Patent Application
20220062904
This invention was made with Government support under Grant Number DGE-1650044 awarded by the National Science Foundation and Grant Number IR21CA191243-01A1 awarded by the NIH. The Government has certain rights in the invention.
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The present disclosure relates generally to intracellular delivery system and methods. Particularly, embodiments of the present disclosure relate to a system for intracellular delivery of molecules from a cell medium into cells via a microchannel.
Intracellular molecular delivery is important in cell manufacturing applications, especially gene transfection and editing. Known methods of intracellular molecular delivery result in very low delivery efficiencies for very large macromolecules (>500 kDa) to the cells, and also precludes delivery to the cell nucleus. Additionally, these methods result in device clogging that decrease the throughput capabilities of such devices
Cells can substantially change their shape without changing volume. Cell manipulations due to significant deformations of up to 85% strain applied across a range of timescales from ˜10
μs to >1 s have described cell deformation and shape change but have not described cell volume change.
In an exemplary embodiment, the present invention is a microchannel and/or a system for intracellular delivery of a plurality of molecules from a cell medium into a plurality of cells, the system comprising a microchannel, wherein the microchannel comprises a first wall, a second wall, and a plurality of ridges that are arranged in a chevron pattern oriented with respect to a central flow axis of the microchannel, the first wall and the second wall being substantially planar to each other, wherein the plurality of ridges protrude outwardly from the first wall and define a plurality of compression gaps between the plurality of ridges and the second wall, and wherein the plurality of compression gaps have a height, measured perpendicular to the second wall, of from 20 to 80% of an average cell diameter of the plurality of cells, and a plurality of relaxation spaces disposed between the plurality of ridges.
A ridge angle formed by at least one ridge of the plurality of ridges can be from 20 degrees to 90 degrees with respect to the central flow axis of the microchannel.
The ridge angle can be different for different ones of the plurality of ridges along the central flow axis of the microchannel.
The ridge angle can be configured to facilitate movement of dead or damaged cells in the plurality of cells to a side wall of the microchannel thereby preventing clogging of the microchannel.
The side wall can extend between the first wall and the second wall.
The system can further comprise a first outlet and a second outlet, wherein the first outlet is positioned along the side wall and is configured to receive the dead or damaged cells, and the second outlet is positioned away from the side wall.
A ridge angle formed by at least one ridge of the plurality of ridges can be 30 degrees or 45 degrees with respect to the central flow axis of the microchannel.
A width of at least one of the plurality of relaxation spaces can be from 100 micrometers to 300 micrometers between two adjacent ones of the plurality of ridges.
The plurality of ridges can comprise 1 ridge to 21 ridges, each ridge defining a separate one of the plurality of compression gaps.
The plurality of compression gaps can be configured to allow simultaneous passing of multiple cells of the plurality of cells through each of the plurality of compression gaps.
The plurality of compression gaps can be configured to compress the plurality of cells as the plurality of cells passes through each of the compression gaps causing the plurality of cells to undergo a loss in intracellular volume (Vloss).
The plurality of relaxation spaces can be configured to allow the plurality of cells to undergo a gain in volume (Vgain) and absorb a portion of the plurality of molecules from the cell medium.
The system can further comprise one or more additional microchannels, sharing a common inlet with the microchannel and providing additional flow paths to the plurality of cells through the system, wherein each of the one or more additional microchannels comprises an additional first wall, an additional second wall, and an additional plurality of ridges that are arranged in the chevron pattern oriented with respect to the central flow axis of the microchannel, the additional plurality of ridges protrude outwardly from the additional first wall and define an additional plurality of compression gaps between the additional plurality of ridges and the additional second wall, and the additional plurality of compression gaps have an additional height, measured perpendicular to the additional second wall, of from 20 to 80% of the average cell diameter of the plurality of cells.
Each of the plurality of ridges can have a rectangular shape, a cylindrical shape, a trapezoidal shape, or a triangular shape.
Each of the plurality of ridges can have a thickness from about 7 to about 30 microns.
The microchannel can further comprise a second plurality of ridges protrude outwardly from the second wall toward the first wall.
The system can further comprise one or more sheath flow inlets for hydrodynamic focusing of the cell medium within the microchannel.
The system can further comprise a cell inlet for delivering the cell medium into the microchannel, wherein the cell inlet is positioned between two of the one or more sheath flow inlets.
The system can further comprise a cell inlet for delivering the cell medium into the microchannel, wherein the cell inlet is surrounded by at least one of the one or more sheath flow inlets.
The system can further comprise a cell inlet for delivering the cell medium into the microchannel, wherein the cell inlet is positioned downstream relative to at least one of the one or more sheath flow inlets.
At least one of the plurality of ridges can comprise a cell adhesion entity or a passivation layer.
In another exemplary embodiment, the present invention is a method for convective intracellular delivery comprising providing a cell medium comprising a plurality of cells and a plurality of molecules to a microchannel, the microchannel comprising a first wall, a second wall, and a plurality of ridges that are arranged in a chevron pattern oriented with respect to a central flow axis of the microchannel, the first and second walls being substantially planar to each other, wherein the plurality of ridges protrude outwardly from the first wall and define a plurality of compression gaps between the plurality of ridges and the second wall, and wherein the plurality of compression gaps have a height, measured perpendicular to the second wall, of from 20% to 80% of an average cell diameter of the plurality of cells, and a plurality of relaxation spaces disposed between the plurality of ridges, flowing the cell medium through the microchannel at a flow velocity, wherein as the cell medium flows through the microchannel, the plurality of cells undergo a convective intracellular delivery process comprising compressing the plurality of cells in a first compression gap of the plurality of compression gaps, wherein the compressing causes the plurality of cells to undergo a loss in intracellular volume Vloss, and passing the plurality of cells to a first relaxation space of the plurality of relaxation spaces, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules from the cell medium, and collecting the plurality of cells in an outlet.
A ridge angle formed by at least one ridge of the plurality of ridges can be from 20 degrees to 90 degrees with respect to the central flow axis of the microchannel.
The plurality of molecules can be selected from the group consisting of macromolecules, nanoparticles, dextran, plasmids, mRNA, antibodies, beads, viruses, and combinations thereof.
The plurality of molecules can comprise macromolecules having an average size of from 3 kDa to 6 MDa.
A width of at least one of the plurality of relaxation spaces can be from 100 microns to 300 microns between two adjacent ones of the plurality of ridges.
At least one of ridge surfaces extending between the plurality of compressive surfaces ridges and the first wall can be substantially orthogonal to a corresponding one of the plurality of compressive surfaces to the first wall.
The flow velocity can be from 3 mm/sec to 500 mm/sec.
The loss in intracellular volume Vloss can be from 5% to 30% of an average cell volume of the plurality of cells.
The loss in intracellular volume Vloss can occur in between 1 microsecond and 1000 milliseconds from when each cell of the plurality of cells first encounter one of the plurality of ridges.
The gain in volume Vgain can be from 25% to 100% of the loss in intracellular volume Vloss.
The gain in volume Vgain of at least 10% of the loss in intracellular volume Vloss can occur in from 4 microseconds to 100 microseconds.
At least one of the gain in volume Vgain and the second gain in volume (Vgain2) can be at least 10% of one of the loss in intracellular volume Vloss and the second loss in intracellular volume (Vloss2).
