, about 3,000 V/cm, about 4,000 V/cm, about 5,000 V/cm, about 6,000 V/cm, about 7,000 V/cm, about 8,000 V/cm, about 9,000 V/cm, about 10,000 V/cm, about 15,000 V/cm, about 20,000 V/cm, about 25,000 V/cm, about 30,000 V/cm, about 35,000 V/cm, about 40,000 V/cm, about 45,000 V/cm, or about 50,000 V/cm.
Systems of the invention typically include a fluid delivery source that is configured to deliver the plurality of cells suspended in the fluid through the first electrode, e.g., the entry zone, to the second electrode, e.g., the recovery zone. Fluid delivery sources typically includes pumps, including, but not limited to, syringe pumps, micropumps, or peristaltic pumps. Alternatively, fluids can be delivered by the displacement of a working fluid against a reservoir of the fluid to be delivered or by air displacement. Other fluid delivery sources are known in the art. In some cases, the fluid delivery source is configured to flow cells suspended in a fluid by the application of a positive pressure. Without wishing to be bound by any particular theory, the flow rate at which cells in a suspension are flowed through devices of the invention and the specific geometry of the electroporation zone of devices of the invention will determine the residence time of the cells in the electric field in the electroporation zone.
In some instances, the volumetric flow rate of fluid delivered from a fluid delivery source has a volumetric flow rate of about 0.001 mL/min to about 1,000 mL/min, e.g., from about 0.001 mL/min to about 0.1 mL/min, about 0.01 mL/min to about 1 mL/min, about 0.1 mL/min to about 10 mL/min, about 1 mL/min to about 50 mL/min, about 10 mL/min to about 100 mL/min, about 25 mL/min to about 200 mL/min, about 50 mL/min to about 400 mL/min, about 100 mL/min to about 600 mL/min, about 300 mL/min to about 800 mL/min, or about 500 mL/min to about 1,000 mL/min, e.g., about 0.001 mL/min, about 0.002 mL/min, about 0.003 mL/min, about 0.004 mL/min, about 0.005 mL/min, about 0.006 mL/min, about 0.007 mL/min, about 0.008 mL/min, about 0.009 mL/min, about 0.01 mL/min, about 0.02 mL/min, about 0.03 mL/min, about 0.04 mL/min, about 0.05 mL/min, about 0.06 mL/min, about 0.07 mL/min, about 0.08 mL/min, about 0.09 mL/min, about 0.1 mL/min, about 0.2 mL/min, about 0.3 mL/min, about 0.4 mL/min, about 0.5 mL/min, about 0.6 mL/min, about 0.7 mL/min, about 0.8 mL/min, about 0.9 mL/min, about 1 mL/min, about 2 mL/min, about 3 mL/min, about 4 mL/min, about 5 mL/min, about 6 mL/min, about 7 mL/min, about 8 mL/min, about 9 mL/min, about 10 mL/min, about 15 mL/min, about 20 mL/min, about 25 mL/min, about 30 mL/min, about 35 mL/min, about 40 mL/min, about 45 mL/min, about 50 mL/min, about 55 mL/min, about 60 mL/min, about 65 mL/min, about 70 mL/min, about 75 mL/min, about 80 mL/min, about 85 mL/min, about 90 mL/min, about 95 mL/min, about 100 mL/min, about 150 mL/min, about 200 mL/min, about 250 mL/min, about 300 mL/min, about 350 mL/min, about 400 mL/min, about 450 mL/min, about 500 mL/min, about 550 mL/min, about 600 mL/min, about 650 mL/min, about 700 mL/min, about 750 mL/min, about 800 mL/min, about 850 mL/min, about 900 mL/min, about 950 mL/min, or about 1,000 mL/min. In particular embodiments, the flow rate is from 10 mL/min to about 100 mL/min, e.g., about 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min, 50 mL/min, 60 mL/min, 70 mL/min, 80 mL/min, 90 mL/min, or 100 mL/min.
In some instances, a Reynolds number of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 0.04 and 2.43×104, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some instances, a maximum velocity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 5×10−5 m/s and 32.7 m/s, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some instances, shear rates of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone are between 0.1 1/s and 2×106 1/s, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some instances, a peak pressure of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1×10−3 Pa and 9.5×104 Pa, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some instances, an average velocity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1.5×10−5 m/s and 15.9 m/s, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet. In some instances, a kinematic viscosity of a liquid and/or the plurality of cells in suspension delivered from a fluid delivery source from the first lumen to the electroporation zone is between 1×10−6 m2/s and 15×10−4 m2/s, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet.
The residence time of cells in the electroporation zone of devices of the invention may be from about 0.5 ms to about 50 ms, e.g., from about 0.5 ms to about 5 ms, about 1 ms to about 10 ms, about 5 ms to about 15 ms, about 10 ms to about 20 ms, about 15 ms to about 25 ms, about 20 ms to about 30 ms, about 25 ms to about 35 ms, about 30 ms to about 40 ms, about 35 ms to about 45 ms, or about 40 ms to about 50 ms, e.g., about 0.5 ms, about 0.6 ms, about 0.7 ms, about 0.8 ms, about 0.9 ms, about 1 ms, about 1.5 ms, about 2 ms, about 2.5 ms, about 3 ms, about 3.5 ms, about 4 ms, about 4.5 ms, about 5 ms, about 5.5 ms, about 6 ms, about 6.5 ms, about 7 ms, about 7.5 ms, about 8 ms, about 8.5 ms, about 9 ms, about 9.5 ms, about 10 ms, about 10.5 ms, about 11 ms, about 11.5 ms, about 12 ms, about 12.5 ms, about 13 ms, about 13.5 ms, about 14 ms, about 14.5 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, or about 50 ms. In some embodiments, the residence time is from 5-20 ms (e.g., from 6-18 ms, 8-15 ms, or 5-14 ms).
Systems of the invention typically feature a housing that contains and supports the device(s) of the invention and any necessary electrical connections, e.g., electrode connections. The housing may be configured to hold and energize a single device of the invention, or alternatively, may be configured to hold and simultaneously energize a plurality of devices of the invention. For example, in the embodiment of a system of the invention shown in
In some embodiments, the housing (e.g., cartridge) is configured for use with and/or insertion into an automated closed system that is used to deliver cell therapies to patients in a clinical or hospital setting.
In some embodiments, the housing (e.g., cartridge) further includes a cooling/heating area/enclosure for cell suspension and/or buffer storage during, before and after electroporation of the specimen. In some embodiments, the system (e.g., device and housing) is externally powered.
