Intracellular delivery of biomolecules mediated by porous surfaces

Abstract

Providing intracellular delivery of biomolecules mediated by a surface having pores. [Solution] The present disclosure relates to a method and device for delivering a compound into a cell, comprising the step of passing a cell suspension through a surface containing pores, wherein the pores deform the cell, thereby causing a cellular perturbation that allows the compound to enter the cell, and the cell suspension comes into contact with the compound. The surface may be a filter, a membrane, or a microsieve. The cellular perturbation may be a cellular tear that allows a substance from outside the cell to move into the cell.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority under U.S. Provisional Application No. 62 / 214,820, filed on 4 September 2015, and U.S. Provisional Application No. 62 / 331,363, filed on 3 May 2016, all of which are incorporated herein by reference in their entirety.

[0002] Field of Invention This disclosure generally relates to a method for delivering compounds into cells by passing a cell suspension through a pore-containing surface. [Background technology]

[0003] background Intracellular delivery is a central step in the research and development of novel therapeutics. Existing technologies aimed at intracellular delivery of molecules rely on electric fields, nanoparticles, or pore-forming chemicals. However, these methods suffer from numerous cumbersome problems, including nonspecific molecular delivery, modification or damage of payload molecules, high cell death, use of substances that may not be considered generally safe (GRAS) substances, low throughput, and / or difficulty of implementation. Furthermore, these intracellular delivery methods are not effective in delivering molecules to susceptible cell types such as primary immune cells and stem cells. Thus, there is a need for intracellular delivery techniques that are highly effective in delivering a given range of molecules to various cell types, but this need remains unmet. Moreover, techniques that enable rapid, high-throughput intracellular delivery can be more effectively applied to large-scale clinical, manufacturing, and drug screening applications. References describing methods using channels to deliver compounds to cells include International Publication Nos. 2013 / 059343 and International Publication Nos. 2015 / 023982. All references cited herein, including patent applications and publications, are incorporated in their entirety as references. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2013 / 059343 [Patent Document 2] International Publication No. 2015 / 023982 [Overview of the project]

[0005] Brief summary of the invention Certain embodiments of this disclosure include a method for delivering a compound into cells, comprising the step of passing a cell suspension through a surface containing pores, wherein the pores deform the cells, thereby causing a perturbation of the cells so that the compound enters the cells, and the cell suspension comes into contact with the compound. In some embodiments, the surface is a membrane; in some embodiments, the surface is a filter; in some embodiments, the surface is a microsieve; in some embodiments, the surface is the surface of a meandering pathway; in some embodiments, the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, and ceramic. In some embodiments, the entrance to the pores is wider than the pores, narrower than the pores, or the same width as the pores. In some embodiments, the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, micropore punching, polymer sponge, direct foam molding, extrusion, and hot embossing.

[0006] In some embodiments that can be combined with previous embodiments, the pore size correlates with the cell diameter. In some embodiments, the pore size is about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cell diameter. In some embodiments, the width of the surface cross-section is in the range of about 1 mm to about 1 m. In some embodiments, the width of the pore cross-section is in the range of about 0.01 μm to about 300 μm. In some embodiments, the width of the pore cross-section is in the range of about 0.01 to about 35 μm. In some embodiments, the pore size is about 0.4 μm, 4 μm, 5 μm, 8 μm, 10 μm, 12 μm, or about 14 μm. In some embodiments, the pore size is about 200 μm. In some embodiments, the pore size is heterogeneous. In some embodiments, the size of the heterogeneous pores varies within a range of 10 to 20%. In some embodiments, the size of the pores is homogeneous.

[0007] In some embodiments, which can be combined with previous embodiments, the pores have the same inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the shape of the cross-section of the pore is selected from annular, circular, quadrilateral, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal shapes. In some embodiments, the shape of the cross-section at the inlet to the pore is different from the shape of the cross-section at the outlet from the pore (e.g., annular at the inlet and quadrilateral at the outlet). In some embodiments, the shape of the cross-section of the pore is selected from cylindrical or conical shapes. In some embodiments, the edges of the pore are smooth. In some embodiments, the edges of the pore are sharp. In some embodiments, the passage of the pore is straight. In some embodiments, the passage of the pore is curved. In some embodiments, the pores constitute about 10 to 80% of the total surface area. In some embodiments, the surface totals about 1.0 × 10 5 From approximately 1.0 x 10 30 It contains 1 pore. In some embodiments, the surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 1015 It contains 100 μm of pores. In some embodiments, the pores are distributed in parallel. In some embodiments, the surfaces having the pores are stacked on top of each other. In some embodiments, the surfaces are distributed in series. In some embodiments, the distribution of the pores is regular. In some embodiments, the distribution of the pores is random. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is not uniform. In some embodiments, the surface is about 0.01 μm to about 5 μm thick. In some embodiments, the surface is about 10 μm thick. In some embodiments, the surface is less than 1 μm thick.