The method can further comprise, while flowing the cell medium through the microchannel, sorting the plurality of cells based on one or more of viscoelasticity, stiffness, elasticity, and adhesion.
The convective intracellular delivery process can further comprise compressing the plurality of cells in a second compression gap of the plurality of compression gaps, wherein the compressing in the second compression gap causes the plurality of cells to undergo a second loss in intracellular volume Vloss2, and passing the plurality of cells to a second relaxation space of the plurality of relaxation spaces, wherein the plurality of cells undergo a second gain in volume Vgain2 and absorb a second portion of the plurality of molecules from the cell medium while in the second relaxation space.
The plurality of ridges can comprise 5 ridges to 50 ridges, each ridge defining a separate one of the plurality of compression gaps.
The plurality of compression gaps can be configured to allow simultaneous passing of multiple cells of the plurality of cells through each of the plurality of compression gaps.
The ridge angle can be configured to affect trajectories of the plurality of cells within the microchannel.
The ridge angle can be configured to facilitate movement of dead or damaged cells in the plurality of cells to a side wall of the microchannel thereby preventing clogging of the microchannel, and the side wall can extend between the first wall and the second wall.
A ridge angle formed by at least one ridge of the plurality of ridges can be 30 degrees with respect to the central flow axis of the microchannel. A ridge angle formed by at least one ridge of the plurality of ridges can be 45 degrees with respect to the central flow axis of the microchannel.
In other exemplary embodiments of the present disclosure, a method for convective intracellular delivery comprises providing a cell medium and a plurality of molecules to a microchannel, the cell medium comprising a plurality of cells and the microchannel comprising a first wall and a second wall, the walls being substantially planar to each other and the first wall having a plurality of compressive surfaces, wherein each compressive surface protrudes outwardly from the first wall and defines a compression gap between the compressive surface and the second wall, wherein the compression gap has a height of between 20 and 80% of the average cell diameter, and a plurality of relaxation spaces disposed between the compressive surfaces, flowing the cell medium through the microchannel at a flow velocity, wherein as the cell medium flows through the microchannel, the plurality of cells undergo a convective intracellular delivery process comprising compressing the plurality of cells in a first compression gap, wherein the compressing causes the plurality of cells to undergo a loss in intracellular volume Vloss, and passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules, and collecting the plurality of cells in an outlet.
In other exemplary embodiments of the present disclosure, a method for convective intracellular delivery comprises applying a plurality of cells and a plurality of molecules to a microchannel, the microchannel having a first orthogonal surface defining a first compression gap, a second orthogonal surface defining a second compression gap, and a relaxation space disposed between the first and second orthogonal surfaces, flowing the plurality of cells to the microchannel at a flow velocity of 100 to 500 mm/s, applying at the first orthogonal surface a compressive force to the cells, wherein the compressive force causes the cells to undergo a first loss in volume (Vloss1), passing the plurality of cells through a first relaxation space, wherein the plurality of cells undergo a first gain in volume (Vgain1), applying at the second orthogonal surface a compressive force to the cells, wherein the compressive force causes the cells to undergo a second loss in volume Vloss2, and collecting the plurality of cells at a collection point, wherein as the plurality of cells are collected they undergo a second gain in volume Vgain2, wherein as the plurality of cells undergo at least one of Vgain1 and Vgain2 they absorb a portion of the plurality of molecules.
In other exemplary embodiments of the present disclosure, a system for intracellular delivery comprises a microchannel comprising a first wall and a second wall, the walls being substantially planar to each other and the first wall having a plurality of compressive surfaces, wherein each compressive surface protrudes outwardly to the first wall and defines a compression gap between the compressive surface and a surface of the second wall, and a plurality of relaxation spaces disposed between the compressive surfaces, and a cell medium comprising a plurality of cells and a plurality of molecules, the cell medium flowing through the microchannel at a flow velocity, wherein as the cell medium flows through the microchannel, the plurality of cells undergo a convective intracellular delivery process comprising compressing the plurality of cells in a first compression gap, wherein the compressing causes the cells to undergo a loss in volume Vloss, passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules, and wherein the compression gap has a height of from 20 to 80% of the average cell diameter.
Embodiments of the present disclosure can include a cell comprising a plurality of macromolecules having an average diameter of 3 kDa to 6 MDa the cell formed by a process comprising providing a cell medium and a plurality of molecules to a microchannel, the cell medium comprising the cell and the microchannel comprising a first wall and a second wall, the walls being substantially planar to each other and the first wall having a plurality of compressive surfaces wherein each compressive surface protrudes normal to the first wall and defines a compression gap between the compressive surface and the second wall, wherein the compression gap has a height of from 20 to 80% of the cell diameter, and a plurality of relaxation spaces disposed between the compressive surfaces, flowing the cell medium through the microchannel at a flow velocity, wherein as the cell medium flows through the microchannel, the cell undergoes a convective intracellular delivery process comprising compressing the cell in a first compression gap, wherein the compressing causes the cell to undergo a loss in volume Vloss, passing the cell to a first relaxation space, wherein the cell undergoes a gain in volume Vgain and absorbs a portion of the plurality of molecules, and collecting the cell in an outlet.
In some embodiments, the compressive surface(s) of any of the above-described systems or methods can comprise a plurality of ridges that are diagonally oriented with respect to a central flow axis of the microchannel. In some embodiments, an angle formed by a ridge can be 30 degrees with respect to the central axis of the microchannel. In other embodiments. In other embodiments, an angle formed by a ridge can be 45 degrees with respect to the central axis of the microchannel. In other embodiments, an angle formed by the ridge can be from 20 to 90 degrees with respect to the central axis of the microchannel. In some embodiments of any of the above-described systems or methods, the micro-channel can include a plurality of ridges and the plurality of ridges can be arranged in a chevron pattern within the microchannel. In some embodiments, the plurality of compressive surfaces can comprise from 1 to 21 ridges. In other embodiments, the plurality of compressive surfaces can comprise from 1 to 7 ridges.
In some embodiments, the molecules of any of the above-described systems or methods, can include at least one of macromolecules, nanoparticles, dextran, plasmids, mRNA, antibodies, beads, or viruses. In some embodiments, the macromolecules can have an average size of from 3 kDa to 6 MDa.
In some embodiments, the microchannel of any of the above-described systems or methods, can include at least one inlet. In some embodiments, the plurality of relaxation spaces of any of the above-described systems or methods, can be from 100 to 300 microns. In some embodiments, the plurality of compressive surfaces of any of the above-described systems or methods, can be substantially orthogonal. In some embodiments, the plurality of compression gaps can be from 20 to 80% an average cell diameter.
In some embodiments, the flow velocity of any of the above-described systems or methods, can be from 100 to 500 mm/sec.
In some embodiments, the Vloss of any of the above-described systems or methods can be from 5% to 30% the average cell volume. In other embodiments, Vloss can be 25% the average cell volume. In some embodiments, the Vloss of any of the above-described systems or methods can occur in about 10 μsis measured from when the cell first encounters a compressive surface. In some embodiments, the Vgain of any of the above-described systems or methods can be 25% to 100% Vloss. In some embodiments, Vgain of approximately 100% Vloss can occur in from 4 to 100 ms.
Although preferred embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the preferred embodiments, specific terminology will be resorted to for the sake of clarity.
It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Also, in describing the preferred embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.
Ranges can be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.
By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.
It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.
