Patent Application
20220056393
Intracellular delivery can be used in many different applications, such as gene transfection, editing, cell labeling, and cell interrogation. However, conventional delivery methods, such as microinjection, electroporation, and sonoporation, may have low delivery efficiencies, especially for large molecules, such as molecules with a size of at least 2000 kilodaltons (kDa), and large particles, such as particles with the size of at least 100 nanometers.
Provided are cell processing apparatuses and systems as well as methods of using these systems and producing intracellular delivery. The intracellular delivery may be caused by rapid compression of cells, resulting in a volume loss. The subsequent recovery of the cells may be performed in a media, comprising reagents. The cells may recover and increase their volume by absorbing the media and the reagents. The cells may be suspended in the media and flow through a cell processing apparatus, which may comprise a plurality of compressive elements such as ridges. The compression may occur when the cells pass through gaps formed by the compressive elements (e.g., ridges), which may be smaller than the cell size. The compressive elements (e.g., ridges) may divert less compressible cells into a channel, thereby sorting the cells based on their compressibility, and/or size, and/or preventing clogging of the apparatus. This approach may allow processing of cells with varying sizes including large cells (such as cells with a size of at least about 20 micrometers in diameter) with high delivery efficiencies.
An aspect of the present disclosure provides a cell processing apparatus comprising: a first wall comprising a first surface, wherein the first wall extends along a flow direction; a second wall comprising a second surface, wherein the second wall extends along the flow direction; a plurality of ridges connected to the first wall, wherein the plurality of ridges extends from the first surface toward the second surface, and wherein a ridge of the plurality of ridges comprises a ridge surface that forms a gap with the second surface; and a diversion channel extending along the flow direction, which diversion channel is at least partially defined by at least a subset of the plurality of ridges.
In some embodiments, the cell processing apparatus further comprises two or more outlets, wherein at least one of the two or more outlets is aligned with the diversion channel, and wherein at least an additional one of the two or more outlets is positioned away from the diversion channel. In some embodiments, the cell processing apparatus further comprises an intermediate outlet fluidically coupled and open to the diversion channel and disposed between a pair of the plurality of ridges. In some embodiments, the cell processing apparatus further comprises side walls each connected to at least one of the first wall and the second wall, wherein each of the plurality of ridges extends between the side walls at an angle between 10° and 80° relative to the flow direction. In some embodiments, all ridges of the plurality of ridges are parallel to one another. In some embodiments, at least two ridges of the plurality of ridges are oriented at different angles relative to the flow direction. In some embodiments, each of the plurality of ridges has a ridge thickness of between 5 micrometers and 100 micrometers. In some embodiments, the ridge surface of the ridge of the plurality of ridges is parallel to the second surface. In some embodiments, the plurality of ridges each has a ridge surface that forms a gap with the second wall, and a height of the gaps varies along the flow direction. In some embodiments, the plurality of ridges comprises a first ridge set and a second ridge set, and the first ridge set and the second ridge set form a chevron pattern. In some embodiments, the first ridge set and/or the second ridge set comprises a plurality of leading edges, positioned at different distances from one of the side walls. In some embodiments, the diversion channel is positioned between the first ridge set and the second ridge set. In some embodiments, the first ridge set and the second ridge set overlap and are offset relative to each other along the flow direction, thereby forming a tortuous path of the diversion channel. In some embodiments, a first ridge of the plurality of ridges comprises a first ridge surface which forms a first gap with the second surface, and a second ridge of the plurality of ridges comprises a second ridge surface which forms a second gap with the second surface, and the first gap has a different height than the second gap. In some embodiments, a height of the gap is adjustable. In some embodiments, at least one of the side walls is flexible such that the first wall is movable relative to the second wall. In some embodiments, a first ridge of the plurality of ridges comprises a first ridge surface which forms a first gap with the second surface, and a second ridge of the plurality of ridges comprises a second ridge surface which forms a second gap with the second surface, and the first gap has a different width than the second gap. In some embodiments, the cell processing apparatus further comprises a recovery space positioned between two adjacent ridges of the plurality of ridges, wherein a distance of the recovery space, along the flow direction, is between 100 micrometers and 1000 micrometers. In some embodiments, a width of the diversion channel, measured perpendicular to the flow direction, is variable along the flow direction. In some embodiments, the cell processing apparatus further comprises an additional diversion channel, wherein the diversion channel is positioned between each of the plurality of ridges and one of the side walls, and wherein the additional diversion channel is positioned between each of the plurality of ridges and an additional one of the side walls.
Another aspect of the present disclosure provides a system for cell processing, the system comprising: a cell processing apparatus comprising: a first wall comprising a first surface; a second wall comprising a second surface; a plurality of ridges connected to the first wall, wherein the plurality of ridges extends from the first surface toward the second surface, and wherein a ridge of the plurality of ridges comprises a ridge surface that forms a gap with the second surface; and a diversion channel extending along a flow direction of the cell processing apparatus, which diversion channel is defined at least partially by at least a subset of the plurality of ridges; a pressure source fluidically coupled to the cell processing apparatus; one or more sensors operably coupled to the cell processing apparatus; and a system controller, operably coupled to the pressure source and to the one or more sensors, and configured to control an operation of the pressure source based on one or more inputs from the one or more sensors.
In some embodiments, the system further comprises a temperature controlling module, thermally coupled to at least one of the first wall and the second wall, and operably coupled to the system controller, wherein the temperature controlling module is configured to control a temperature of a medium flown through the cell processing apparatus. In some embodiments, the one or more sensors comprise a cell counter positioned in the diversion channel. In some embodiments, the system further compries a gap adjuster, mechanically coupled to at least one of the first wall and the second wall, and operably coupled to the system controller, wherein the gap adjuster is configured to adjust a height of the gap between the ridge surface and the second surface. In some embodiments, the one or more inputs comprise at least one of: a pressure inside the cell processing apparatus, a cell count at an inlet of the cell processing apparatus, a cell count at an outlet of the cell processing apparatus, a temperature inside the cell processing apparatus, a flow rate inside the cell processing apparatus, an optical image from the cell processing apparatus, and a position of the first wall relative to the second wall. In some embodiments, the system further comprises an additional cell processing apparatus comprising an additional plurality of ridges, wherein a ridge of the additional plurality of ridges comprises an additional ridge surface which forms an additional gap with an additional second surface of an additional second wall of the additional cell processing apparatus, and wherein the additional gap has a different height than the gap; and a cell sorter positioned upstream and fluidically coupled to the cell processing apparatus and the additional cell processing apparatus, such that the cell processing apparatus and the second cell processing apparatus are configured to operate in parallel. In some embodiments, the system further comprises an additional cell processing apparatus, wherein the cell processing apparatus and the additional cell processing apparatus are connected in sequence. In some embodiments, the system further comprises an inlet fluidically coupled to and positioned between the cell processing apparatus and the additional cell processing apparatus. In some embodiments, the system further comprises two or more outlets, wherein: at least one of the two or more outlets is aligned with the diversion channel, and at least an additional one of the two or more outlets is positioned away from the diversion channel. In some embodiments, the system further comprises side walls each connected to at least one of the first wall and the second wall, wherein each of the plurality of ridges extends between the side walls of the cell processing apparatus at an angle between 10° and 80° relative to the flow direction. In some embodiments, the pressure source is a pump.