In some embodiments, devices of the invention include a touchscreen user interface or other alternative user interface(s) that enables the user to select parameters such as flow rate, waveforms, applied potential, volume to transfect, time delay, cooling features, heating features, electroporation or transfection status, progress and other parameters used to optimize the electroporation or electro-mechanical protocol. In some embodiments, the user interface also contains pre-formulated parameter selections that enable the user to operate the system at specific parameters and conditions that have previously been validated by user or as recommended by the manufacturers. In some embodiments, the user interface may be connected to programming that allows for automated running of the system and/or running an algorithm to optimize electroporation for a given sample of a known cell type. In some embodiments, the optimization algorithms have the ability to adjust electro-mechanical parameters independently or autonomously if the user selects this functionality. In some embodiments, the optimization algorithms allow for continuous adjustment of the parameters used in the electroporation or electro-mechanical process that may depend on the cell type, conductivity of cell suspensions, volume of cell suspensions, viscosity, lifetime of the electroporating cartridge(s), the physical state of the suspension, or the state of the transfection device(s).
In some embodiments, the optimization algorithms have the ability to perform predictive analysis based on known input cell-type parameters and to adjust electro-mechanical parameters accordingly. In some embodiments, the optimization algorithms adjust electro-mechanical parameters based on electrical signals within any of the devices of the invention. In some embodiments, the optimization algorithms adjust electro-mechanical parameters based on detected flow parameters within any of the devices of the invention. In some embodiments, the optimization algorithms adjust electroporation parameters based on unique dimensionless input parameters. In some embodiments, the optimization algorithms have the ability to adjust electroporation parameters based on unique multivariate combinations of parameters that are predictive of high viability results, high efficiency results, or matched viability and efficiency results.
Systems of the invention may include one or more outer structures that are configured to cover the electrodes of one or more devices of the invention, e.g., to reduce end user exposure to live electrical connections. Typically, a device of the invention (e.g., a FLOWFECT™ device) will include one outer structure that covers its electrodes and electroporation zone. The outer structure may be a non-conductive material, e.g., a non-conductive polymer, that includes structural features for electro-mechanically engaging the parts of the device, e.g., the electrodes or electroporation zone. The outer structure may include one or more recesses, cutouts, or similar openings within the structure to accommodate the device. The outer structure may be configured to be a component that can be removed from the device. For example, the outer structure may include two separate components connected by a hinge, e.g., a living hinge, such that it can be folded over the device of the invention. Alternatively, the outer structure may be one or more separate pieces that can be connected together using suitable mating features to form a single structure. In these embodiments, the outer structure may be affixed to the device of the invention using any suitable fastener, e.g., snaps, latches, button, or clips, which may be integrated into the outer structure or externally connected to the outer structure. Other suitable fastener types are known in the art. In some embodiments, the outer structure includes one or more alignment features, e.g., pins, divots, grooves, or tabs, that ensure correct alignment of the one or more pieces of the outer structure. In some cases, the outer structure is configured to be permanently connected to the devices of the invention.
In some embodiments, the housing (e.g., cartridge, e.g., outer structure) encapsulates one or more of the previously stated inventions or one or more electroporating devices used for continuous flow electroporation. In some embodiments, the housing (e.g., cartridge) is configured to allow use with and/or insertion into an automated closed system that delivers cell therapies to patients. In some embodiments, the housing further includes a cooling/heating area/enclosure for cell suspension and/or buffer storage during, before and after electroporation of the specimen. In some embodiments, the system (e.g., one or more devices and housing) is externally powered.
In some embodiments, the system also includes optimization algorithms that have the ability to adjust electro-mechanical parameters independently or autonomously if the user selects this functionality. These optimization algorithms allow for continuous adjustment of the parameters used in the electroporation process that may depend on the cell type, conductivity, volume of suspensions, viscosity, lifetime of the electroporating cartridge, the physical state of the suspension or the state of the electroporation device.
In any of the embodiments of the outer structure described herein, the outer structure provides for electrical connection between an external source of electric potential and the electrodes of the devices of the invention. For example, the outer structure may include one or more electrical inputs for electrical connections, e.g., spades, banana plugs, or bayonet, e.g., BNC, connectors, that facilitate electrical connection between the source of electric potential and the electrodes of the devices of the invention inside the outer structure.
Devices and outer structures of the invention may be combined with additional external components, such as reagents, e.g., buffers, e.g., transfection or recovery buffers, and/or samples, in a kit. In some instances, a transfection buffer includes a composition appropriate for cell electroporation. In some instances, the transfection buffer includes a suitable concentration of one or more salts (e.g., potassium chloride, sodium chloride, potassium phosphate, potassium dihydrogen phosphate) or sugars (e.g., dextrose or myo-inositol), or any combination thereof, at a concentration from 0.1 to 200 mM (e.g., from 0.1 to 1.0 mM, from 1.0 mM to 10 mM, or from 10 mM to 100 mM).
Devices and systems of the invention may be used with transfection buffer or with cell culture growth medium that contains additives to support transfection. Certain additives may be added to control the conductivity of transfection buffer and/or cell culture growth medium used, including KCl, MgCl2, NaCl, glucose, Na2HPO4, NaH2PO4, Ca(NO3)2, mannitol, succinate, dextrose, hydroxyethyl piperazineethanesulfonic acid (HEPES), trehalose, CaCl2), dimethyl sulfoxide (DMSO), K2HPO4, KH2PO4, ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), KOH, NaOH, K2SO4, Na2SO4, histidine buffer, citrate buffer, phosphate-buffered saline (PBS), ATP-disodium salt, and NaHCO3. Certain additives may be added to control the viscosity of transfection buffer and/or cell culture growth medium used, including Ficoll, dextran, polyethylene glycol (PEG), methylcellulose (MethoCel), collagen I, and Matrigel.
The invention features methods of introducing a composition, e.g., transfection, into at least a portion of a plurality of cells suspended in a fluid, using the electroporation devices described herein. The methods described herein may be used to greatly increase the throughput of the delivery of compositions into cell types, often considered to be a bottleneck in the research fields of genetic engineering and therapeutic fields of gene-modified cell therapies. In particular, the methods described herein have significantly increased number of recovered cells, transfection efficiency and cell viability after transfection with applications to more cell types than typical methods of transfection, e.g., lentiviral transfection, or commercially available cell transfection instruments, e.g., the NEON® Transfection System (Thermo Fisher, Carlsbad, Calif.) or the 4D-NUCLEOFECTOR (Lonza, Switzerland).
A composition is introduced into at least a portion of a plurality of cells suspended in a fluid by passing the fluid with the suspended cells, also containing the composition to be introduced into the cells, through a device of the invention, e.g., an electroporation device, as described herein. The composition and the cells suspended in the fluid can be delivered through the device of the invention by the application of a positive pressure, e.g., from a pump connected to a source of fluid, e.g., a peristaltic pump, a digital pipette, or automated liquid handling source. The composition and the cells suspended in the fluid pass from the first electrode, e.g., including and entry zone, to an electroporation zone fluidically connected to the first electrode, and then to the recovery zone, which is fluidically connected to electroporation zone. As the composition and cells suspended in the fluid flow through the first electrode to the electroporation zone, a potential difference is applied to the first and second electrodes, producing and thus exposing the cells to an electric field in the electroporation zone. The exposure of the cells to the generated electric field enhances temporary permeability of the plurality of cells, thus introducing the composition into at least a portion of the plurality of cells.