Embodiments of the present disclosure can achieve a cell behavior of transient (up to 30%) cell volume change in response to large magnitude deformations at ultrafast timescales without impairing cell viability. Known methods for intracellular delivery of molecules and/or particles to cells rely on diffusive delivery of molecules and/or particles to cells, which does not rely on cell volume change. Instead, such diffusive delivery uses shear and/or compressive forces to create micro-pores in the cell membrane. The cell membrane is susceptible to manipulation for intracellular delivery, yet in order to maintain high cell viability and avoid cell damage or death, such manipulation is limited in both scale (e.g., amount of force, size of pores, and number of compressions) and the types of molecules that can be delivered (e.g., smaller molecule delivery, not macromolecule delivery above a few hundred kiloDalton). Additionally, absent an external concentration difference between inside and outside the cell, diffusion will not occur in a way to facilitate intracellular delivery. A mechanism for obtaining volume flow and corresponding “convective” volume reuptake is not described. In fact, it is expected in the art and that forcing substantial volume (such as liquids within the cell, e.g., cell cytoplasm) out of a cell is not possible without substantial cell damage and cell death. Surprisingly, embodiments of the present disclosure can achieve bulk volume transfer in substantial amounts, which can facilitate improved intracellular molecular deliver with high cell viability.
Additionally, known microfluidic devices for obtaining diffusive delivery are prone to clogging, due to the need to use narrow constrictions in order to operate at high shear to facilitate cell membrane pore creation and obtain diffusive delivery. Attempts to reduce cell clogging by using high shear force combined with high flow rate, can result in cell damage and cell death. Also, repeated exposure to high shear force can result in cell damage and cell death.
Embodiments of the present disclosure can obtain abrupt volume decrease and fluid transfer during recover results in high-throughput delivery of a variety of macromolecules with high cell viability. Additionally, embodiments of the present disclosure can be used to deliver a variety of macromolecules to a variety of different cell types. Embodiments of the present disclosure have the benefit of providing higher throughput delivery with less clogging. Also, embodiments of the present disclosure can achieve delivery molecules and/or particles with less specialized equipment than microinjection and nanoneedle injection, sometimes used for macromolecule delivery. Embodiments of the present disclosure can have less risk of cell death and aggregation than that of microfluidic devices based on diffusive delivery.
Embodiments of the present disclosure can include methods, systems, and devices for convective intracellular delivery of molecules. Methods for convective intracellular delivery can comprise one or more of the following steps: 1) providing a cell medium and a plurality of molecules to a microchannel, the cell medium comprising a plurality of cells, 2) flowing the cell medium through the microchannel at a flow velocity, 3) applying a convective intracellular delivery process as cell medium flows through the microchannel, 4) compressing the plurality of cells in a first compression gap, wherein the compressing causes the plurality of cells to undergo a loss in intracellular volume Vloss, 5) applying at the first orthogonal surface a compressive force to the cells, wherein the compressive force causes the cells to undergo a first loss in volume Vloss1, passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume (Vgain or Vgain1), 6) applying a compressive force to the cells, wherein the compressive force causes the cells to undergo a second loss in volume Vloss2, 7) collecting the plurality of cells at an outlet, and 8) collecting the plurality of cells at an outlet, wherein as the plurality of cells are collected they undergo a second gain in volume Vgain2.
In some embodiments, the methods for convective intracellular delivery can comprise: providing a cell medium and a plurality of molecules to a microchannel, the cell medium comprising a plurality of cells, flowing the cell medium through the micro-channel at a flow rate, wherein as the cell medium flows through the micro-channel, the plurality of cells undergo a convective intracellular delivery process comprising: compressing the plurality of cells in a first compression gap, wherein the compressing causes the plurality of cells to undergo a loss in intracellular volume Vloss, and passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules, and collecting the plurality of cells in an outlet.
In some embodiments, the methods for convective intracellular delivery of molecules can comprise providing a plurality of cells to a microchannel, flowing the plurality of cells to the microchannel, applying a compressive force to the cells, wherein the compressive force causes the cells to undergo a first loss in volume Vloss1, passing the plurality of cells through a first relaxation space, wherein the plurality of cells undergo a first gain in volume Vgain1, applying a compressive force to the cells, wherein the compressive force causes the cells to undergo a second loss in volume Vloss2 and collecting the plurality of cells at an outlet, wherein as the plurality of cells are collected they undergo a second gain in volume Vgain22.
Embodiments of the present disclosure may also include one or more systems for intracellular delivery of molecules. In some embodiments, the system may comprise a micro-channel and a cell medium comprising a plurality of cells and a plurality of molecules, the cell medium flowing through the micro-channel at a flow rate, wherein as the cell medium flows through the micro-channel, the plurality of cells undergo a convective intracellular delivery process comprising: compressing the plurality of cells in a first compression gap, wherein the compressing causes the cells to undergo a loss in volume Vloss, passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules.
Any of the above-described systems and methods may also include a cell comprising a plurality of macromolecules having an average diameter of 1 nm to 150 nm, the cell formed by any of the methods or systems described previously. In any of the above-described systems and methods, a plurality of macromolecules can be delivered to the plurality of cells. The plurality of macromolecules can be of uniform size or varying size. For instance, any molecule of the plurality of macromolecules can be from 1 nm to 100 nm, from 5 nm to 100 nm, from 10 nm to 100 nm, from 20 nm to 100 nm, from 30 nm to 100 nm, from 40 nm to 100 nm, from 50 nm to 100 nm, from 60 nm to 100 nm, from 75 nm to 100 nm, from 80 nm to 100 nm, from 85 nm to 100 nm, from 90 nm to 100 nm, from 110 nm to 120 nm. In some embodiments the macromolecules can range in size from 3 kDa to 6 MDa, from 10 kDa, to 6 MDa, from 15 kDa to 6 MDa, from 20 kDa to 6 MDa, from 25 kDa to 6 MDa, from 30 kDa to 6 MDa, from 40 kDa to 6 MDa, from 50 kDa to 6 MDa, from 60 kDa to 6 MDa, from 70 kDa to 6 MDa, from 75 kDa to 6 MDa, from 80 kDa to 6 MDa, from 90 kDa to 6 MDa, from 100 kDa to 6 MDa, from 250 kDa to 6 MDa, from 500 kDa to 6 MDa, from 750 kDa to 6 MDa, 1 MDa to 5 MDa, 2 MDa to 4 MDa, 3 MDa.