Another aspect of the present disclosure provides a cell processing method comprising: (a) directing cells into a cell processing apparatus comprising: a plurality of ridges, wherein a ridge of the plurality of ridges comprises a ridge surface that forms a gap with a surface of the cell processing apparatus, and wherein the gap is configured to reduce a cell volume of cells flowing therethrough, a recovery space between two adjacent ridges of the plurality of ridges, which recovery space is configured to recover at least a portion of the cell volume reduced by the gap, and a diversion channel extending along a flow direction of the cell processing apparatus, which diversion channel is defined at least partially by at least a subset of the plurality of ridges; (b) flowing a first subset of the cells through the gap and the recovery space to generate one or more processed cells, wherein the first subset of the cells has a reduced cell volume upon flowing through the gap, and wherein the reduced cell volume is recovered at least partially by absorbing a surrounding medium upon the first subset of the cells flowing through the recovery space, thereby generating the one or more processed cells; and (c) directing a second subset of the cells not passing through the gap or the recovery space into the diversion channel.
In some embodiments, an average duration of the first subset of the cells passing through the gap is less than about 1 second. In some embodiments, cells of the first subset of cells have an average volume reduction of at least about 10%, as compared to an original cell volume. In some embodiments, the cell processing method further comprises, sorting the cells prior to (a). In some embodiments, the cell processing method further comprises, sorting the cells after (b). In some embodiments, the cell processing method further comprises flowing the first subset of cells through one or more gaps formed by one or more ridges of the plurality of ridges, thereby resulting in one or more volume reductions of the first subset of the cells. In some embodiments, a linear flow rate of the cells flowing through the cell processing apparatus is adjustable. In some embodiments, the linear flow rate is adjusted between 10% and 50% of an average linear flow rate with a frequency between 0.1 Hertz (Hz) and 100 Hz. In some embodiments, the linear flow rate is adjusted in response to an input received from one or more sensors. In some embodiments, the cell processing method further comprises reversing a direction of fluid flow of the cells through the cell processing apparatus. In some embodiments, a flow rate of the cells flowing through the cell processing apparatus is between about 5 millimeters per second and 200 millimeters per second. In some embodiments, (a) comprises vibrating the cell processing apparatus. In some embodiments, the cell processing apparatus is vibrated with an amplitude of greater than 1 micrometer. In some embodiments, the cell processing apparatus is vibrated with a frequency of greater than 1 Hz.
Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.
Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.
Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.
Provided herein are methods and systems for intracellular delivery. The methods may comprise providing a fluidic device (e.g., a microfluidic device). The methods and systems may facilitate delivery of one or more reagents or substances (such as therapeutic reagents, gene-editing reagents) into cells. The methods and systems may comprise the use of a fluidic device. The fluidic device may comprise one or more compression elements.
The fluidic device may comprise a plurality of channels (e.g., microchannels). A plurality of the channels may be one or more microchannels. The number of the channels in the fluidic device may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more channels. Increasing the number of channels in the fluidic device may increase an exit flow rate or the throughput of a process. Two or more fluidic channels may be connected in parallel, in series, or a combination of series and parallel. The channels may be connected by various configurations.
The fluidic device may comprise a principal axis. The principal axis may be parallel to a flow direction of the device. The principal axis may be parallel to the plurality of microchannels. The method may further comprise subjecting one or more cells to flow through the microchannel of the fluidic device. As the cells flow through the microchannel, the cells may be in contact with the compressive element comprised in the microchannel. The microchannel may have a cross-sectional dimension that is greater than or equal to about 1 micrometers (μm), 5 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1,000 μm, or more. In some cases, the cross-sectional dimension of the channel may be less than or equal to about 2,000 μm, 1,500 μm, 1,000 μm, 850 μm, 700 μm, 550 μm, 400 μm, 300 μm, 200 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, 10 μm, or less. In some cases, the cross-sectional dimension of the channel may fall within any of the two values described above, e.g., between about 20 μm and about 1,000 μm, or between about 50 μm and about 100 μm.
The microchannel may comprise a plurality of compressive elements. The plurality of compressive elements may be one or more compressive elements. Each microchannel in the microfluidic device may include one or more compressive elements. The plurality of compressive elements may comprise ridges. The compressive elements may comprise compressive surfaces. Compressive surfaces may have different shapes and/or curvatures, such as rectangular, triangular, cylindrical, spherical, or other shapes and/or curvatures. The compressive elements may have different sizes, such as different surface areas. As a cell flows through the fluidic device, the cell may be in contact with the compressive element. The compressive element may result in a cell volume reduction. After the compression, the cell may recover part or all of its reduced volume by absorbing media surrounding the cell.
The compression element may comprise a plurality of compressive surfaces, e.g., greater than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 compressive surfaces, or more. The compressive surfaces may be ridges. The compressive surfaces may or may not extend parallel with respect to one another. In some cases, at least a subset of the compressive surfaces extends parallel with respect to one another. The compressive surfaces may have regular or irregular cross-sectional shapes. In some cases, the compressive surfaces have rectangular cross-sections.
Dimensions of the compressive surfaces may vary, depending upon various factors, such as cell flow rate, cell type, cell size, cell stiffness, cell adhesiveness, substance type, channel material and/or channel size. For example, in some cases, the compressive surfaces have an average width that is greater than or equal to about 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, or more. In some cases, the compressive surfaces have an average width that is less than or equal to about 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 150 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, or less. In some cases, the compressive surfaces have an average width that falls between any of the two values described above, for example, between about 20 μm and 250 μm.
So that the cells may pass through the channel, the compressive element may have a dimension (e.g., a height) that is smaller than a cross-sectional dimension of the channel. Consequently, there may be a gap between a surface the compressive element (e.g., a ridge surface) and an interior surface of the channel or the device. The gap may have a size that is adjustable. The gap size may be adjusted based upon a variety of factors, such as cell size, cell type, cell stiffness, cell adhesiveness, flow rate, channel material, channel size, temperature, substance type, and/or substance size. In some cases, the gap size may be greater than or equal to about 0.1 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 22 μm, 24 μm, 26 μm, 28 μm, 30 μm, or more. In some cases, the gap size may be less than or equal to about 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 18 μm, 16 μm, 14 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm, 2 μm, 1 μm, or less. In some cases, the gap size may fall within a range of any of the two values described above, for example, between about 1 μm and about 20 μm, or between about 3 μm and 15 μm.
The gap size may be smaller than a cell size. For example, the gap size may be less than or equal to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% of an average diameter of a cell, or less. In some cases, the gap size may be less than or equal to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% of a diameter of a given cell comprised in the cells that pass through the channel.
In cases where multiple compressive elements (e.g., compressive surfaces) are comprised in the channel, each compressive element may have the same or a different dimension. As a result, gap sizes between each compressive element and an interior surface of the channel may or may not differ. In some cases, at least a subset (e.g., at least about 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, or more) of the compressive elements have different dimensions.