In some instances of the methods, the phenotype of the cells may or may not be altered relative to a baseline measurement of cell phenotype upon exiting the electroporation zone of devices of the invention. In some cases, the phenotype of the cells is altered from 0% to about 25% relative to a baseline measurement of cell phenotype upon exiting the electroporation zone of devices of the invention, e.g., from about 0% to about 2.5%, from about 1% to about 5%, from about 1% to about 10%, from about 5% to about 15%, from about 10% to about 20%, from about 15% to about 25%, or from about 20% to about 25%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%. In particular instances, the plurality of cells has no phenotypic change upon exiting the electroporation zone. For example, a baseline or control measurement to establish the cell phenotype may be the measurement of the expression of a cell surface marker on cells that have not been transfected using devices of the invention. A corresponding identical measurement of the expression of the same cell marker on cells that have been transfected using devices of the invention can be used to assess changes in cell phenotype. The cell phenotype is assessed via flow cytometry analysis of cell surface marker expression to ensure that the cell phenotype is minimally changed or unchanged after electroporation. Examples of the cell surface markers to evaluate include, but are not limited to, CD3, CD4, CD8, CD19, CD45RA, CD45RO, CD28, CD44, CD69, CD80, CD86, CD206, IL-2 receptor, CTLA4, OX40, PD-1, and TIM3. Cell morphology is assessed using bright field or fluorescent microscopy to confirm lack of phenotypic changes after electroporation.
In some instances, the after introduction of the composition into at least a portion of the plurality of cells, the plurality of cells are stored in a recovery buffer. The recovery buffer is configured to promote the final closing of the pores that were formed in the plurality of cells. Recovery buffers typically include cell culture media that may include other ingredients for cell nourishment and growth, e.g., serum, minerals, etc. A skilled artisan can appreciate that the choice of recovery buffer will depend on the cell type undergoing electroporation.
In some embodiments of the method described herein, the volume of fluid with the suspended cells (e.g., displacement volume) and the composition to be introduced to the cells that are flowed through the electroporation zone of devices of the invention may be from about 0.001 mL to about 2000 mL, about 0.001 mL to about 1000 mL, e.g., 0.001 mL to about 1000 mL, e.g., from about 0.001 mL to about 0.1 mL, about 0.01 mL to about 1 mL, about 0.01 mL to about 750 mL, about 0.01 mL to about 1500 mL, about 0.1 mL to about 5 mL, about 0.1 mL to 500 mL, about 0.1 mL to about 2000 mL, about 1 mL to about 10 mL, about 1 mL to about 1000 mL, about 2 mL to about 2000 mL, about 2.5 mL to about 20 mL, about 5 mL to about 40 mL, about 10 mL to about 60 mL, about 10 mL to 1000 mL, about 20 mL to about 2000 mL, about 30 mL to about 80 mL, about 50 mL to about 200 mL, about 100 mL to about 500 mL, or 250 mL to about 750 mL, about 500 mL to about 1000 mL, about 500 mL to 2000 mL, about 750 mL to 1500 mL, or about 1000 mL to 2000 mL, e.g., about 0.01 mL to 100 mL, about 0.1 mL to about 99 mL, about 1 mL to about 97 mL, or about 10 mL to about 95 mL, e.g., about 0.0025 mL to about 10 mL, about 0.01 mL to about 1 mL, or about 0.025 mL to about 0.1 mL, e.g., about 0.001 mL, about 0.0025 mL, about 0.005 mL, about 0.0075 mL, about 0.01 mL, about 0.025 mL, about 0.05 mL, about 0.075 mL, about 0.1 mL, about 0.25 mL, about 0.5 mL, about 0.75 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 150 mL, about 200 mL, about 250 mL, about 300 mL, about 350 mL, about 400 mL, about 450 mL, about 500 mL, about 550 mL, about 600 mL, about 650 mL, about 700 mL, about 750 mL, about 800 mL, about 850 mL, about 900 mL, about 950 mL, about 1000 mL, about 1050 mL, about 1100 mL, about 1150 mL, about 1200 mL, about 1250 mL, about 1300 mL, about 1350 mL, about 1400 mL, about 1450 mL, about 1500 mL, about 1550 mL, about 1600 mL, about 1650 mL, about 1700 mL, about 1750 mL, about 1800 mL, about 1850 mL, about 1900 mL, about 1950 mL, or about 2000 mL.
In some embodiments, the volume of fluid that is flowed through the electroporation zone of devices of the invention (e.g., displacement volume), displacement rate, or other controlled parameters may or may not affect the transfection efficiency of a plurality of cells. In some embodiments, the devices of the invention are configured for use with an automated fluid handling platform that can process a plurality of cells in volumes less than 100 μL that can accumulate to volumes ranging from more than 100 μL to about 2000 mL. In some embodiments, the automated fluid handling platform is configured for use with one or more fluid delivery sources (e.g., pumps, e.g., syringe pumps, micropumps, or peristaltic pumps) that deliver the volume of fluid that is flowed through the electroporation zone of devices of the invention. In some embodiments, the volume of fluid that is flowed through the electroporation zone of devices of the invention can be delivered by the displacement of a working fluid against a reservoir of the fluid to be delivered or by air displacement. In some embodiments, the fluid delivery source is configured to flow cells suspended in a fluid by the application of a positive pressure.
In certain aspects, the electrical conductivity of the fluid where the cells are suspended can affect the electroporation of, and thus the delivery of a composition to, the cells in the suspension. The conductivity of the fluid with the suspended cells may be from about 0.001 mS to about 500 mS, e.g., from about 0.001 mS to about 0.1 mS, about 0.01 mS to about 1 mS, about 0.1 mS to about 10 mS, about 1 mS to about 50 mS, about 10 mS to about 100 mS, about 25 mS to about 200 mS, about 50 mS to about 400 mS, or about 100 mS to about 500 mS, e.g., about 0.01 mS to about 100 mS, about 0.1 mS to about 50 mS, or about 1 to 20 mS, e.g., about 0.001 mS, about 0.002 mS, about 0.003 mS, about 0.004 mS, about 0.005 mS, about 0.006 mS, about 0.007 mS, about 0.008 mS, about 0.009 mS, about 0.01 mS, about 0.02 mS, about 0.03 mS, about 0.04 mS, about 0.05 mS, about 0.06 mS, about 0.07 mS, about 0.08 mS, about 0.09 mS, about 0.1 mS, about 0.2 mS, about 0.3 mS, about 0.4 mS, about 0.5 mS, about 0.6 mS, about 0.7 mS, about 0.8 mS, about 0.9 mS, about 1 mS, about 2 mS, about 3 mS, about 4 mS, about 5 mS, about 6 mS, about 7 mS, about 8 mS, about 9 mS, about 10 mS, about 15 mS, about 20 mS, about 25 mS, about 30 mS, about 35 mS, about 40 mS, about 45 mS, about 50 mS, about 55 mS, about 60 mS, about 65 mS, about 70 mS, about 75 mS, about 80 mS, about 85 mS, about 90 mS, about 95 mS, about 100 mS, about 150 mS, about 200 mS, about 250 mS, about 300 mS, about 350 mS, about 400 mS, about 450 mS, or about 500 mS.