Any of the above-described systems and methods can achieve convective intracellular delivery of molecules into a variety of cell types. These cell types may include, but are not limited to cells of the reproductive system, e.g., oocytes, spermatozoa, leydig cells, embryonic stem cells, amniocytes, blastocysts, morulas, and zygotes, leukocytes, e.g., peripheral blood leukocytes, spleen leukocytes, lymph node leukocytes, hybridoma cells, T cells (cytotoxic/suppressor, helper, memory, naive, and primed), B cells (memory and naive), monocytes, macrophages, granulocytes (basophils, eosinophils, and neutrophils), natural killer cells, natural suppressor cells, thymocytes, and dendritic cells, cells of the hematopoietic system, e.g., hematopoietic stem cells (CD34+), proerythroblasts, normoblasts, promyelocytes, reticulocytes, erythrocytes, pre-erythrocytes, myeloblasts, erythroblasts, megakaryocytes, B cell progenitors, T cell progenitors, thymocytes, macrophages, mast cells, and thrombocytes, stromal cells, e.g., adipocytes, fibroblasts, adventitial reticular cells, endothelial cells, undifferentiated mesenchymal cells, epithelial cells including squamous, limbal cells, cuboid, columnar, squamous keratinized, and squamous non-keratinized cells, and pericytes, cells of the skeleton and musculature, e.g., myocytes (heart, striated, and smooth), osteoblasts, osteoclasts, osteocytes, synoviocytes, chondroblasts, chondrocytes, endochondral fibroblasts, and perichonondrial fibroblasts, cells of the neural system, e.g., astrocytes (protoplasmic and fibrous), microglia, oligodendrocytes, and neurons, cells of the digestive tract, e.g., parietal, zymogenic, argentaffin cells of the duodenum, polypeptide-producing endocrine cells (APUD), islets of langerhans (alpha, beta, and delta), hepatocytes, and kupfer cells, cells of the skin, e.g., keratinocytes, langerhans, and melanocytes, cells of the pituitary and hypothalamus, e.g., somatotropic, mammotropic, gonadotropic, thyrotropic, corticotropin, and melanotropic cells, cells of the adrenals and other endocrine glands, e.g., thyroid cells (C cells and epithelial cells), adrenal cells, and tumor cells.
The cells may be Burkitt lymphoma cells, choriocarcinoma cells, adenocarcinoma cells, non-Hodgkin's B and T cell lymphoma cells, fibrosarcoma cells, neuroblastoma cells, plasmacytoma cells, rhabdomyosarcoma cells, carcinoma cells of the pharynx, renal adenocarcinoma, hepatoma cells, fibrosarcoma cells, myeloma cells, osteosarcoma cells, teratoma cells, teratomal keratinocytes, lung carcinoma cells, colon adenocarcinoma cells, lung adenoma cells, renal carcinoma cells, rectum adenocarcinoma cells, chronic myelogenous leukemia cells, ileocecal adenocarcinoma cells, hairy cell leukemia cells, acute myelogenous leukemia cells, colon carcinoma cells, cecum carcinoma and adenocarcinoma cells, leukemia-cecum adenocarcinoma cells, pancreatic carcinoma, Wilm' s tumor cells, prostate adenocarcinoma cells, renal leimyooblastoma cells, bladder carcinoma cells, plasmacytoma cells, teratocarcinoma cells, breast carcinoma, epidermoid carcinoma of the cervix, ovarian teratocarcinoma, myeloma cells, T and B cell lymphoma cells, amalanotic melanoma cells, cervical carcinoma cells, rhabdomyosarcoma, hepatoma, medullary Thyroid carcinoma cells, malignant melanoma cells, glioblastoma cells, plasma cell leukemia, endometrial adenocarcinoma, squamous cell carcinoma, pancreatic adenocarcinoma, astrocytoma, gastric adenocarcinoma, pulmonary mucoepidermoid carcinoma cells, myeloid leukemia cells, EBV-transformed B cells, renal cell adenocarcinoma, acute leukemia, B cell plasmacytoma, acute lymphocytic leukemia, cutaneous T lymphoma, T cell leukemia, acute lymphoblastic leukemia, HIV+T cells, medulloblastoma, B cells from sickle cell disease, acute monocytic leukemia, adrenocortical carcinoma, Bowes Melanoma and hepatocellular carcinoma.
The plurality of cells in any of the above-described systems and methods may include any of the above cells or derivatives thereof. While the presently described systems and methods are described in terms of biological cells, it is understood that these presently disclosed systems and methods can be achieved using a variety of materials other than biological cells, in some embodiments, the above-described systems and methods can be achieved with a variety of particles, including nanoparticles, intracellular probe sensors (e.g., molecular beacons and SmartFlares), viruses (e.g., lentivirus), and quantum dots.
Additionally, any of the above-described systems and methods can include any of the above-described cells suspended in a fluid, such as a cell medium. The cell medium can be any liquid in which a plurality of cells can be suspended and can include additional substances including one or more of a carbon source (e.g., glucose) water, various salts, a source of amino acids and nitrogen (e.g., beef, yeast extract). Additionally, the medium may include other nutrients such as plant count agar, nutrient agar, or trypticase soy agar.
Any of the above-described systems and methods can include flowing cells and/or cell medium through a microchannel. In some embodiments of the above-described systems and methods, the microchannel can be defined by a first wall and a second wall, the walls being substantially planar to each other. The microchannel may comprise a plurality of compressive surfaces protruding from the first wall. In some embodiments, the plurality of compressive surfaces can protrude outwardly from the first wall and towards a second wall. In some embodiments, the plurality of compressive surfaces can protrude outwardly from the second wall and towards a first wall. In some embodiments, the plurality of compressive surfaces can protrude outwardly from one of the first and second wall and towards one of the first and second wall. For instance, in some embodiments, the compressive surfaces can protrude normal from one or both of the walls. In other embodiments, the plurality of compressive surfaces can protrude from the first and/or second wall at an angle. In some embodiments, the plurality of compressive surfaces can protrude outwardly from both the first wall and the second wall. For example, each of the plurality of compressive surfaces can protrude outwardly from the first wall towards a second plurality of compressive surfaces protruding outwardly from the second wall. Additionally, in some embodiments of the above-described systems and methods, the plurality of compressive surfaces can define a plurality of compressive gaps. In some embodiments, the plurality of compressive surfaces may be a plurality of orthogonal surfaces. As such, in some embodiments, the microchannel of the above-described systems and methods can comprise a first orthogonal surface defining a first compression gap, and a second orthogonal surface defining a second compression gap.
An exemplary microchannel 100 for achieving convective intracellular delivery of any of the above-described systems and methods is illustrated in
The microchannel can comprise a plurality of compressive surfaces 130. In some embodiments, the plurality of compressive surfaces 130 can comprise a plurality of ridges, as illustrated at
The plurality of compressive surfaces 130 may define a compression gap 170 between a compressive surface 130 and a surface of an opposing wall 120. For instance, in an embodiment wherein the plurality of compressive surfaces 130 protrudes from the first planar wall 110, the plurality of compressive surfaces 130 may define a compression gap 170 between a compressive surface 130 and a surface across from the compressive surface 130 on the second planar wall 120. As used herein, a surface may include the closest or nearest portion of the opposing wall, for example where the wall does not otherwise have corresponding ridges or protrusions. In some embodiments, the second planar wall 120 can comprise a plurality of compressive surfaces 130, and the opposing surface can be, for example, an opposing compressive surface 130. The compression gap 170 can therefore be defined as the space formed between a compressive surface 130 and a surface of the second wall 120, or the space between opposing compressive surfaces on opposing walls. In some embodiments, the opposing ridges can be aligned with each other.
While the first and second walls of the microchannel are described with respect to
The size of the compression gap 170 can be increased or decreased as desired, based on device design. In some embodiments, the size of the compression gap 170 can be defined in terms of the average diameter of a cell. As will be understood, the diameter of the cell can be defined as the largest distance between two points on a cell. In some embodiments, the height of the compression gap may be defined based on a percentage of the average cell diameter. For instance, the compression gap 170 may have a height of about 10% to about 80% an average cell diameter, about 10% to about 50% an average cell diameter, about 10% to about 40% an average cell diameter, about 10% to about 30% an average cell diameter, about 10% to about 20% an average cell diameter, about 20% to about 30% an average cell diameter, about 30% to about 40% an average cell diameter, about 40% to about 50% an average cell diameter, about 50% to about 60% an average cell diameter. In some embodiments, the height of the compression gap 170 can be about 15%, about 20% about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%, about 65%, about 70%, about 75%, or about 80% an average cell diameter. The average cell size can refer to average of the largest cross-sectional dimension of the cells flowed through the sorting device, and can be calculated using. In some embodiments, the average cell diameter can be measured using a variety of tools now known or later discovered including but not limited to optical microscopy, confocal microscopy, coulter counter, and flow cytometry.