The compressive elements may be spaced apart from one another. Such configuration may facilitate periodic compression and expansion of the cells. For example, as a cell passes through the channel, the cell may be compressed while in contact with a compressive element. Following the compression and prior to being subjected to contact with a subsequent compressive element, the cell may flow into an area (e.g., a recovery space) between two adjacent compressive elements where the cell may expand and recover some or all of the volume lost during the compression. Dimensions or shapes of spaces between each pair of adjacent compressive elements may or may not be the same. In some cases, the compressive elements are equally distant. In some cases, a space between each pair of adjacent compressive elements progressively increases or decreases along a flow direction of the cells. The flow direction may be the main flow direction of a majority of the cells. The flow direction may be in alignment with a principal axis of the channel. The flow direction may be a direction from an inlet of the channel to an outlet of the channel.
The compressive element may comprise an angled compressive element, such as an angled surface relative to the principal axis of the microfluidic device. All compressive elements in the microfluidic device may be parallel to one another (i.e., oriented at the same angle relative to the principal axis of the device). Alternatively, different compressive elements may have different angles relative to the principal axis of the device. In some examples, the angles may be from 10 to 50 degrees, or from 20 to 80 degrees, or from 30 to 60 degrees. For example, the angles may by greater than or equal to about 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, 80 degrees, 85 degrees, or more. Angled compressive elements may facilitate the removal of unwanted substances from the microchannel. The unwanted substances may comprise, for example, nonviable cells, aggregates, clogging agents, excess contrasting agents, excess amounts of other reagents, or more. The removal of unwanted substances may contribute to increasing the throughput and/or efficiency of cell processing.
A plurality of cells may be directed to pass through the fluidic device. The plurality of cells may comprise greater than or equal to about 20 million, 50 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million cells, or more. In some cases, the plurality of cells may be less than or equal to about 2,000 million, 1,500 million, 1,000 million, 800 million, 600 million, 400 million, 200 million, 100 million cells, or less.
In some examples, the methods may comprise rapid compression of cells which may result in a volume loss by the cells. The compression may occur when the cells pass through the fluidic device comprising the compression elements (e.g., ridges). The compression may be rapid. The compression may occur within a short time period. For example, the compression may occur in less than or equal to about 2 seconds (s), 1.8 s, 1.6 s, 1.4 s, 1.2 s, 1 s, 900 milliseconds (ms), 800 ms, 700 ms, 600 ms, 500 ms, 400 ms, 350 ms, 300 ms, 280 ms, 260 ms, 240 ms, 220 ms, 200 ms, 180 ms, 160 ms, 140 ms, 120 ms, 100 ms, 90 ms, 80 ms, 70 ms, 60 ms, 50 ms, 40 ms, 30 ms, 20 ms, 10 ms, 5 ms, 1 ms, or less. In some cases, the compression may occur within a time period that falls between any of the two values described above, for example, between about 10 ms and about 300 ms. As an example, the compression may occur in less than 1 second, such as within 10 microseconds to 300 milliseconds. The compression time may depend on the flowrate, cell size, compressive element (e.g., ridge) geometry, and various other factors, such as the factors further described above or elsewhere herein.
During the compression, the cells may change their volume (e.g., experience volume loss of at least 10% or even at least 30% of the pre-compression volume) rather than simply changing their shapes (e.g., adapting to the compression gap without significant volume change). A combination of the compression speed and the volume loss may distinguish the methods and systems of the present disclosure from conventional microfluidic techniques, in which cells may simply be reshaped. In some examples, the cells may change shapes and without substantially changing the volume (e.g., less than or equal to about 25%, 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2% volume change relative to the original volume, or less). In other examples, the cells may change their volume without substantially changing their shapes. In some examples, both cell shape (e.g., morphology) and cell volume may change.
The volume reduction (or loss) may be temporary. The compression may cause a cell to lose at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of its volume, or more. The compressed state may be a non-natural state for the cells and the cells may attempt to recover to the original volume. As a result, the compression may be followed by cell expansion and recovery. During the recovery, the cells may increase their volume by absorbing media surrounding the cells. The media which comprise various reagents as described above or elsewhere herein.
The cells may attempt to recover to their original volume as soon as no further compression forces are applied on them. In some cases, the methods of the present disclosure may comprise releasing the cells into a recovery space after compression by the compressive elements. In some cases, the cells may be immediately released into the recovery space. The recovery space may be inside the same cell processing apparatus. The recovery process may allow the cells to recover at least partially and to increase the volume by absorbing the surrounding media. The media may comprise one or more reagents, which may be introduced into the cells as a part of this recovery process. Examples of such reagents may comprise plasmids and magnetic nanoparticles (e.g., introduced into stem cells or other types of cells for various applications) and mRNA (e.g., introduced into primary peripheral blood mononuclear cells or other types of cells for various applications). Other reagents and cell types may be used.
As provided herein, one or more reagents may be directed into an interior of cells including large cells. The reagents may comprise large molecules. In some examples, the plurality of substances may have an average molecular weight greater than or equal to about 0.5 megadaltons (MDa), 0.6 MDa, 0.7 MDa, 0.8 MDa, 0.9 MDa, 1.0 MDa, 1.1 MDa, 1.2 MDa, 1.3 MDa, 1.4 MDa, 1.5 MDa, 1.6 MDa, 1.7 MDa, 1.8 MDa, 1.9 MDa, 2.0 MDa, 2.1 MDa, 2.2 MDa, 2.3 MDa, 2.4 MDa, 2.5 MDa, 2.6 MDa, 2.7 MDa, 2.8 MDa, 2.9 MDa, 3.0 MDa, 3.5 MDa, 4.0 MDa, 4.5 MDa, 5.0 MDa, or more. In some cases, each of the substances may has a molecular weight that is greater than or equal to about 0.5 megadaltons (MDa), 0.6 MDa, 0.7 MDa, 0.8 MDa, 0.9 MDa, 1.0 MDa, 1.1 MDa, 1.2 MDa, 1.3 MDa, 1.4 MDa, 1.5 MDa, 1.6 MDa, 1.7 MDa, 1.8 MDa, 1.9 MDa, 2.0 MDa, 2.1 MDa, 2.2 MDa, 2.3 MDa, 2.4 MDa, 2.5 MDa, 2.6 MDa, 2.7 MDa, 2.8 MDa, 2.9 MDa, 3.0 MDa, 3.5 MDa, 4.0 MDa, 4.5 MDa, 5.0 MDa, or more.
The reagents may or may not comprise a charged substance. For example, the reagents may comprise a drug, a nucleic acid molecule, an antigen, a polypeptide, an antibody, an antigen, a hapten, an enzyme, or combinations thereof. The nucleic acid molecule may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), or combinations thereof.
In some examples, the reagents comprise reagents greater than or equal to about 5 kilobases (kB), 6 kB, 7 kB, 8 kB, 9 kB, 10 kB, 11 kB, 12 kB, 13 kB, 14 kB, 15 kB, 16 kB, 17 kB, 18 kB, 19 kB, 20 kB, 21 kB, 22 kB, 23 kb, 24 kB, 25 kB, 26 kB, 27 kB, 28 kB, 29 kB, 30 kB, or larger. One example reagent may be DNA plasmids with size larger than 10 kB.