Methods of the invention can deliver compositions to a variety of cell types including, but not limited to, mammalian cells, eukaryotes, prokaryotes, synthetic cells, human cells, animal cells, plant cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, or activated cells immune cells, stem cells (e.g., primary human induced pluripotent stem cells, e.g., iPSCs, embryonic stem cells, e.g., ESCs, mesenchymal stem cells, e.g., MSCs, or hematopoietic stem cells, e.g., HSCs), blood cells (e.g., red blood cells), T cells (e.g., primary human T cells), B cells, antigen presenting cells (APCs), natural killer (NK) cells (e.g., primary human NK cells), monocytes (e.g., primary human monocytes), macrophages (e.g., primary human macrophages), and peripheral blood mononuclear cells (PBMCs), neutrophils, dendritic cells, human embryonic kidney (e.g., HEK-293) cells, or Chinese hamster ovary (e.g., CHO-K1) cells. Typical cell numbers that can be electroporated may be from about 104 cells to about 1012 cells, (e.g., about 104 cells to about 105 cells, about 104 cells to about 106 cells, about 104 cells to about 107 cells, about 5×104 cells to about 5×105 cells, about 105 cells to about 106 cells, about 105 cells to about 107 cells, about 2.5×105 cells to about 106 cells, about 5×105 cells to about 5×106 cells, about 106 cells to about 107 cells, about 106 cells to about 108 cells, about 106 cells to about 1012 cells, about 5×106 cells to about 5×107 cells, about 107 cells to about 108 cells, about 107 cells to about 109 cells, about 107 cells to about 1012 cells, about 5×107 cells to about 5×108 cells, about 108 cells to about 109 cells, about 108 cells to about 1010 cells, about 108 cells to about 1012 cells, about 5×108 cells to about 5×109 cells, about 109 cells to about 1010 cells, about 109 cells to about 1011 cells, about 1010 cells to about 1011 cells, about 1010 cells to about 1012 cells, or about 1011 cells to about 1012 cells, e.g., about 104 cells, about 2.5×104 cells, about 5×104 cells, about 105 cells, about 2.5×105 cells, about 5×105 cells, about 106 cells, about 2.5×106 cells, about 5×106 cells, about 107 cells, about 2.5×107 cells, about 5×107 cells, about 108 cells, about 2.5×108 cells, about 5×108 cells, about 109 cells, about 2.5×109 cells, about 5×109 cells, about 1010 cells, about 5×1010 cells, about 1011 cells, or about 1012 cells).
Cell concentrations, i.e., number of cells per mL of fluid, for achieving cell poration numbers of about 104 cells to about 1012 cells typically ranges from about 103 cells/mL to about 1011 cells/mL, e.g., about 103 cells/mL to about 104 cells/mL, about 5×103 cells/mL to about 5×104 cells/mL, about 105 cells/mL to about 105 cells/mL, about 5×105 cells/mL to about 5×106 cells/mL, about 106 cells/mL to about 107 cells/mL, about 5×106 cells/mL to about 5×107 cells/mL, about 107 cells/mL to about 108 cells/mL, about 5×107 cells/mL to about 5×108 cells/mL, about 108 cells/mL to about 109 cells/mL, about 5×108 cells/mL to about 5×109 cells/mL, about 109 cells/mL to about 109 cells/mL, about 5×109 cells/mL to about 5×1010 cells/mL, or about 1010 cells/mL to about 1011 cells/mL, e.g., about 103 cells/mL, about 5×103 cells/mL, about 104 cells/mL, about 5×104 cells/mL, about 105 cells/mL, about 5×105 cells/mL, about 106 cells/mL, about 5×106 cells/mL, about 107 cells/mL, about 5×107 cells/mL, about 108 cells/mL, about 5×108 cells/mL, about 109 cells/mL, about 5×109 cells/mL, about 1010 cells/mL, about 5×1010 cells/mL, or about 1011 cells/mL.
Methods of the invention described herein may deliver any composition to the cells suspended in the fluid. Compositions that can be delivered to the cells include, but are not limited to, therapeutic agents, vitamins, nanoparticles, charged molecules, e.g., ions in solution, uncharged molecules, nucleic acids, e.g., DNA or RNA, CRISPR-Cas complex, proteins, polymers, a ribonucleoprotein (RNP), engineered nucleases, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing nucleases, meganucleases (MNs), megaTALs, enzymes, peptides, transposons, or polysaccharides, e.g., dextran, e.g., dextran sulfate. Exemplary compositions that can be delivered to cells in a suspension include nucleic acids, oligonucleotides, antibodies (or an antibody fragment, e.g., a bispecific fragment, a trispecific fragment, Fab, F(ab′)2, or a single-chain variable fragment (scFv)), amino acids, peptides, proteins, gene therapeutics, genome engineering therapeutics, epigenome engineering therapeutics, carbohydrates, chemical drugs, contrast agents, magnetic particles, polymer beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotic agents, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, anti-inflammatory agents, anti-microbial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacteriophages, adjuvants, minerals, and combinations thereof. A composition to be delivered may include a single compound, such as the compounds described herein. Alternatively, the composition to be delivered may include a plurality of compounds or components targeting different genes.