As shown in
The plurality of compressive surfaces 130 may comprise an angle (a), as illustrated at
The angle may be increased or decreased, based on device design. For instance, in some embodiments, the angle can be from 20 to 90 degrees, from 20 to 75 degrees, from 30 to 60 degrees, from 30 to 45 degrees, from 45 to 60 degrees, at least 30 degrees, at least 45 degrees, at least 60 degrees, at least 75 degrees. The angle of each respective compressive surface may also be the same or different along a length of the microfluidic device. In instances where a compressive surface 130 is not linear, the angle can be measured based on a line that is a linear fit to the non-linear ridge.
The number of compressive surfaces 130 in the microchannel 110 can be increased or decreased as desired. In some embodiments, the microchannel 110 can comprise 1 to 100 compressive surfaces 130. In some embodiments, the microchannel 110 can comprise at least 3 compressive surfaces 130, at least 4 compressive surfaces 130, at least 5 compressive surfaces 130, at least 6 compressive surfaces 130, at least 7 compressive surfaces 130, at least 8 compressive surfaces 130, at least 9 compressive surfaces 130, or at least 10 compressive surfaces 130. In some embodiments, the microchannel 110 can comprise up to 100 compressive surfaces 130, up to 75 compressive surfaces 130, up to 50 compressive surfaces 130, or up to 40 compressive surfaces 130. In some embodiments, the microchannel 110 can include 5 to 50 compressive surfaces 130, 7 to 40 compressive surfaces 130, or 7 to 21 compressive surfaces 130. In some embodiments, the microchannel 110 can comprise about 14 compressive surfaces 130.
The plurality of compressive surfaces 130 can be described by a thickness. The thickness can be defined as the linear measurement of the compressive surface in the direction of primary flow. The thickness can be increased or decreased as desired. In some embodiments, the thickness can be from about 7 to about 30 microns, from about 7 to about 20 microns, from about 7 to about 18 microns, from about 7 to about 16 microns, from about 7 to about 11 microns, from about 7 to about 9 microns, from about 20 to about 30 microns, from about 22, to about 28 microns, from about 24 to about 28 microns, from about 18 to about 21 microns, from about 16 to about 22 microns, or from about 8 to about 11 microns. In some embodiments, the thickness can be at least about 9 microns, at least about 11 microns, and at least about 16 microns.
The microchannel 100 can have one or more inlets 140. The one or more inlets 140 may be located on a first side wall of microchannel 100. In some embodiments, the microchannel 100 can have a cell inlet 140 and a sheath flow inlet 145A, 145B. In some embodiments, the cell inlet can be located between a first sheath flow inlet 145A and a second sheath flow inlet 145B, or can be surrounded by a first sheath flow inlet 145Aa. In some embodiments, the cell inlet 140 can be downstream from one or more sheath flow inlets 145A, 145B, or can be aligned with one or more sheath flow inlets 145A, 145B. A sheath fluid can allow for hydrodynamic focusing of the cell medium. The one or more sheath flow inlets 145A, 145B can be located proximate the cell flow inlet 147, or upstream of the cell flow inlet 147. Focusing the cells in the inlet can comprise providing a sheath fluid to the sheath flow inlets 145A, 145B until the sheath fluid reaches laminar flow and then subsequently introducing the cell medium cell medium through the cell inlet 147. The cells can be introduced into the cell inlet 147 by injection, for example by syringe pumps.
The described microchannel 100 can be constructed in a variety of ways. In one exemplary non-limiting embodiment, the microchannel can be made using a replica molding of polydimethylsiloxane (PDMS) on a permanent mold. The mold can be created by two-step photolithography patterning of a photoresist on a 4-inch-diameter silicon wafer. After the removal of PDMS from the mold, inlet and outlet holes can be punched in the side walls of the PDMS, and the PDMS can be subsequently bonded to a glass substrate to form the microfluidic channel. Additionally, in some embodiments as illustrated at
The plurality of cells 180 can be flowed into the microchannel 100 at a flow velocity. The flow velocity of any of the systems and methods described previously can be increased or decreased as desired. As used herein, the flow velocity can describe the velocity of the cell medium at an inlet or at an outlet. The flow velocity can be from about 3 to about 1000 mm/s, from about 3 to about 500 mm/s, from about 3 to about 250 mm/s, from about 3 to about 100 mm/s, from about 3 to about 50 mm/s, from about 3 to about 25 mm/s. The flow velocity can be at least about 3 mm/s, at least about 20 mm/s, at least about 50 mm/s, at least about 100 mm/s, or at least about 500 mm/s. The flow velocity can be about 3 mm/s, about 20 mm/s, about 500 mm/s, or about 1000 mm/s. The flow velocity can also be adjusted as a function of the length of the channel, and/or the size of the relaxation space, based on design preferences. For instance, increasing the length of the channel can allow for a greater flow velocity. Increasing the velocity in similarly sized devices can result in increased pressure within the device. By increasing the length of the microchannel, the increased pressure can be accounted for while permitting higher flow velocity. For instance, increasing the relaxation space can permit increasing the flow velocity as the greater space allows the cells a longer distance over which to travel and be subjected to secondary flow in the ridge channels. As such, increased relaxation space can permit an increased relaxation time and positive lateral displacement for certain cells despite greater flow velocity. In some embodiments, the plurality of cells 180 can be flowed into the microchannel 100 at a flow rate. The flow rate can be 3 to 1500 mm/s.
The microchannel can comprise a plurality of outlets 150a, 150b for collecting portions of the plurality of cells. As illustrated at
Any of the above-described outlets can include a well or chamber for pooling and/or pipetting them in the direction of a chamber or directly to a chamber. In other embodiments, the outlets can be further integrated with additional processing steps, as described below, through an integrated chip or through a capillary. Additionally, after cells are collected, any of the above-mentioned systems and methods can include an additional step of analyzing the cells using any analysis tool now known or later discovered including but not limited, flow cytometry, fluorescence microscopy, functional assays (e.g., apoptosis, cell cycle, viability, proliferation, angiogenesis), spectroscopy, immunoassays, and microplating. Additionally, in some embodiments, the microchannel and cells can be analyzed with electrode counters and microscopy.
As discussed previously, any of the above-described systems and methods can comprise flowing a plurality of cells through the microchannel 100 at a flow velocity. As the cells flow through the microchannel 100, the cells can undergo a convective intracellular delivery process. This process can be characterized by one or more compressions of the plurality of cells followed by a relaxation period. For instance, as described previously, the convective intracellular delivery process can comprise compressing the plurality of cells in a first compression gap, wherein the compressing causes the plurality of cells to undergo a loss in intracellular volume Vloss and passing the plurality of cells to a first relaxation space, wherein the plurality of cells undergo a gain in volume Vgain and absorb a portion of the plurality of molecules. As will be understood by those skilled in the art, depending on the number of compressive surfaces and relaxation spaces, the convective intracellular delivery process can occur one or more times.