In some cases, large reagents, such as reagents at least about 10 kB in size may not be introduced by conventional microfluidic methods, such as squeezing cells in narrow pores to improve the membrane poration characteristics, followed by the slow diffusion of reagents through the temporary membrane pores. Since the diffusion is slower for larger reagents, such reagents may not be effectively delivered using these diffusion methods. Furthermore, the degree of cell membrane poration, proposed by conventional methods, may be limited to ensure cell viability and avoid cell damage or death. In some examples, conventional microfluidic devices for intracellular delivery may be prone to clogging because narrow channels may be used for achieving high membrane shear and opening membrane pores. Increasing flowrates, e.g., to reduce clogging, may result in cell damage.
Referring to apparatuses and methods described herein, in some examples, a sequence of compression (volume reduction) and recovery (reagent absorption) may be performed multiple times, e.g., once for each compressive element (e.g., compression ridge) provided inside a cell processing apparatus. Furthermore, in some examples, this processing sequence may be repeated in a different manner, e.g., using a different gap height (level of compression), a different compressive element geometry, a different gap length (compression duration), a different flowrate (compression and recovery duration), different reagents and/or reagents concentrations. For example, the ridges with different gaps and/or different widths and profiles may affect the degree, speed, and/or duration of cell compressions. Furthermore, in some examples, a cell processing apparatus may comprise a diversion channel for removal of less compressible cells. For example, the compressive elements (e.g., ridges) may be angled (not perpendicular) relative to the flow direction, which may be referred to as diagonal ridge orientation. A diversion channel may be positioned at the end of these ridges, e.g., along one of the side walls and/or away from the side walls. Cells, which are not sufficiently compressible and cannot pass through the gap formed by these compressive elements (e.g., ridges), may be pushed (by the flow) along these compressive elements and into the diversion channel. In some examples, these cells may then be collected separately from cells that have undergone one or more compression-recovery sequences.
In general, the methods and systems described herein may be used to deliver a variety of reagents (e.g., macromolecules) to a variety of different cell types. In some cases, the intracellular delivery may be achieved with high throughput and minimal clogging, while posing lower risk of cell death and aggregation than conventional methods. In some cases, the substances may be delivered into the plurality of cells at an efficiency of greater than or equal to about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. The efficiency of the method may be higher than diffusion-based methods and/or endocytosis. The efficiency of the method may be maintained while increasing the throughput and/or rate of cell processing.
In some examples, first wall 110 and/or second wall 112 may be formed from one or more transparent materials. For example, transparent materials of these walls may allow for integration of optical sensors into the cell processing apparatus 100 and/or other types of process control. On the other hand, nontransparent materials for the walls may be used to deliver light-sensitive reagents. Some examples of wall materials may comprise, but not be limited to, polydimethylsiloxane (PDMS), injection molded plastics, silicon, glass, and other polymers.
Referring to
Each of plurality of ridge 130 may comprises ridge surface 131, forming gap 132 with second interior surface 113. The height (H) of gap 132 may be smaller than the size/diameter (D) of cells 230, which may cause cells 230 to compress as cells 230 pass through gap 132. The compression may also depend on the flowrate and the length of ridge surface 131 (in the X direction), which may be also referred to as a ridge thickness. In some examples, the length of the ridge surface 131 and/or the ridge thickness may be between about 5 micrometers (μm) and 100 micrometers or, between about 20 micrometers and 50 micrometers. The length of the ridge surface 131 may be at least about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm 8 μm, 9 μm, 10 μm, 12 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 millimeter (mm), or more. In some examples, the length of the ridge surface 131 may be at most about 1 mm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 15 μm, 10 μm, 5 μm, or less.
In some examples, all ridges (or a subset) of the plurality of ridges 130 may have the same length of ridge surface 131 and/or ridge thickness. Alternatively, the length of ridge surface 131 and/or ridge thickness may vary among the ridges. For example, upstream ridges (initial ridges along the flow direction) may have a shorter length of ridge surface 131 than downstream ridges. As such, the compression duration provided by these downstream ridges may be longer than that provided by the upstream ridges. The compression duration may also be impacted by the linear flow rates, which may be controllable by the cross-sectional areas of the cell processing apparatus 100, as further described below.
In some cases, when the length of ridge surface 131 is smaller than the cell size (D), the cell compressions can be compromised due to the cell ability to deform around the ridges, e.g., at least partially remain in uncompressed state when portions of the cell extend outside of gap 132. On the other hand, when the length of ridge surface 131 is much larger than the cell size, such as 10 times or more than the cell diameter, the cells may be prone to accumulation in gaps 132, which can lead to clogging.
Referring to
In some examples, ridge surface 131 may be parallel to the second interior surface 113. In other words, gap 132 may be defined by two parallel surfaces, one being ridge surface 131 and another one being a portion of second interior surface 113, and the gap thickness may be constant. Such parallel compressive surfaces may allow for a uniform compression for the entire cell. In some examples, the compression surfaces can be converging and/or diverging. Converging surfaces may allow for increasing the cell compression as the cells pass through the compressive space. Diverging surfaces can be used to allow cell expansion that accelerates cell motion and prevents clogging.
In some examples, the surface roughness of ridge surface 131 may be configured to increase cell membrane poration. For some materials, the surface roughness can be controlled using vapor etching. In some examples, the surface roughness with a mean size of between 10 nanometers (nm) and 1000 nm may be used. In some cases, the surface roughness may have a mean size of at least about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 20 nm, 50 nm, 100 nm, 300 nm, 500 nm, 800 nm, 1000 nm, 1200 nm, nm, 1300 nm, 1500 nm, or more. In some cases, the surface roughness may have a mean size of less than or equal to about 2000 nm, 1500 nm, 1200 nm, 1000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 20 nm, 10 nm, 5 nm, 1 nm, or less.
In some examples, plurality of ridges 130 may be flexible (e.g., compliant). Flexible ridges may help to reduce cell damage. The ridge flexibility/compliance may be configured by selecting ridge material. In some examples, materials with modulus from 1 to 100 kPa may be used. Furthermore, ridge compliance may be configured using surface coatings with desired elasticity modulus.
Further referring to
Referring to
Referring to
In some examples, inlet 180 may be a self-focusing inlet (e.g. with no sheath focus). The self-focusing inlet may use hydrodynamic focusing, such as Dean's flow effect. For example, inlet 180 may incorporate a focusing section, such as a serpentine channel, focusing ridges, focusing posts, focusing flow splitters, curved geometry using Dean's flow effect, inertial migration effect, and other methods leading to cross-stream cell migration. The focusing section may concentrate cells 230 at desired transverse location within the cell processing apparatus 100. Among other factors, the focusing location depends on the geometry of ridges 130 and ridge surface 131, which may be also referred to as compressive surfaces. For chevron ridges (e.g., shown in
In some examples, a single inlet may be used to reduce an amount of reagents 220 that otherwise can be diluted by focusing a sheath fluid. At outlet 190, processed and unprocessed cells can be mixed for collection. An additional sorting device and operation can be used to separate unprocessed cells from mixture 200 after mixture 200 exists cell processing apparatus 100.