Typical concentrations of the composition in the fluid may be from about 0.0001 μg/mL to about 1000 μg/mL, (e.g., from about 0.0001 μg/mL to about 0.001 μg/mL, about 0.001 μg/mL to about 0.01 μg/mL, about 0.001 μg/mL to about 5 μg/mL, about 0.005 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 0.1 μg/mL, about 0.01 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 1 μg/mL, about 0.1 μg/mL to about 5 μg/mL, about 1 μg/mL to about 10 μg/mL, about 1 μg/mL to about 50 μg/mL, about 1 μg/mL to about 100 μg/mL, about 2.5 μg/mL to about 15 μg/mL, about 5 μg/mL to about 25 μg/mL, about 5 μg/mL to about 50 μg/mL, about 5 μg/mL to about 500 μg/mL, about 7.5 μg/mL to about 75 μg/mL, about 10 μg/mL to about 100 μg/mL, about 10 μg/mL to about 1,000 μg/mL, about 25 μg/mL to about 50 μg/mL, about 25 μg/mL to about 250 μg/mL, about 25 μg/mL to about 500 μg/mL, about 50 μg/mL to about 100 μg/mL, about 50 μg/mL to about 250 μg/mL, about 50 μg/mL to about 750 μg/mL, about 100 μg/mL to about 300 μg/mL, about 100 μg/mL to about 1,000 μg/mL, about 200 μg/mL to about 400 μg/mL, about 250 μg/mL to about 500 μg/mL, about 350 μg/mL to about 500 μg/mL, about 400 μg/mL to about 1,000 μg/mL, about 500 μg/mL to about 750 μg/mL, about 650 μg/mL to about 1,000 μg/mL, or about 800 μg/mL to about 1,000 μg/mL, e.g., about 0.0001 μg/mL, about 0.0005 μg/mL, about 0.001 μg/mL, about 0.005 μg/mL, about 0.01 μg/mL, about 0.02 μg/mL, about 0.03 μg/mL, about 0.04 μg/mL, about 0.05 μg/mL, about 0.06 μg/mL, about 0.07 μg/mL, about 0.08 μg/mL, about 0.09 μg/mL, about 0.1 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL, about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1 μg/mL, about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about 3 μg/mL, about 3.5 μg/mL, about 4 μg/mL, about 4.5 μg/mL, about 5 μg/mL, about 5.5 μg/mL, about 6 μg/mL, about 6.5 μg/mL, about 7 μg/mL, about 7.5 μg/mL, about 8 μg/mL, about 8.5 μg/mL, about 9 μg/mL, about 9.5 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, about 100 μg/mL, about 200 μg/mL, about 250 μg/mL, about 300 μg/mL, about 350 μg/mL, about 400 μg/mL, about 450 μg/mL, about 500 μg/mL, about 550 μg/mL, about 600 μg/mL, about 650 μg/mL, about 700 μg/mL, about 750 μg/mL, about 800 μg/mL, about 850 μg/mL, about 900 μg/mL, about 950 μg/mL, or about 1,000 μg/mL).
In some cases, the temperature of the fluid with the suspended cells and the composition is controlled using a thermal controller that is incorporated into a housing that supports the device(s) of the invention. The temperature of the fluid is controlled to reduce the effects of Joule heating originating from the electric field generated in the electroporation zone, as too high a temperature may compromise cell viability post-electroporation. The temperature of the fluid may be from about 0° C. to about 50° C., e.g., from about 0° C. to about 10° C., about 1° C. to about 5° C., about 2° C. to about 15° C., about 3° C. to about 20° C., about 4° C. to about 25° C., about 5° C. to about 30° C., about 7° C. to about 35° C., about 9° C. to about 40° C., about 10° C. to about 43° C., about 15° C. to about 50° C., about 20° C. to about 40° C., about 25° C. to about 50° C., or about 35° C. to about 45° C., e.g., about 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C. about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C.
Cells transfected using the methods of the invention are more efficiently transfected and have higher viability than using typical methods of transfection, e.g., lentiviral transfection, or commercially available cell transfection instruments, e.g., the NEON® Transfection System (Thermo Fisher, Carlsbad, Calif.) or 4D-NUCLEOFECTOR (Lonza, Switzerland). For example, the transfection efficiency, i.e., the efficiency of successfully delivering a composition to a cell, for the methods described herein, may be from about 0.1% to about 99.9%, e.g., from about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, or about 50% to about 99.9%, e.g., from about 10% to about 90%, from about 25% to about 85%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99.9%.
The cell viability, i.e., the number or percentage of cells that have survived electroporation, of the cells suspended in the fluid after having a composition introduced using methods of the invention described herein may be from about 0.1% to about 99.9%, e.g., from about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, or about 50% to about 99.9%, e.g., from about 10% to about 90%, from about 25% to about 85%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99.9%.
The number of recovered cells, i.e., the number of live cells collected after electroporation, may be from about 104 cells to about 1012 cells, e.g., about 104 cells to about 105 cells, about 104 cells to about 106 cells, about 104 cells to about 107 cells, about 5×104 cells to about 5×105 cells, about 105 cells to about 106 cells, about 105 cells to about 107 cells, about 2.5×105 cells to about 106 cells, about 5×105 cells to about 5×106 cells, about 106 cells to about 107 cells, about 106 cells to about 108 cells, about 106 cells to about 1012 cells, about 5×106 cells to about 5×107 cells, about 107 cells to about 108 cells, about 107 cells to about 109 cells, about 107 cells to about 1012 cells, about 5×107 cells to about 5×108 cells, about 108 cells to about 109 cells, about 108 cells to about 1010 cells, about 108 cells to about 1012 cells, about 5×108 cells to about 5×109 cells, about 109 cells to about 1010 cells, about 109 cells to about 1011 cells, about 1010 cells to about 1011 cells, about 1010 cells to about 1012 cells, or about 1011 cells to about 1012 cells, e.g., about 104 cells, about 2.5×104 cells, about 5×104 cells, about 105 cells, about 2.5×105 cells, about 5×105 cells, about 106 cells, about 2.5×106 cells, about 5×106 cells, about 107 cells, about 2.5×107 cells, about 5×107 cells, about 108 cells, about 2.5×108 cells, about 5×108 cells, about 109 cells, about 2.5×109 cells, about 5×109 cells, about 1010 cells, about 5×1010 cells, about 1011 cells, or about 1012 cells.
The recovery yield, i.e., the percentage of live engineered cells collected after electroporation, may be from about 0.1% to about 500%, e.g., from about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, about 50% to about 99.9%, from about 75% to about 150%, from about 100% to about 200%, from about 150% to about 250%, from about 200% to about 300%, from about 250% to about 350%, from about 300% to about 400%, from about 350% to about 450%, or from about 400% to about 500%, e.g., about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99.9%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500%.
A skilled artisan will appreciate that optimal conditions may vary depending on cell type or other factors. For each new cell type, the following parameters can be adjusted as necessary: waveform, electric field, pulse duration, buffer exposure time, buffer temperatures, and post-electroporation conditions.
A continuous flow electroporation device and related system were designed and fabricated to allow for a plurality of devices to be used in parallel to enhance or maximize the number of cell electroporation events occurring in a fixed time window, thereby enhancing or maximizing throughput of cell engineering and/or accelerating biological discovery. The electroporation device is configured to be compatible with current automated fluid handling systems, e.g., pipette tip-based dispensers, robotic fluid pumps, etc.
In the embodiment shown in
The electroporation devices of the invention have been designed to meet the requirements of injection and insert molding manufacturing techniques, both of which are scalable in nature, and are shown in
A housing can be configured to energize a plurality of electroporation devices, e.g., 96 electroporation devices in parallel in an industry standard 96-well pipette tip tray with grid electrodes, to energize all of the electroporation devices simultaneously with an identical applied voltage pulse such that the electric field within each electroporation device is identical. A single power supply can be used to deliver the electrical energy. Thus, a mechanism may be needed to distribute the power to each electroporation device. One method to implement this is shown in
Experiments have been conducted to study the physical and biological parameters influencing electroporation of the Jurkat immortalized T cell line using devices of the current invention. Using industry standard flow cytometry methods, both cell viability (measured by 7-AAD dye exclusion) and transfection efficiency (measured by GFP expression) of engineered Jurkat cells were assessed using our devices, both of which are common measures of electroporation success in the field of gene delivery.