Any of the above-described systems and methods can include bulk volume flow across the cell membrane, such that when the cells are compressed by a compressive surface the cells abruptly undergo a compressive force such that cell cytoplasm is transported in bulk volume flow out of the cell. This transfer out of the cell may be characterized as Vloss. In some embodiments, Vloss can be characterized in terms of an initial cell volume before compression. Initial cell volume before compression can be measured using a variety of tools now known or later discovered including but not limited to optical microscopy, confocal microscopy, coulter counter, and flow cytometry. In an embodiment, Vloss may be 30%, 25%, 20%, 15%, or 10% the initial volume of the cell. In some embodiments, Vloss can be at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% the initial volume of the cell. The transfer of volume back into the cell may be characterized as Vgain. In any of the above-described systems and methods, Vgain can be described in terms of Vloss such that Vgain can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 100% Vloss. In any of the above-described systems and methods, Vloss can occur in at least 1 microsecond, at least 2 microseconds, at least 3 microseconds, at least 4 microseconds, at least 5 microseconds, at least 6 microseconds, at least 7 microseconds, at least 8 microseconds, at least 9 microseconds, at least 10 microseconds, at least 15 microseconds, at least 20 microseconds, at least 25 microseconds, at least 30 microseconds, at least 45 microseconds, at least 50 microseconds. Additionally, any of the above-described systems and methods can cause a cell to undergo multiple losses in volume and multiple gains in volume based on the number of compressive surfaces. In some embodiments, a cell may regain 100% Vloss in from 1 to 100 ms, from 4 to 100 ms, from 10 to 100 ms, from 15 to 100 ms, from 20 to 100 ms, from 25 to 100 ms, from 30 to 100 ms, from 40 to 100 ms, from 50 to 100 ms, from 60 to 100 ms, from 75 to 100 ms, from 80 to 100 ms, and from 90 to 100 ms, Additionally, it is contemplated that the time taken to regain 100% Vloss may vary with the cell type, therefore the time may be more or less depending on the type of cell. Therefore, the plurality of cells may undergo Vloss1, Vloss2, Vloss3, Vloss7, Vloss14, Vloss21, up to VlossN. As such the plurality of cells may undergo Vgain1 , Vgain2, Vgain3, Vgain7, Vgain14, Vgain1, up to VgainN depending on the number of relaxation spaces which corresponds to the number of compressive surfaces.
Additionally, the convective intracellular delivery process of any of the above-described methods can include intracellular delivery of extracellular molecules. Molecules may comprise a variety of entities including but not limited to particles, macromolecules, nanoparticles, dextran, plasmids, mRNA, or beads.
While the above-mentioned embodiments are described with respect to compressing cells within a microchannel, it is understood that any of the above-described systems and methods can obtain transient volume change through other approaches including inertial contact of a cell with a wall (including slamming, thrusting, or otherwise forcing contact between a cell and a wall), rapid compressions with non-solid force fields including fluid or acoustic fields.
The present disclosure is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the disclosure may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. The disclosure is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
The microfluidic device design used constrictions in the form of angled ridges in a single large channel for the rapid processing of high numbers of cells. The large channel allowed a multitude of cells to pass simultaneously under each ridge while hydrodynamic drag forces maintained cell velocity through the constrictions, allowing cell processing to continue rapidly even after many constrictions. The angled ridges also served as an escape mechanism for nonviable cells and cell aggregates that would otherwise clog the device or dilute the processed cell population. Therefore, this design functioned effectively even with localized clogs, and rapidly self-cleared. A multi-channel design of this device successfully processed 50 million cells in 10 minutes without clogging.
The microfluidic features of this device were molded onto polydimethylsiloxane (PDMS) and plasma bonded to a glass slide. A reusable SU-8 mold was made using standard two-step photolithography on a silicon wafer. Constriction gaps of 50-60% of the average relaxed cell diameter (14.5±1.5 μm) were used for optimal delivery, but gaps of 40-130% of the average cell diameter were also studied. The cell inlet flow directed cells through the constrictions, preventing the cells from preferentially flowing around the ridges without compressing. To fabricate the devices, a 10:1 ratio of PDMS and crosslinking agent was mixed and poured onto the SU-8 mold to form the microfluidic channel features by replica molding. The PDMS was then degassed in a vacuum chamber and cured for 6 hrs at 60° C. The cooled PDMS was then removed from the molds and outlets and inlets were punched using biopsy punches. The PDMS was then bonded to sonicated glass slides using a plasma bonder (PDC-32G Harrick) followed by 1 hr in a 60° C. oven. After cooling, the channels were passivated using 1% bovine serum albumin (BSA) for an overnight incubation at 4° C.
K562 cells (CCL-243) from ATCC were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. PC3 prostate cancer cells (CRL-1435) were cultured in F-12K with 10% FBS and 1% penicillin-streptomycin. The cells were incubated at 37° C. with 5% CO2. The PC3 cells were passaged using 0.25% Trypsin-EDTA. Primary leukocytes were isolated from whole donor blood by density gradient centrifugation. Whole donor blood was centrifuged at 700 RCF for 10 mins with Ficoll density centrifugation media and the concentrated leukocyte band (buffy coat) was collected.
A cell flow buffer consisting of DPB S (-/-) with 0.1% BSA, 0.04% EDTA, and trace Tween 20 was used to maintain a single-cell suspension throughout the experiment. Experiments performed using pure DPBS (-/-) and serum-free RPMI-1640 without BSA, EDTA, or Tween determined that these agents had no observable effect on molecular delivery. Transfection and RNA probe delivery experiments were done using Opti-MEM and serum-free RPMI-1640, respectively. The cells were isolated from culture media and resuspended in buffer at ˜5×106 cells/mL with the desired concentration of target molecules. The cell-buffer suspension was infused into the microfluidic device at a controlled rate using syringe pumps. Following collection from the outlets, the cells were washed 2× with DPBS (-I-) to remove residual molecules external to the cells.
The experiments were carried out on the stage of an inverted bright-field microscope (Eclipse Ti, Nikon), with a high-speed camera attachment (Phantom v7.3, Vision Research). High speed (˜5,000 fps) videos were taken of cells during processing at various segments of the device.
To measure the cell volume inside the device, measurements were taken of the cell area from video data and applied volume assumptions based on a cell deformation model. A custom cell tracking algorithm was used to automatically track the trajectory and area of cells in the video, with manual measurements used to verify. For each tracked cell, the algorithm identified all video frames where the cell was visible, and extracted the position and number of pixels it occupied (area). For each manual measurement, the ellipse that fit to the pixels of the sharpest gray scale intensity gradient to represent the maximum projected cell boundary was taken. The length scales of each image were calibrated based on known ridge dimensions, which enabled translation of the number of pixels into an area measure. For each cell, the area before it entered the ridge region of the device was measured to determine its uncompressed volume and the area when completely under each ridge to determine the compressed volumes. The volume of the unperturbed cell was taken as an ellipsoid where the projected area of the ellipse was revolved about the major axis, resulting in the minimum reasonable volume for the unperturbed cell. To take the compressed cell measurements, the same revolved ellipsoid procedure was applied to the compressed cell area and cut equal caps that represent the volume of the ellipsoid that intersected with the constraints of the ridge and channel bottom. This was considered the maximum reasonable volume for the compressed cell as it approached the cylindrical case for smaller gap sizes and collapsed back to the unperturbed ellipsoid case for larger gap sizes.