In some examples, cell processing apparatus 100 may comprise intermediate inlet 182, e.g., to introduce different reagents and reagent combinations for multistage cell processing. For example, intermediate inlet 182 may be used to introduce an additional mixture into recovery spaces 140 between adjacent ones of plurality of ridges 130. The composition of this additional mixture may be different from mixture 200, introduced upstream through inlet 180, which may be also referred to as a primary inlet.
In some examples, multiple outlets (e.g., outlet 190 and additional outlet 192) may be used for collecting different types of cells 230. As noted above, cell processing apparatus 100 may have cell sorting capabilities such that different types of cells 230 may flow into different portions of cell processing apparatus 100. Referring to
In some examples, cell processing apparatus 100 may comprise intermediate outlet 193 as, for example, shown in
Referring to
In some examples, all of plurality of ridges 130 may have the same angle relative to principal axis 101 (e.g., α=β, referring to
Referring to
The shape of each of plurality of ridge 130 may affect or determine the compression profile of cells 230 as they pass through gap 132, created by the ridge, and are compressed by the ridge. Some examples of different ridge cross-sectional shapes of plurality of ridge 130 are illustrated in
Referring to
Returning to
In some examples, the diversion channel 170 may have the constant width (in the Y direction) along the flow path as, e.g., is shown in
Ridge designs may be selected to reduce an amount of unprocessed cells and enhances the delivery efficiency and uniformity. In some examples, when stiffer cells or cells with larger size encounter a diagonal ridge, they may experience a force that can displace the cells along the ridge. This can result in the separation of cells by stiffness and size with the larger and stiffer cells being displaced in the diversion channel without sufficient processing by compressive surfaces. In this case, the use of overlapping chevron pattern can reintroduce the rejected cells to the trajectories that follow through compressive spaces for additional processing. This in turn can improve the processing of heterogeneous cell populations. It should be note that ridges 130 may have the side of gap 132 that may vary along the width of the ridge. For example, the gap 132 may gradually increase along the width of ridge 130, e.g., toward the diversion channel 170. In this example, the cells 230 that cannot pass through a smaller gap size (e.g., due to their size) are diverted along the ridge and may be able to eventually pass under the ridge as the gap size increases (e.g., closer to diversion channel 170).
In addition to compression of the cells 230, thereby inducing intracellular delivery of the reagent 220 into the cells 230, the ridges 130 may also produce hydrodynamic mixing within the liquid media 210. This hydrodynamic mixing may be due to the various angles of the ridges 130 relative to the flow directions. The hydrodynamic mixing may redistribute the reagent 220 within the liquid media 210 as the reagent 220 is being consumed by the cells 230 during intracellular delivery.
Referring to
Furthermore, the gap height (H) may be also defined relative to the cell size (D), which may be defined as an average largest cross-sectional dimension of the cells 230. The ratio of the gap height (H) relative to the cell size (D), i.e., H/D, may define the compression level of the cells 230 as they pass through the gap. In some examples, this H/D ratio may be between 15% and 75%, or between 30% and 60%. In some cases, the ratio of the gap height (H) relative to the cell size (D), i.e., H/D, may be at least about 5%, 8%, 10%, 15%, 20%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some cases, the ratio of the gap height (H) relative to the cell size (D), i.e., H/D, may be at most about 99%, 90%, 80%, 75%, 70%, 60%, 65%, 60%, 50%, 30%, 20%, 15%, 10%, or less. Furthermore, in some examples, the cell size (D) may be between 4 micrometers and 20 micrometers or, or in some examples, between 6 micrometers and 15 micrometers. In some cases, the cell size (D) may be at least about 1 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 12 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or more. In some cases, the cell size (D) may be at most about 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, 10 μm, or less.
While
In some examples, this gap height adjustment may be performed dynamically while flowing liquid media 210 through the cell processing apparatus 100 and even while performing intracellular delivery. For example, the cell processing apparatus 100 may be brought to the second processing stage shown in
In some examples, the interior height (IH) of recovery spaces 140 may change along the length of the cell processing apparatus 100/X-axis/flow direction 240 as, e.g., is shown in
In some examples, the ridges 130 may be attached to both opposite walls (e.g., first wall 110 and second wall 112) as illustrated in
The gap height may determine the level of cell deformation that may be needed to pass through the gap. Furthermore, the gap height may determine the flowrate through the gap. For example, when two consecutive ridges in the same cell processing apparatus 100 have different gap heights, the flowrate through the gap having a smaller gap height may be greater.
Intracellular delivery may be controlled and/or adjusted according to the cell compression rate, which may be a rate of volume loss by example cells as they pass through the gap formed by a ridge. The cell compression rate can be determined by flowrate, ridge geometry, a ratio of the gap height to the cell size, the ridge width, ridge angle, and compressive surface coating. Furthermore, the volume loss (Vloss) may increase with the increase in the cell compression rate. Various processing and device characteristics may be specifically selected to achieve desired cell compression rates.
In some example, gaps 132 formed by two ridges 130 in the same cell processing apparatus 100 have different widths as, for example, schematically shown in
Another flow control may be achieved by varying the cross-sectional area of diversion channel 170. The cross-sectional area may depend on the width and height of diversion channel 170. Varying the width of diversion channel 170 is described above with reference to
One or more pumps 330 may be configured to deliver a mixture 200 comprising cells 230, liquid media 210, and/or reagents 220 to the cell processing apparatus 100. The pumps may be fluidically coupled to the inlet 180 of the cell processing apparatus 100. The one or more pumps 330 may control the flowrate, pressure, and other characteristics of the fluid flow, e.g., according to an input provided by the system controller 310.
The system controller 310 may be configured to receive various inputs and/or to control various operations of different components of the system 300. For example, the system controller 310 may receive various sensor data. The system controller 310 may instruct the one or more pumps 330 to increase or decrease one or more flowrates. In some examples, the system controller 310 may instruct the cell processing apparatus 100 to adjust the gaps 132 formed by the compressive elements (e.g., ridges 130).
Optionally, a temperature controlling module 340 may be provided. The temperature controlling module may be thermally coupled to or integrated into the cell processing apparatus 100. For example, the temperature controlling module 340 may be thermally coupled to at least one of the first wall 110 or the second wall 112 of the cell processing apparatus 100 and, in some cases, may be communicatively or operably coupled to the system controller 310. The temperature controlling module 340 may be used to maintain a target temperature. Maintaining a set temperature may improve cell viability and increase the delivery efficiency. Desired temperatures may vary for different processes and/or applications.
The method and systems may further comprise providing a gap adjuster 350, which may be configured to controllably apply a force between the first wall 110 and the second wall 112, which may change the distance between these walls, thereby also changing the gaps formed by the ridges 130 (or other compressive elements). Furthermore, flow regulators, flow sensors, and/or valves may be used for controlling flow conditions. In some examples, actuators may be used to redirect processed and/or unprocessed cells, change flow parameters in the channel(s), and/or induce fluid mixing in the channel to improve delivery.