Unless specified otherwise, experimental results shown below were generated by electroporating a population of Jurkat cells at a concentration of 1×106 cells in 100 μL of buffer with 5 μg of plasmid (e.g., GFP expression plasmid). Electroporation experiments were performed at 100 Hz with square waveforms and a pulse duration of 9.5 ms. After 24-hour incubation, cells were stained with 7-AAD stain and analyzed via flow cytometry to measure viable cells and live GFP expressing cells. Experiments were performed in triplicate, with error bars representing the standard error of the mean (SEM). Table 1 below presents a summary of the parameters used for transfection using devices of the invention. Most of the parameters listed in Table 1 are independent of one another, with the notable exceptions falling within two distinct groups. The first group of dependent parameters are those which vary according to the physical channel dimensions and process parameters such as the Reynolds number, Velocity, Average Velocity, Shear Rates, and Peak Pressure. A skilled artisan will appreciate that the Reynolds number is a function of the Average Velocity, Kinematic Viscosity, and Channel Diameter. The Velocity (which can vary locally within the channel), Average Velocity, Shear Rates, and Peak Pressure are functions of the Flow Rate and Channel dimensions. The second group of dependent parameters are those related to the biological outcome of the transfection, including the Recovered cells, Cell Viability, Transfection Efficiency, and Yield from input cells. These parameters are dependent upon a host of factors including the cell type being transfected and the combination of the physical parameters indicated in Table 1.
Devices of the invention show peak transfection performance when the flow rate is maximized through the electroporation channel (
Transfection efficiency using devices of the invention is influenced by the electric field strength.
Devices of the invention showed ˜20% increases in both cell viability and transfection efficiency by chilling the sample on ice to minimize any potential deleterious thermal effects that may affect cell viability due to increased temperature during the electroporation (
Electroporation using devices of the invention showed no significant changes in performance when electroporation was performed across a range of pulse durations with matched frequencies (
Electroporation using devices of the invention showed no significant changes in performance when electroporation was performed across a range of volumes and cell densities (
Electroporation using devices of the invention showed no significant changes in performance when electroporation was performed across a range of cross-sectional dimensions of the electroporation zone (
Viability and efficiency depended on the voltage pulse waveform shapes, as shown in
The therapeutic application of primary human T-cells has shown significant advancement in the field of immuno-oncology by targeting the patient's immune system to be effective at fighting cancer. A number of technologies, including chimeric antigen receptors and engineered T-cell receptors, have shown clinical success in recent years. However, applications of genetically modifying the patient's immune system remains somewhat limited to treating blood cancers since the tumor microenvironment of solid tumors inhibit T-cell function at the tumor site. To overcome some of the biological challenges of tumor microenvironment suppression, there is a desire to further modify the T-cells to be more effective by knocking-out genes that express regulatory ligands on the T-cell surface. Identification of these genes is often achieved through CRISPR screens, in which Cas9 and guide RNA libraries are delivered into the T-cells to knock-out a wide range of endogenous genes to achieve functional enhancements against specific tumors. However, delivery of these libraries remains a hurdle for the identification of genes in “hard to transfect” cell types, such as primary T-cells and Natural Killer Cells. Typically, in these instances, the CRISPR libraries are delivered as lentiviral particles that will infect the cells and transduce the Cas9/guide RNA sequences into the cellular genome, which will then knock-out the gene of interest in a sequence-specific manner. These libraries are very laborious to produce, requiring cloning of viral expression plasmids and purification of the viral particles for delivery. Additionally, this methodology leaves the unwanted “baggage” of genetically incorporated Cas9/guide RNA sequences at random genomic insertion sites, which may interrupt other functional genes. The use of non-viral delivery for Cas9 ribonucleoprotein complexes is an attractive method to overcome these hurdles, enabling researchers to screen a large number of knock-outs in the absence of viral incorporation using a transient delivery of Cas9 protein complexed with the guide RNA molecules.
In a related experiment, electroporation using devices of the invention shows significantly increased performance compared to NEON® in the THP-1 monocyte cell line (ATCC number TIB-202) using published NEON® transfection system monocyte electroporation protocols (
Electroporation using devices of the invention showed increased performance compared to NEON® transfection system in primary human monocytes using published NEON® transfect system monocyte electroporation protocols (
Electroporation using devices of the invention showed increased performance compared to NEON® transfection system in independent experiments and for the successful delivery of 40 kDa dextran molecules into Natural Killer Cell Lines of the NK-92 (ATCC) (
SIRPalpha mRNA Delivery to Primary Monocytes
In another study, transient expression of SIRPalpha in primary human monocytes was achieved using devices of the invention (
CXCR4-Targeting Cas9-RNP Delivery to Primary Macrophages
eGFP labeled Cas9-RNP has also been successfully delivered to monocyte-derived human macrophages using devices of the invention. Delivery of the eGFP labeled Cas9-RNP to the nucleus was confirmed via microscopy and flow cytometry. eGFP expression was observed in up to 21.4% of differentiated macrophages 24 hours after transfection, which dropped to 5.1% within five days. While no gene editing was observed at the 24-hour time point, by 48 hours, a 13.9% KO efficiency was observed. Knock-out efficiency, as determined by flow cytometry, then increased to 16.5% by day five.
Naive T Cell Engineering with Delivery of mRNA
Isolated naïve T cells (CD45RA+/CD45RO−) were electroporated with mRNA encoding GFP using the device of the invention. After 24 hours, cells were analyzed for viability and efficiency metrics. The naïve cell counts and viabilities for electroporated cells were equivalent to nontreated cells, and ˜40% delivery efficiency was observed (
When configured as an automated system, the sample of the specimen of interest is aspirated in another location on the liquid handling platform by the devices of the invention. The sample is then transported over to the EDM where the electrode contacts are suspended over the surface of the sample plate. The devices of the invention are then lowered into the device in order to establish contact with the electrode contacts of the EDM. The mechanism depicted in
In the device
The pipette tips 25.5 hover over the surface of a fluidic chip-based electroporation device 25.6. The fluidic chip-based electroporation device includes two components: an electroporation plate that contains an encapsulated arrangement of electrodes and a cover plate that has embedded microfluidic channels that enable the user to modulate the pulse of the electric field that is delivered to the cells. The electroporation plate enables flow through electroporation of multiple samples simultaneously or individually if desired. After the electroporation of the specimen occurs in the electroporation plate the sample flows towards the bottom of the collection plate 25.7. This system uses industry standard liquid handling components, e.g., 1-5,000 μL pipette tips, facilitating integration into industry standard liquid handling manifolds.