The BD Accuri C6 Flow Cytometer was used to characterize cell uptake of fluorescent target molecules. Samples processed with FITC-dextran or GFP RNA or plasmid were excited with a 488 nm wavelength laser and emission was detected with a 533/30 filter. Samples with cyanine-3 were excited with a 488 nm laser and detected by a 585/40 filter. The viability of the cells was tested by staining with 2 μM EthD-1 solution 34, 35. EthD-1 stained cells were excited at 640 nm and detected with a 670 LP filter.
Confocal microscopy of cells with tetramethylrhodamine (TRITC)-dextran was performed using the Zeiss LSM 700 to determine the intracellular localization of the delivered dextran molecules. The Zeiss 710 NLO with a 40× water lens was used to image K562 cells with Cy3-plasmid and 100 nm nanoparticles. These cells were stained with DiO membrane stain and Hoechst nucleus stain per manufacturer protocol.
Measurements of the viscous relaxation of individual cells during repeated compressions were performed using an MFP-3D AFM in concert with an inverted optical microscope (Nikon Ti) to optically align the AFM probe with the center of each cell. The probes used in this study were MLCT-O10-D probes with a nominal spring constant of 0.03 N/m. The AFM cantilever interacted with the cells via a 15 μm diameter PMMA microsphere. Cantilever calibration was performed using the thermal vibration method against a glass surface. K562 cells in culture media were adhered to the surface of a glass Fluorodish using Cell-Tak. The indentation depth was chosen to be 10 μm to simulate the strain imposed by a 5 μm gap in a microfluidic channel. The cell relaxation constant was extracted from the decay of viscous forces acting on the probe while maintaining constant indentation for 2 seconds after compression.
Cell deformation was caused by microfluidic flow through ridges with rectangular cross-section that were repeated within a microchannel to precisely exert abrupt and brief compressions upon cells. Hydrodynamic forces maintained high cell velocity throughout multiple constrictions, while the angled ridges removed dead cells and clusters of cells which could cause occlusions.
Using a computational cell deformation model combined with area analysis of high speed videos of individual cells in the microfluidic channel, the change in cell volume was evaluated at several points in the channel (
From the known gap and modeled cell shape, the cell volume before and during compressions was determined. An overlay of cell area measurements at the various positions shows subtle area change, suggesting that the vertical constraint from the ridge mainly accounts for the volume change (
While the volume was observed to decrease by up to 30% during compressions, cells were quickly restored to their initial size with little impact on cell integrity, viability, and related gene expression. After microfluidic processing, cell culturing and expansion was successfully conducted with no change in cell growth rate. Flow cytometry analysis <30 min after processing showed that the compression experiments have negligible impact on forward scatter measurements of cells (
The volume reduction of compressed cells indicated that a portion of cytosol was expelled from the cell interior. Cell volume recovery, on the other hand, requires extracellular fluid to enter the cell. Since the video analysis does not allow for evaluation of cell volume in between the ridges, the dynamics of volume exchange was further characterized by transmembrane fluid transfer using fluorescently labeled dextran (Sigma-Aldrich) as a tracker molecule. Dextran of various sizes was added to the cell suspension immediately before compression experiments. It was hypothesized that the cell relaxations after each compression will cause the extracellular fluid to enter the cell interior transporting dispersed fluorescent molecules, and that the molecules will partially remain in the cell interior after consecutive compressions serving as an indicator of volume exchange.
Confocal imaging determined that molecular delivery by the presently-disclosed systems and methods was dispersed throughout the cell interior, suggesting non-endocytic delivery (
Based on the correlation between volume loss and molecule delivery, it was hypothesized that altering the time that the cell relaxes as it moves between consecutive constrictions can affect the volume uptake and, therefore, molecular delivery. The relaxation time between ridges was controlled either by varying the ridge spacing or the flow rate. It was observed that that increased flow rate resulted in decreased delivery, while the 200 μm spacing between ridges consistently resulted in higher delivery than the 100 μm spacing (
Therefore, the increased relaxation time between ridges led to greater delivery (
To further confirm that intracellular delivery occurs due to cell volume change, the methods were tested to see if they were affected by the size of the molecule. Since diffusion rate is inversely proportional to molecule size, diffusive delivery typically shows lower efficiency for larger macromolecules. In contrast, the described methods demonstrated intracellular delivery with high efficiency (˜90% of cells uptake molecules) regardless of molecule size for the range tested (
The use of multiple ridges greatly increased volume exchange and molecular delivery to the cells. A positive and non-linear correlation between the number of ridges and molecule delivery was observed, which saturated at 14 ridges for these experimental conditions (
To further explore the hypothesis that the described systems and methods causes the cytosol to reach equilibrium with extracellular molecule concentration, the previously dextran-positive cells were processed with dextran-free buffer to remove the dextran from within the cells. 2000 kDa FITC-dextran was first delivered to K562 cells using the described systems and methods, then resuspended these delivered cells in FITC-free buffer and processed them in the device again for the Removal group. It was discovered that the Removal group had a mean fluorescence intensity that matches the No Device group, indicating that this method is highly effective in removing previously delivered molecules (
To determine the time scale at which delivery occurs during the described systems and methods, an experiment was designed to analyze the relative amount of delivery that occurs during the brief time (<0.1 s) of cell compressions inside the device channel and immediately after leaving the device. Delivery inside the channel was determined by flowing K562 cells through the channel with the target delivery molecules, 2000 kDa FITC-dextran, and then inhibiting delivery after the channel by immediately diluting the outlet sample into a molecule-free bath. Delivery after the channel was isolated by flowing cells through the channel in the absence of target molecules, then exposing the cells to a molecule-rich bath immediately after leaving the channel. Molecules were delivered to over 80% of cells during their <0.1 s transit through the channel, while only ˜33% of cells exhibited delivery when provided dextran immediately after transit through the compressions, even after incubation in the outlet well for >10 minutes. A threshold of 10% of the No Device control was used to define the lower bound of fluorescence for positive delivery (
To better understand the relationship between volume change and intracellular delivery, a simple mathematical model of molecular delivery due to repeated volume exchange events was constructed. The model assumes the cell interior and exterior are well mixed and incompressible liquids. Therefore, as cells are compressed to volume VC under the ridge, a corresponding volume of liquid exits the cell carrying out a mass of target molecules dictated by the intracellular concentration of that species. Conversely, as a cell recovers lost volume, any species outside the cell are drawn in at a rate set by their external concentration and the time-dependent cell volume recovery V(t) (
Cell volume recovery was modeled after each compression as V(t)=(Vc−V0)·eτ+V0, where V0 is the original (uncompressed) volume of the cell, Vc is the cell volume under a ridge (which is assumed to be independent of ridge number), τ is the time for cells to recover ˜66.7% of lost volume, and t is the amount of time that has passed after the most recent compression. The cell with volume Vc is approximated to be a truncated sphere with height equal to the ridge gap. Based on the experimental results (
For the variable relaxation model, relaxation time τ(Z)=(τ0−τ∞)·e−ζZ+τ∞ is a function of the number of compressions (Z), the cell initial relaxation constant (τ0), and final relaxation constant (τ∞) characterizing relaxation after many compressions. When fit to experimental data for molecule delivery, τ decreases with increasing number of ridges, as represented by the decay constant ζ.