In some examples, the system 300 may comprise one or more vibrator(s) 390, which may be mechanically coupled to or integrated into the cell processing apparatus 100. Some examples of vibrators 390 may comprise an electromagnetic vibrator, a piezoelectric vibrator, a magnetic vibrator, and a mechanical vibrator. The one or more vibrators 390 may be configured to produce intermittent or continuous vibrations, e.g., at an amplitude of greater than 1 micrometer (μm) and/or at a frequency of greater than 1 Hertz (Hz). The amplitude of the vibrations may be at least about 0.5 micrometers (μm), 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, or more. In some cases, the amplitude of the vibrations may be at most about 200 μm, 150 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, 10 μm, 5 μm, 1 μm, or less. In some cases, the frequency of the vibrations may be greater than about 0.5 Hz, 1 Hz, 2 Hz, 3 Hz, 5 Hz, 10 Hz, 20 Hz, or more, In some cases, the frequency of the vibrations may be at most about 100 Hz, 80 Hz, 50 Hz, 20 Hz, 10 Hz, 5 Hz, 2 Hz, 1 Hz, 0.5 Hz, or less. In some examples, the one or more vibrators 390 may be communicatively or operably coupled to and/or controlled by the system controller 310, e.g., based on an input from one or more sensors 320.
In some examples, the system 300 may comprise multiple cell processing apparatuses connected in series, in parallel, or a combination thereof, and/or other configurations to each other as, for example, is schematically shown in
Multiplexed devices may be used to deliver multiple types of molecules independently to different cells, either in serially connected single channels, or in parallel connected multiple channels with unique molecule inputs to each channel, or in their combination. Multiplexed devices can be used to produce mixtures of cells with different delivered reagents and different combinations of delivered reagents. This can be used to perform multistep delivery into cells. Inline (e.g., series) and/or in parallel, other configurations, and/or combinations thereof of devices can be used to achieve multiplexed delivery.
For example, drug discovery may be accelerated using multiplexed devices where cells may be initially separated into subpopulations that may be used to deliver different reagents to rapidly screen the effects of these reagents on cells to identify reagents which may the most significant efficient on cell functions, or to answer other research questions, or accomplish other goals. Multiple cell sample inlets may be used to process different cells to examine the effect of reagent or reagent-combination on cell function. Furthermore, cells may receive different combinations of reagents. In this case, the device may include several stages of processing where cells may be sequentially delivered different combinations of reagents or different amounts of reagents to evaluate their effects on cell function. To achieve such device functionality, multilayered devices may be used, where different layers of the device(s) may be utilized to perform delivery of specific set of reagents. The processing channels between layers may be connected using in-plane and/or out-of-plane connectors.
In some examples, a two-phase droplet generator may be positioned before the inlet 180, as described below with reference to
In some examples, the presorting operation (block 410) may involve separation using different microfluidic methods such as using gaps formed by diagonal ridges (or other geometries and architectures of ridges and/or microfluidic features) leading to distinct trajectories of cells with different mechanical properties and their separation within the microchannel. In some examples, the separated cells may be then collected at different channel outlets. In some cases, the multiple outlets may be integrated into the system, for example into the microfluidic channels in which intracellular delivery takes place. Presorting may be achieved using microfluidic sorters that may rely on inertial effects leading to equilibration of different cells at different locations within the microfluidic channel, and/or in the flow. Presorting and cell separation may be accomplished using various presorting mechanisms, for example, acoustic streaming effects may discriminate cells with different properties and may lead to different cell positions within the flow. Presorting and separation may be achieved using magnetic forces using magnetic particles attached to the outer membrane of the cells or located inside the cell interior, where the cells may be separated based on the different magnitude of the magnetic force that may be proportional to the amount of the magnetic particles. Cell presorting and separation may be achieved based on cell electrical properties using electrical forces such as using alternating current dielectrophoresis.
In some examples, cell sorting may be used after the intracellular delivery operation and may be referred to post-sorting (block 450). For example, sorting by size and mechanical properties after convective intracellular delivery can be used to remove abnormal cells and nonviable cells from the processed cells. In some examples, post-sorting may be achieved using diagonal ridges that may redirect cells with different properties to follow different trajectories within the cell processing apparatus 100. Other microfluidic and non-microfluidic sorting methods, such as magnetic, acoustic, and electric sorting methods, may be used in other examples. The post-sorting step (block 450), when present, may be used to concentrate the cells 230 and separate them from the reagent 220. In some examples, the separated reagent 220 may be reused for processing additional cells 230 to reduce processing cost.
Intracellular delivery (block 420) may comprise compressing the cells (by passing the cells through the gaps formed by compressive elements (e.g., ridges)) and optionally allowing the cells to recover in between compression stages and after the last compression or at other times or in different sequences. The compressing stage may cause the cells to undergo a loss in intracellular volume (Vloss). The recovery stages may allow the cells to gain in volume (Vgain) and absorb reagents from the surrounding liquid medium. The volume loss and gain may correspond to bulk volume flows across the cell membrane. The volume loss (Vloss) may depends on the flowrate, gap, cell properties, and any other characteristics. These characteristics may also be interdependent with the compression time. In some examples, the volume loss (Vloss) during one compression stage may be different than the volume loss (Vloss) in another compression stage. In some examples, the volume loss (Vloss) may be at least 10% or 30% of the initial cell volume. The volume loss (Vloss) depends on the flowrate, gap, cell properties, and other characteristics. These characteristics may be interdependent with the compression time. In some examples, the volume loss (Vloss) during one compression stage may be different than the volume loss (Vloss) in another compression stage. In some cases, the volume loss (Vloss) may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or more. In some cases, the volume loss (Vloss) may be at most about 100%, 90%, 80%, 70%, 60%, 50%, 45%, 40%, 30%, 20%, 15%, 10%, or less
In some cases, the cell may gain volume. Volume gain may be achieved after volume loss. For example, a cell may lose some of its volume due to compression by the compressive element and may further gain some or all of its volume back. In some cases, the cell may even gain more volume than its initial volume. The volume gain (Vgain) can be characterized in terms of the volume loss (Vloss). The volume gain (Vgain) may depend on the cell properties, recovery time, experimental or operational conditions, flowrate, fluid properties, temperature, pressure, the size and age and type of the cells, the reagent, the device architecture, other features, and other factors. In some examples, the volume loss (Vloss) during one compression stage may be different from the volume loss (Vloss) in another compression stage. As an example, volume gain may be at least about 30%, or at least 70% of the volume loss (Vloss). In some cases, volume gain may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 80%, 90%, 100%, 110%, 120%, 140%, 150%, 300%, 400% or more of the volume loss. In some examples, volume gain may be at most about 300%, 200%, 100%, 90%, 80%, 70%, 60%, 59%, 30%, 20%, 15%, 10%, or less of the volume loss.
In some examples, intracellular delivery (block 420) may be performed one or more additional times using previously processed cells as schematically shown by the decision block 430 in
In some examples, the degree of intracellular delivery may increase by using osmotic effects, leading to cell swelling caused by an increase in the internal cell pressure. Osmotic effects can be controlled using, e.g., the difference of pH levels between the cell interior and media 210.