The cylindrical electrodes 26.7 and 26.8 in this embodiment are made of conductive porous material that allows the fluid to travel through its pores 26.6 into the cavity of the device. The pores 26.6 in the cylindrical electrode 26.7, 26.8 allow a buffer solution to stabilize the chemical reactions on the surface of the cylindrical electrodes 26.7, 26.8 and minimize the pH transition observed due to the application of an electrical potential during the electroporation process. The buffer introduced by the porous cylindrical electrodes 26.7, 26.8 allows for a change in the fluid flow to create a “lubricating” or sheath flow on the internal surface of the cylindrical electrodes 26.7, 26.8 or to induce other fluid dynamics elements to the electroporation process (such as rotation of the suspension with cells) as it is electroporated. The reduction of the pH transition reduces the negative effects of high variations in the pH of the suspended specimens used during electroporation. Cylindrical electrodes 26.7 and 26.8 complete the external circuit requirement and allow the system to be energized using an external power supply. In an alternative embodiment, the outlet 26.2 of the electroporation device can be used to remove a highly conductive buffer, e.g., a growth media or PBS, and inlet 26.1 can be used to introduce low electrical conductivity buffer to minimize heating of the liquid sample as it flows through the electroporation zone 26.4. This buffer exchange will result in a higher cell viability and higher transfection efficiency that ultimately will generate a greater number of successfully engineered cells. The low conductivity buffer can then be extracted in the outlet after the electroporation zone and supplemented with growth media upon contact with the inlet after the electroporation zone.
A Flowfect device with a particular electrode configuration to help increase the transformation/transfection efficiency of flowing cells has been designed and computationally modeled.
Using the gating strategy described herein, performance data for Jurkat cells, activated primary human T-cells, THP-1 monocytes, primary human monocytes, and differentiated primary human macrophages are shown below in Table 2. In Table 2, Yield represents the ratio of the numbers of cells that are viable and expressing the payload of interest to the input number of cells that entered the process. For example, Yield of 0.5× means that one half of the input cells are viable and express the desired payload at the time of analysis. For perspective, a cell therapy product is administered to a patient if the yield with viral delivery is greater than approximately 0.1× at the time of harvest.
Electroporation of the CHO-K1 (Chinese hamster ovary cells) and HEK-293T (human embryonic kidney cells) cell lines has been conducted. Devices of the invention can be used for electroporation of adherent cells that have been lifted and resuspended in an electroporation buffer. CHO-K1 (
Studies in primary T-cells have been conducted. Fluorescent reporters that have been primarily utilized for analysis of electroporation efficiency include fluorescent small molecules (e.g., FITC-labeled dextran), genes expressed from plasmid DNA (e.g., GFP), and genes expressed from mRNA (e.g., GFP). Delivery and expression of these reporters is determined using flow cytometry, in which the live cells are pre-gated using the gating strategy as described herein to determine fluorescent detection on a single-cell basis. These assays demonstrate intercellular detection of the fluorescent reporter, and in some cases, direct nuclear delivery. Due to the gentle nature of electroporations performed with devices of the invention, higher cells counts are achieved after transfection compared to commercial systems, e.g., the Lonza 4D-NUCLEOFECTOR system or NEON® transfection system (Thermo Fisher, Carlsbad, Calif.).
a. Expanded T-Cell Demonstrations
Transfection using devices of the invention to deliver fluorescently labeled (FITC) dextran molecules (40 kDa) into primary human T-cells (starting at cell density of 106 cells/experimental condition) was performed, and analysis of four metrics against a commercially available bench-top electroporation device (e.g., a Thermo Fisher NEON® transfection system) was conducted: total cell count (post EP), cell viability, transfection efficiency, and total number of live transfected cells. Results are shown in
b. Delivery of mRNA with Platform Comparison
Delivery of mRNA to cells was also demonstrated using devices of the invention. These experiments were performed with a commercially sourced mRNA at two operating cell densities. The experiments were then completed on two commercially available systems (Lonza 4D-NUCLEOFECTOR and Thermo Fisher NEON® Transfection System) and the devices of the invention for comparison as shown in
Each of the payloads described in Examples 13 and 14 are transient upon delivery. To demonstrate delivery of reagents stable genome modification (i.e., CRISPR gene knock-out), experiments were performed with Cas9 ribonucleoprotein complexes (RNPs) for CRISPR knock-out in primary cells. As is shown in
THP-1, an immortalized monocyte cell line, was further used for comparison studies with both monocytes and macrophages. Activation of THP-1 cells with LPS (lipopolysaccharide) endotoxin induces macrophage-like THP1-Mac immortalized cells. As shown in
Primary human monocyte cells, a notoriously challenging cell type to transfect through conventional means, have been successfully transfected using devices of the invention. As is shown
Further, primary monocytes electroporated using devices of the invention retained the ability to differentiate into macrophages, as shown in
Devices of the invention can outperform commercial transfection system for the electroporation of primary monocytes. As shown in
Devices of the invention can be used for the electroporation of large volumes and high cell number suspensions in a truly continuous flow manner. Existing technologies, such as the Lonza 4D-NUCLEOFECTOR™ LV Unit and the Maxcyte Scalable Transfection Systems (STX, VLX, or GT) rely on fluid flow to load the samples into their NUCLEOCUVETTE™ cartridge or processing assembly, respectively. However, during electrical pulse delivery, the cell and payload suspensions are stationary. Commercially available electroporation systems treat static or stationary cell suspensions, which is a critical difference from the devices of the invention. Devices of the invention allow for continuous flow of the cell and payload suspension during the exposure to the electric fields. Specifically, rapidly flowing cells are exposed to sufficient electric field to disrupt the cell membrane and internalize the genetic payload of interest but are immediately dispensed into their growth media for cell recovery. Additionally, any heat that is generated during the electroporation process is dissipated due to convective heat transfer that is facilitated by the flowing samples directly into recovery media. This study expands significantly on the data generated, both in cell type and in scale of the electroporations.