[0008] In some embodiments, which can be combined with previous embodiments, the surface is coated with a material. In some embodiments, the material is Teflon®. In some embodiments, the material includes an adhesive coating that binds to cells. In some embodiments, the material includes a surfactant. In some embodiments, the material includes an anticoagulant. In some embodiments, the material includes a protein. In some embodiments, the material includes an adhesion molecule. In some embodiments, the material includes an antibody. In some embodiments, the material includes a factor that modulates cellular function. In some embodiments, the material includes a nucleic acid. In some embodiments, the material includes a lipid. In some embodiments, the material includes a carbohydrate. In some embodiments, the material includes a complex. In some embodiments, the complex is a lipid-carbohydrate complex. In some embodiments, the material includes a transmembrane protein. In some embodiments, the material is covalently bonded to the surface. In some embodiments, the material is non-covalently bonded to the surface. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged.

[0009] In some embodiments, which can be combined with previous embodiments, the cell suspension comprises mammalian cells. In some embodiments, the cell suspension comprises a mixed population of cells. In some embodiments, the cell suspension is whole blood. In some embodiments, the cell suspension is lymph. In some embodiments, the cell suspension comprises peripheral blood mononuclear cells. In some embodiments, the cell suspension comprises a purified population of cells. In some embodiments, the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, erythrocytes, or platelets. In some embodiments, the immune cells are lymphocytes, T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. In some embodiments, the cells are cells from a mouse, dog, cat, horse, rat, goat, or rabbit. In some embodiments, the cells are human cells.

[0010] In some embodiments that can be combined with previous embodiments, the compound comprises a nucleic acid. In some embodiments, the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. In some embodiments, the compound is a peptide nucleic acid. In some embodiments, the compound comprises a transposon. In some embodiments, the compound is a plasmid. In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound comprises a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid encoding a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound comprises a nuclease, TALEN protein, zinc finger nuclease, meganuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase. In some embodiments, the compound is an antibody. In some embodiments, the compound is a transcription factor. In some embodiments, the compound is a small molecule. In some embodiments, the compound is a nanoparticle. In some embodiments, the compound is a chimeric antigen receptor. In some embodiments, the compound is a fluorescently tagged molecule. In some embodiments, the compound is a liposome. In some embodiments, the compound is a virus.

[0011] In some embodiments, which can be combined with previous embodiments, the cell suspension comes into contact with the compound either simultaneously with, or after, its passage through the pores. In some embodiments, the delivered compound is coated on the surface. In some embodiments, the method is performed between 0°C and 45°C. In some embodiments, the cells pass through the pores by positive or negative pressure. In some embodiments, the cells pass through the pores by constant or variable pressure. In some embodiments, the pressure is applied using a syringe. In some embodiments, the pressure is applied using a pump. In some embodiments, the pressure is applied using decompression. In some embodiments, the cells pass through the pores by capillary pressure. In some embodiments, the cells pass through the pores by hydrostatic pressure. In some embodiments, the cells pass through the pores by blood pressure. In some embodiments, the cells pass through the pores by g-force. In some embodiments, the cells pass through the pores under pressures ranging from about 0.05 psi to about 500 psi. In some embodiments, the cells pass through the pores under pressures of about 2 psi. In some embodiments, the cells pass through the pores under pressures of about 2.5 psi. In some embodiments, the cells pass through the pores under pressures of about 3 psi. In some embodiments, the cells pass through the pores under pressures of about 5 psi. In some embodiments, the cells pass through the pores under pressures of about 10 psi. In some embodiments, the cells pass through the pores under pressures of about 20 psi. In some embodiments, the cells pass through the pores by fluid flow. In some embodiments, the fluid flow is turbulent before the cells pass through the pores. In some embodiments, the fluid flow through the pores is laminar. In some embodiments, the fluid flow is turbulent after the cells have passed through the pores. In some embodiments, the cells pass through the pores at a uniform cell velocity. In some embodiments, the cells pass through the pores at a variable cell velocity. In some embodiments, the cells pass through the pores at a velocity ranging from about 0.1 mm / second to about 20 m / second. In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe.