In some examples, heat shock may be used to temporary alter the state of the cells 230, which can enhance the intracellular delivery. The heat shock may be generated by one or more heating elements integrated into cell processing apparatus 100. In some cases, each heating element may provide localized heating as the cells 230 flow though the cell processing apparatus 100. Heating may be configured such that the heat shock experienced by the cells 230 may not exceed the critical time leading to cell damage. In some examples, the heat shock may be less than 1 second. The heat shock may be less than 30 seconds (s), 25 s, 20 s, 15 s, 10 s, 9 s, 8 s, 7 s, 6 s, 7 s, 5 s, 4 s, 3 s, 2 s, 1.5 s, 0.9 s, 0.8 s, 0.7 s, or less. In other examples, the heat shock may be more than about 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1 s, 1.5 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, or more.
In some examples, cooling may be used to reduce the temperature of the cells 230 and/or the reagent 220 and/or to maintain a reduced temperature of the cells 230 and the reagent 220. In some examples, the temperature reduction may be configured to suppress adverse cell response to the reagents and/or to increase cell viability. Different methods may be used to achieve temperature control in the channel. For example, incorporating of a Peltier element into the cell processing apparatus 100 may be used to achieve thermoelectric cooling of cells 230 and reagent 220. The temperature may be between 1° C. to 50° C., be between 1° C. to 40° C., be between 1° C. to 35° C., be between 1° C. to 30° C., be between 1° C. to 25° C., be between 1° C. to 20° C., be between 1° C. to 15° C., be between 1° C. to 10° C., be between 3° C. to 30° C., be between 5° C. to 30° C., be between 5° C. to 20° C., or be between 10° C. to 20° C. In some examples, the temperature may be between 5° C. and 30° C. In some cases temperature may be between 10° C. to 20° C.
In some examples, the state of the cells 230 may be altered using light, which in some cases, can enhance the intracellular delivery. In some examples, a light source may be integrated into the cell processing apparatus 100 (e.g., using transparent walls of the cell processing apparatus 100). Light sources with different wavelength may be used to modify the cell state and the cell response to the reagents.
In some examples, electric fields and/or magnetic fields may be used to enhance the transport of the reagents 220 into the interior of cells 230. For example, electrical fields may be generated by electrodes integrated into the walls of the cell processing apparatus 100. Electric fields may induce the motion of charged molecules, e.g., to increase the delivery of reagents into the cell interior. In some examples, permanent magnets and/or electric magnets may be used to generate magnetic fields of desired strengths which may depend on the magnetic properties of the reagents.
In some examples, immune suppressing reagents may be used to suppress the adverse cell response to delivered reagents and/or to improve the incorporation of reagents into the cells 230 and the cell viabilities.
In some examples, intracellular delivery (block 420) may comprise self-cleaning (block 422) of the cell processing apparatus 100.
In some examples, self-cleaning may be achieved using an axillary fluid flow along ridges 130. For example, a temporary reverse flow may be used to separate cells from the ridges 130. Furthermore, other forces, such as electric forces, magnetic forces may be used to transport the cells to the channel 170.
Intercellular delivery (block 420) may be performed at various flowrates such as between 1 millimeter per second (mm/s) and 500 millimeters per second or, in some cases 5 millimeters per second and 200 millimeters per second, where the velocity is the average velocity of the fluid. Flowrates may be at least about 1 mm/s, 2 mm/s, 3 mm/s, 4 mm/s, 5 mm/s, 6 mm/s, 7 mm/s, 8 mm/s, 9 mm/s, 10 mm/s, 15 mm/s, 20 mm/s, 30 mm/s, 40 mm/s, 50 mm/s, 60 mm/s, 70 mm/s, 80 mm/s, 90 mm/s, 100 mm/s, 150 mm/s, 180 mm/s, 200 mm/s, 210 mm/s, 220 mm/s, 230 mm/s, 240 mm/s, 250 mm/s, 300 mm/s, 400 mm/s, 450 mm/s, 500 mm/s, or more. In some examples, flowrates may be at most about 1 mm/s, 2 mm/s, 3 mm/s, 4 mm/s, 5 mm/s, 6 mm/s, 7 mm/s, 8 mm/s, 9 mm/s, 10 mm/s, 15 mm/s, 20 mm/s, 30 mm/s, 40 mm/s, 50 mm/s, 60 mm/s, 70 mm/s, 80 mm/s, 90 mm/s, 100 mm/s, 150 mm/s, 180 mm/s, 200 mm/s, 210 mm/s, 220 mm/s, 230 mm/s, 240 mm/s, 250 mm/s, 300 mm/s, 400 mm/s, 450 mm/s, 500 mm/s. Flowrates may controls and/or affect the cell compression rates. Furthermore, the flowrate may affect and/or controls the pressure at the inlet and within the cell processing apparatus 100.
In some example, a variable flowrate may be used during intercellular delivery (block 420), which may be also referred to as an unsteady flow condition. For example, the flowrate may change at a predefined, or random pattern during the intracellular delivery. For example, pulsating flow may be used such as the flowrate can oscillate with amplitude between 10% and 50% of the average flowrate with the frequency between 0.1 Hz and 100 Hz. The flowrate may oscillate with an amplitude of at least about 5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 25%, 27%, 30%, 35%, 40%, 50%, 52%, 55%, 60%, or more of the average flowrate. In some cases, the flowrate may oscillate with an amplitude of at most about 70%, 60%, 55%, 50%, 45%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the average flowrate. The oscillation frequency in each case may be at least about 0.05 Hz, 0.1 Hz, 0.2 Hz, 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 15 Hz, 20 Hz, 25 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 95 Hz, 100 Hz, 105 Hz, 110 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 200 Hz, 300 Hz, or more. In some cases, the flowrate may oscillate with a frequency of at most about 400 Hz, 350 Hz, 300 Hz, 250 Hz, 200 Hz, 150 Hz, 120 Hz, 110 Hz, 100 Hz, 92 Hz, 90 Hz, 85 Hz, 80 Hz, 75 Hz, 70 Hz, 65 Hz, 60 Hz, 50 Hz, 40 Hz, 30 Hz, 25 Hz, 20 Hz, 15 Hz, 10 Hz, 5 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, 0.09 Hz, 0.08 Hz, 0.07 Hz, 0.06 Hz, 0.05 Hz, or less.
In another example, the flowrate can be reduced to substantially zero and then restored to the about the full flow velocity. Flowrate may change according to other patterns. The variable flowrate may be used to temporary increase the hydrodynamic force applied on the cells as these cells pass through the gaps formed by the ridges 130, e.g., to cause faster cell compressions. Furthermore, in some cases, the unsteady flow may improve the removal of abnormal cells and cell clusters from ridges 130 and into diversion channel 170, e.g., to prevent clogging of the cell processing apparatus 100. For example, the flowrate may increase by a magnitude between 20% and 100% for a period of time between about 0.1 second to 5 second. In another example, the flowrate can be reversed with the magnitude between 20% and 200% of the forward velocity for a period of time from 0.1 second to 5 second. The flowrate may increase or decrease by a magnitude of at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.99%, or more. The flowrate may increase or decrease by a magnitude of at most about 100%, 99.9%, 99%, 98%, 95%, 90%, 80%, 85%, 80%, 75%, 70%, 65%, 60%, 50%, 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1% or less. The change in the flowrate or the direction of fluid flow in each case may be temporary or stable according to the desired conditions. For example, in each of the conditions, the increased or decreased flowrate may last for a time period of at least about 0.05 seconds (s), 0.1 s, 0.15 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 15 s, 20 s, 30 s, or more. For example, in each of the conditions, the increased or decreased flowrate may last for a time period of at most about 0.005 seconds (s), 0.05, 0.1 s, 0.15 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s, 15 s, 20 s, 30 s, or less. In some cases, the flowrate increase/decrease may be in the form of a pulse of fluid flow.