To calculate the amount of molecules delivered to a cell after a certain number of ridges, the contribution of each ridge was considered in order. The cell increase in volume between the first two ridges was then calculated as ΔV
inc=V(t=t_transit,z=0)-V_c, where t_transit is the time it takes the cell to travel between the two ridges, as calculated by the ridge spacing and fluid flow rate. The amount of molecules taken up by the cell between the two first ridges is then given as
Δn
_gain=C_0·inc, where C0 is the external concentration of molecules. As the cell encounters the second ridge, it is compressed to Vc and some amount of molecules is forced out of the cell
Δn
_loss=n/V(t=t_transitz=0)·
ΔV
_inc where n is the total amount of molecules in the cell at the current ridge. This procedure is then repeated for each subsequent ridge (while incrementing z) to determine the intracellular concentration after multiple compressions.
To allow comparison between the model and experimental data, values of τ0, τ∞, and ζ, were estimated by performing a nonlinear regression against the experimental data presented in
A model was considered in which cells behave as a Kelvin-Voit viscoelastic material and expand after compression to exponentially to approach their original volume V0. The asymptotic recovery was expressed using an exponential function V(t)=(Vc−V0)·e−t/τ30 V0 where t is the time elapsed after the most recent compression (
A model in which volume exchange increases with consecutive compressions (
The application of the described systems and methods can address important limitations of microfluidic delivery platforms, particularly those that primarily use diffusive transport. To demonstrate the capabilities of the use of the described systems and methods as a highly efficient delivery platform for transfection agents, non-coding plasmids labeled with cyanine-3 (Minis) into K562 cells were successfully delivered. The cells were stained with DiO membrane stain and Hoechst nucleus stain to visualize the intracellular localization of the Cy3-plasmids (
The described systems and methods were tested for potential applications for intracellular labeling and analysis by delivering SmartFlare Live Cell RNA probes (Millipore) to detect GAPDH RNA in K562 cells and adherent PC3 prostate cancer cells. Delivery to PC3 cells was competitive with the established method of 24 hr endocytosis, and was completed in less than 30 mins (
100 nm diameter fluorescent beads were also successfully delivered to K562 cells as a demonstration of this method's ability to deliver extremely large particles (
By using microfluidics to precisely induce rapid, brief, large strain compressions, surprising phenomenon of temporary cell volume exchange that maintains cell integrity, viability, and function was elucidated. A behavior wherein cells initially undergo sudden volume loss followed by fast volume recovery was discovered. Additionally, it was discovered that induced volume change is greater for larger strains imposed through smaller constrictions. It was also found that increased volume exchange required multiple ridges spaced such that there was sufficient time for cells to recover lost volume between each ridge. This effect of volume change and relaxation was used as a new approach to deliver molecules to cells. Specifically, rapid compression-driven volume loss worked in conjunction with cell relaxation to convectively drive volume and molecules into the cell interior.
The physical cause of this surprising cell behavior can be explained by considering the relevant forces imposed on the cell by the ridges. The sudden inertial compression under a ridge with stepwise profile is equivalent to a high velocity (˜1 m/s) vertical impact on the cell to disrupt the membrane in a manner akin to a droplet splatter upon a surface. The subsequent physical constriction of the cell under the ridge results in rapid transfer of momentum to the liquid of the cell interior to drive fluid volume out of the cell. The brief nature of this compression causes cells to relax on a rapid time scale to uptake volume after compression. The observed rapid recovery is consistent with rapid, poroelastic recovery behavior of the cytoplasm at short time scales (<0.5 s) after brief compression. The ability of the cytoskeleton to regulate cell volume and retain solutes could explain the minimal impact of the described systems and methods on cell viability despite the initial volume loss.
In the described studies, it was found that the described systems and methods utilize an advection-dominated molecular driving mechanism to efficiently deliver molecules of a wide range of sizes and structures for many cell types, while maintaining high viability. The microfluidic approach avoids many of the prohibitive drawbacks of detrimental changes to cell state associated with using chemical, viral, or electrical processing. The simplicity of use and successful delivery of an array of biologically relevant macromolecules to various cell types demonstrated great potential for a wide range of highly valuable biomedical applications.
In this study, a new cell behavior was discovered wherein multiple, rapid, high strain compressions caused cell volume change and relaxation without impacting cell viability. It was found that this volume exchange caused extracellular molecules to be convected into the cell interior. The described systems and methods enable new applications for microfluidic molecular delivery, including high-throughput delivery of large macromolecules and particles. The described systems and methods have elucidated a new cell phenomenon with great potential to serve as a nearly universal intracellular delivery platform for a variety of biotechnology applications.
The described systems and methods were also shown to be distinct from current diffusive mechanoporation platforms, both in mechanism and capability. Diffusive microfluidic mechanoporation methods used gradual constrictions to impart shear stress on cells in a manner that facilitates smooth cell flow and thus slower deformation. The compression creates a shear force on the cell membrane leading to membrane poration and extracellular molecular diffusion into the cell interior. While diffusion is a universal transport mechanism, it imposes constraints on delivery due to the inverse relationship between diffusivity and molecule size. Indeed, diffusive approaches to microfluidic mechanoporation have shown limited efficiency in the delivery of large macromolecules.
A microchannel having a 10.2 micrometer compression gap was used to compare the presently described systems and methods with a diffusive delivery approach.
For instance, as illustrated in
Additionally, it was shown that the described systems and methods result in nearly complete homogenization attained by repeat volume exchange and complete removal, as shown in
Further, the above-described systems and methods include improved clog prevention. Angled ridge design automatically and rapidly sorts out and removes large cell aggregates, non-viable cells, large cells, and non-processed cells.
While several possible embodiments are disclosed above, embodiments of the present disclosure are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the disclosure, but instead were chosen and described in order to explain the principles of the present disclosure so that others skilled in the art may practice the disclosure. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
The embodiments of the present disclosure are also not limited to the particular formulations, process steps, and materials disclosed herein as such formulations, process steps, and materials may vary somewhat. Further, the terminology employed herein is used for the purpose of describing exemplary embodiments only and the terminology is not intended to be limiting since the scope of the various embodiments of the present disclosure will be limited only by the appended claims and equivalents thereof.
The specific configurations, choice of materials, and the size and shape of various elements can be varied according to particular design specifications or constraints requiring a device, system, or method constructed according to the principles of the disclosure. Such changes are intended to be embraced within the scope of the disclosure. The presently disclosed embodiments, therefore, are considered in all respects to be illustrative and not restrictive, and those skilled in the art will understand that variations and modifications can be effected within the scope of the disclosure as defined in the appended claims. The scope of the disclosure is therefore indicated by the following claims, rather than the foregoing description and above-discussed embodiments, and all changes that come within the meaning and range of equivalents thereof are intended to be embraced therein.
This is a divisional application of U.S. application Ser. No. 16/348,170 filed 8 May 2019, which application Ser. No. 16/348,170 is a National Stage Entry of International Application No. PCT/US17/60644 filed 8 Nov. 2017, which Application No. PCT/US17/60644 claims the benefit of U.S. Provisional Application No. 62/419,041 filed 8 Nov. 2016, each of which is incorporated herein by reference in its entirety as if fully set forth below.
| Number | Date | Country | |
|---|---|---|---|
| 62419041 | Nov 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16348170 | May 2019 | US |
| Child | 17524168 | US |