a. Initial Demonstration in Jurkat Cells
A range of cell densities and electroporation volumes were used to demonstrate the scalability of a continuous flow platform relative to a single device platform using devices of the invention. In these experiments, it is demonstrated that the scalable platform of the invention operates across a wide range of Jurkat cell densities, shown in
b. Comparability Studies Between Platforms of the Invention
Follow-up experiments were performed to compare the electroporation performance of the devices of the invention and the continuous flow electroporation platform of the invention using the same delivery conditions for both Jurkat and primary T cells. In these comparative experiments, 5 million cells were processed through the continuous flow platform, showing comparable results to the single channel devices of the invention for Jurkat cells and primary T cells, as shown in
c. Increased Scale of T Cell Electroporation
To test whether the electroporation was dependent on cell density, the electroporation experiments described in
d. Comparability Study with the Lonza Large Volume (LV) System
We performed a comparison of the scalable platform of the invention to the Lonza 4D LV system using primary T cells with both FITC-dextran and EGFP mRNA payloads. The experiments were performed with 50 million T cells. At 24 hours, cell staining revealed that the morphology and phenotype of the Lonza treated cells differed significantly from non-treated cells (shown in the flow cytometry plots of
The continuous flow platform of the invention has shown successful electroporation of payloads into very high density, e.g., 1 billion-cell, suspensions. As shown in
Devices of the invention were tested with both pulse and direct current (DC) power sources, as shown in
To demonstrate the compatibility of devices of the invention with certain T cell expansion protocols, T cells that had been expanded with CD3/CD28 Dynabeads were electroporated using devices of the invention. Electroporation of Dynabead-expanded samples was performed with immediate bead addition (5 min prior to electroporation) to the suspension of 1 million primary human T cells or after an overnight (OVN) treatment, with both time periods demonstrating equivalent efficiency results when the magnetic beads were present to when the beads were not present (
The invention provides an outer structure that fits over and secures to devices of the invention, designed to enhance the ease of use, the efficiency, and the safety during electroporation with the devices of the invention. The outer structure is made from non-conductive polymers on the outer surfaces that shields the users from high voltage exposures and minimize the risk of electrical shock to the user during the electroporation workflow. The outer structure accommodates the current design of the devices of the invention and can be modified to accept future designs variation of the devices of the invention. The outer structure accepts the electrical signal supplied from a power supply or high voltage amplifier and redistributes the signal to the electrodes of the devices of the invention by encapsulating the device within the outer structure. The encapsulation of the electrode of the devices of the invention creates a safer work environment for the user of the devices by minimizing the high voltage surfaces that are exposed. The outer structure also makes it easier to repeatedly do experiments without removal of electrical connections. An embodiment of an outer structure of the invention featuring a clamshell-style hinge and clasp is shown in
62.2 is a second positive/negative electrode through hole for connections to the power supply. 62.3 is the clamshell-style hinge. For example, the hinge may be a living hinge, thus enabling the outer structure to close onto itself and engage the locking mechanism. This enclosure mechanism allows the outer structure to encase the electrodes of the device of the invention, ensuring electrical contact between both devices. 62.4 is a latch or other mechanical fastener used to ensure enclosure of the outer structure during electroporation. This design also enables the outer structure to be reusable by making the latching mechanism temporarily engaged. 62.5 is an alignment pin that ensures the outer structures folds with the correct alignment to minimize any offsets that would distort the electrode connections between the outer structure and the devices of the invention. 62.6 are recesses for the electrodes of the device of the invention. 62.7 and 62.8 are the body of a device of the invention and the first and second electrodes defining the electroporation zone of the device of the invention, respectively.
In use, the outer structure connected to the devices of the invention showed no significant loss in transfection efficiency or viability when performing electroporation using devices of the invention without the outer structure. As shown in
Devices of the invention are constructed from resin formulations produced and sold by Formlabs (Somerville, Mass. USA). In particular, devices of the invention are fabricated from either the “Clear resin” or the Formlabs' marketed “Durable resin”. The major difference between the Durable and Clear resins is the mechanical properties. The Clear resin is more brittle in terms of mechanical behavior and the Durable resin has a greater ductility to the extent that the mechanical performance is more similar to that of polypropylene, the material from which conventional pipette tips are manufactured.
Devices of the invention are 3D printed using stereolithography technology for prototyping purposes. For large scale processing, such as injection molding, device of the invention will be fabricated from other resins, such as the Durable resin which closely simulates polypropylene's mechanical properties. To examine whether the resin material impacts electroporation,
Devices of the invention have enabled rapid, high throughput, and automated engineering of human cells. Applications of this technology are widespread, ranging from fundamental research in cell physiology to the discovery of new targets for cellular therapies. The applications in cell therapies alone can contribute to a growing multi-billion dollar industry. The current state of the art in genetic manipulation at the research scale is manually intensive and difficult to incorporate with automated liquid handling systems. Devices of the invention can be readily incorporated into a diverse array of liquid handling platforms. This integration will allow researchers in academia and industry to quickly explore a wide array of questions related to genetics. The devices of the invention have the potential to facilitate research-scale cell engineering thousands of times faster than the current state of the art, leading to life changing discoveries in healthcare and the fundamental biological sciences.
The experiments on T-cells described herein were originally conducted with single-use devices of the invention. With the automated system incorporating devices of the invention, transfection can be streamlined and configured in a high-throughput manner. Eight independently controlled syringes were programmed to drive the cell suspension into single use devices of the invention. 100 μL samples were aspirated above the electroporation zone of each device and were energized during active dispensing into the recovery growth media. Three automated methods of transfection that used air-displacement (manual electronic pipette) or fluid-displacement (automated system) to drive the samples were compared. The resulting viability remained at high levels (>90%) when using the lymphocyte gate methodology for the 3 systems evaluated (shown in
Co-deliver two mRNA types into T cells was evaluated using devices of the invention. These experiments were performed with two commercially sourced mRNAs encoding either GFP or mCherry. The experiments were completed either in parallel (same day) or in series (two days apart). The devices of the invention were successfully able to deliver both mRNAs as demonstrated by the GFP and mCherry expression observed in
mRNA delivery into primary human mixed cell populations (i.e., PBMCs) was also demonstrated using devices of the invention. These experiments were performed with a commercially sourced mRNA encoding GFP, followed by phenotype staining of surface receptors to identify specific cell populations. Delivery of mRNA to both naïve (CD45RA+) and memory (CD45RO+) T cells was achieved, as shown in
Induced pluripotent stem cells (iPSCs) were transected with eGFP-mRNA, in suspension, using a device of the invention (FLOWFECT™). Cells were assessed 24 hours after transfection for indication of positive transfection using florescent microscopy. Images are depicted as an overlay image of GFP and brightfield to capture adherence, cell morphology, and expression of eGFP-mRNA (representative images shown at 10× magnification;
Isolated NK cells (CD56+) were electroporated with mRNA encoding GFP. After 24 hours, the cells were analyzed for viability and efficiency. The NK counts and viabilities are shown in
Human primary T cells were transfected with a reporter molecule using a device of the invention, ThermoFisher's Neon transfection platform (2100V/1 pulse/20 ms), or Lonza 4D-Nucleofector (Program: EO115) using the manufacturer recommending settings as listed. Shown in
Human primary T cells were transfected with GFP reporter mRNA using multiple flowrate and applied energy combinations. Shown in
Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
14. The device of paragraph 1 or paragraph 2, further comprising an outer structure comprising a housing configured to encase the first electrode, second electrode, and the electroporation zone of the device.
Other embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflicting definition between this and any reference incorporated herein, the definition provided herein applies.
While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
Other embodiments are within the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/058375 | Oct 2019 | US | national |
This invention was made with government support under Phase I SBIR Grant No. 1747096 and Phase II SBIR Grant. No 1853194 from the National Science Foundation (NSF). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/040784 | 7/2/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62869958 | Jul 2019 | US |