In some examples, the intracellular delivery or flowing the mixture through the cell processing apparatus 100 (block 420) may comprise or be combined with inside sorting (block 424), which may occur within the interior of the cell processing apparatus. For example, as the cells with different properties flow through the cell processing apparatus, some cells may not be able to pass through the gaps formed by the ridges, e.g., due to mechanical properties, viscoelastic properties, size, adhesive properties, or other properties or characteristics of the cell, or flow, or other reasons and/or factors. The ridges may direct such cells, or a subset of such cells into a diversion channel as described anywhere herein, for example with reference to
In some examples, intracellular delivery/flowing the mixture through the cell processing apparatus 100 (block 420) may comprise vibrating the cell processing apparatus 100 (block 426). As described above with reference to
The vibrations may be initiated or changed in response to the input received from sensors 320. In some examples, the amplitude of the vibrations may be greater than about 1 micrometer (μm). In the same or other examples, the frequency of the vibrations may be greater than 1 Hz. In some examples, the amplitude of vibrations may be at least about 0.001 μm, 0.005 μm, 0.006 μm, 0.007 μm, 0.008 μm, 0.009 μm, 0.01 μm, 0.02 μm, 0.05 μm, 0.08 μm, 0.09 μm, 1 μm, 1.1 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 3 μm, 5 μm, 10 μm, or more. In some examples, the amplitude of vibrations may be at most about 10 μm, 8 μm, 5 μm, 4 μm, 2 μm, 3 μm, 2 μm, 1.5 μm, 1.2 μm, 1.1 μm, 1 μm, 0.9 μm, 0.5 μm, 0.1 μm, or less. In the same or other examples, the frequency of the vibrations may be at least about 0.05 Hz, 0.1 Hz, 0.2 Hz, 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 15 Hz, 20 Hz, 25 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 95 Hz, 100 Hz, 105 Hz, 110 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 200 Hz, 300 Hz, or more. In some cases, in the same or other examples, the frequency of the vibrations may be at most about 400 Hz, 350 Hz, 300 Hz, 250 Hz, 200 Hz, 150 Hz, 120 Hz, 110 Hz, 100 Hz, 92 Hz, 90 Hz, 85 Hz, 80 Hz, 75 Hz, 70 Hz, 65 Hz, 60 Hz, 50 Hz, 40 Hz, 30 Hz, 25 Hz, 20 Hz, 15 Hz, 10 Hz, 5 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, 0.09 Hz, 0.08 Hz, 0.07 Hz, 0.06 Hz, 0.05 Hz, or less.
In some examples, method 400 may further comprise cell encapsulation (block 415). In some cases, increasing the concentration of reagents around cells during intracellular delivery may increase the amount of reagents entering the cells. However, supplying large amounts reagents can be challenging due to higher costs or other factors. In some examples, the cells may be encapsulated into a shell which may have a high concentration of one or more reagents as, for example, as schematically shown in
The shell 203 may surround the cell 230 and may comprise the reagent 220. The core-shell structure 201 may be suspended in liquid media 210, which in these examples may otherwise be free from reagents (i.e., besides reagent 220 in shell 222). Providing reagent 220 in the shell 203 may allow substantially increasing the concentration of reagent 220 at the surface of shell 203 in comparison to dispersing the same amount of reagent 220 in the entire liquid media 210. In some examples, the shell 203 may be a vesicle or in an oil/water droplet.
In some examples, surfactants may be used to configure the formation of droplets. Surfactant may be added to the oil stream. Surfactants may be fluorinated surfactants. In some cases, the addition of the surfactant to the oil may help stabilize the formed droplets.
The present disclosure provides computer systems that are programmed to implement methods of the disclosure.
The computer system 1001 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1001 also includes memory or memory location 1010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1015 (e.g., hard disk), communication interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025, such as cache, other memory, data storage and/or electronic display adapters. The memory 1010, storage unit 1015, interface 1020 and peripheral devices 1025 are in communication with the CPU 1005 through a communication bus (solid lines), such as a motherboard. The storage unit 1015 can be a data storage unit (or data repository) for storing data. The computer system 1001 can be operatively coupled to a computer network (“network”) 1030 with the aid of the communication interface 1020. The network 1030 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1030 in some cases is a telecommunication and/or data network. The network 1030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1030, in some cases with the aid of the computer system 1001, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1001 to behave as a client or a server.
The CPU 1005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1010. The instructions can be directed to the CPU 1005, which can subsequently program or otherwise configure the CPU 1005 to implement methods of the present disclosure. Examples of operations performed by the CPU 1005 can include fetch, decode, execute, and write back.
The CPU 1005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).
The storage unit 1015 can store files, such as drivers, libraries and saved programs. The storage unit 1015 can store user data, e.g., user preferences and user programs. The computer system 1001 in some cases can include one or more additional data storage units that are external to the computer system 1001, such as located on a remote server that is in communication with the computer system 1001 through an intranet or the Internet.
The computer system 1001 can communicate with one or more remote computer systems through the network 1030. For instance, the computer system 1001 can communicate with a remote computer system of a user (e.g., a scientist or technician). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1001 via the network 1030.
Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1001, such as, for example, on the memory 1010 or electronic storage unit 1015. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1005. In some cases, the code can be retrieved from the storage unit 1015 and stored on the memory 1010 for ready access by the processor 1005. In some situations, the electronic storage unit 1015 can be precluded, and machine-executable instructions are stored on memory 1010.
The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
Aspects of the systems and methods provided herein, such as the computer system 1001, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.
Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
The computer system 1001 can include or be in communication with an electronic display 1035 that comprises a user interface (UI) 1040 for providing, for example, relevant information of intracellular delivery such as parameters of microfluidic devices used for delivery, cell type, molecules to be delivered into the cells, and/or results of the delivery. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1005. The algorithm can, for example, regulate one or more parameters of the methods and/or systems (e.g., flow rate, temperature, pressure, buffer solution, cell type etc.).
In some cases, the computer systems may comprise or be operatively coupled to an imaging system to control cell state, cell deformation, cell volume change, delivery of reagents.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Patent Application 62/775,351, filed on Dec. 4, 2018, which is incorporated herein by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62775351 | Dec 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17339757 | Jun 2021 | US |
| Child | 17521237 | US | |
| Parent | PCT/US2019/064310 | Dec 2019 | US |
| Child | 17339757 | US |