[0012] In some embodiments that can be combined with previous embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization. In some embodiments, the agents that affect actin polymerization are latruncrin A, cytochalasin, and / or colchicine. In some embodiments, the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. In some embodiments, the viscosity of the cell suspension is about 8.9 × 10⁻⁶ -4 From Pa·seconds to approximately 4.0 × 10-3 The range is in the Pa·second range. In some embodiments, the method further includes the step of passing the cells through an electric field generated by at least one electrode adjacent to the surface.

[0013] Certain embodiments of the present disclosure include a device for delivering compounds into cells, comprising a surface containing pores, wherein the pores are configured to allow cells suspended in a solution to pass through, and the pores cause perturbation of the cells, thereby allowing the compounds to enter the cells. In some embodiments, the surface is a membrane. In some embodiments, the surface is a filter. In some embodiments, the surface is the surface of a meandering pathway. In some embodiments, the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, and ceramic. In some embodiments, the entrance to the pores is wider than the pores, narrower than the pores, or the same width as the pores. In some embodiments, the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, microporous punching, polymer sponge, direct foam molding, extrusion, and hot embossing.

[0014] In some embodiments that can be combined with the previous embodiments, the size of the pores correlates with the cell diameter. In some embodiments, the size of the pores is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. In some embodiments, the width of the cross-section of the surface ranges from about 1 mm to about 1 m. In some embodiments, the width of the cross-section of the pores ranges from about 0.01 μm to about 300 μm. In some embodiments, the width of the cross-section of the pores ranges from about 0.01 to about 35 μm. In some embodiments, the size of the pores is about 0.4 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm. In some embodiments, the size of the pores is about 200 μm. In some embodiments, the size of the pores is heterogeneous. In some embodiments, the size of the heterogeneous pores varies in the range of 10 to 20%. In some embodiments, the size of the pores is homogeneous.

[0015] In some embodiments that can be combined with the previous embodiments, the pores have the same inlet angle and outlet angle. In some embodiments, the pores have different inlet angles and outlet angles. In some embodiments, the cross-sectional shape of the pores is selected from the shapes of circular, square, star, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-sectional shape of the pores is selected from cylindrical or conical. In some embodiments, the edges of the pores are smooth. In some embodiments, the edges of the pores are sharp. In some embodiments, the passage of the pores is straight. In some embodiments, the passage of the pores is curved. In some embodiments, the pores constitute about 10 to 80% of the total surface area. In some embodiments, the surface has a total of about 1.0×10 5 to about 1.0×10 30 pores. In some embodiments, the surface has about 10 to about 1.0×10 2 per square millimeter of surface area 15It contains 100 μm of pores. In some embodiments, the pores are distributed in parallel. In some embodiments, the surfaces having pores are stacked on top of each other. In some embodiments, the surfaces are distributed in series. In some embodiments, the distribution of the pores is regular. In some embodiments, the distribution of the pores is random. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is not uniform. In some embodiments, the thickness of the surface is from about 0.01 μm to about 5 μm. In some embodiments, the thickness of the surface is about 10 μm.

[0016] In some embodiments, which can be combined with previous embodiments, the surface is coated with a material. In some embodiments, the material is Teflon®. In some embodiments, the material includes an adhesive coating that binds to cells. In some embodiments, the material includes a surfactant. In some embodiments, the material includes an anticoagulant. In some embodiments, the material includes a protein. In some embodiments, the material includes an adhesion molecule. In some embodiments, the material includes an antibody. In some embodiments, the material includes a factor that modulates cellular function. In some embodiments, the material includes a nucleic acid. In some embodiments, the material includes a lipid. In some embodiments, the material includes a carbohydrate. In some embodiments, the material includes a complex. In some embodiments, the complex is a lipid-carbohydrate complex. In some embodiments, the material includes a transmembrane protein. In some embodiments, the material is covalently bonded to the surface. In some embodiments, the material is non-covalently bonded to the surface. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged.

[0017] In some embodiments, which can be combined with previous embodiments, the cell suspension comprises mammalian cells. In some embodiments, the cell suspension comprises a mixed population of cells. In some embodiments, the cell suspension is whole blood. In some embodiments, the cell suspension is lymph. In some embodiments, the cell suspension comprises peripheral blood mononuclear cells. In some embodiments, the cell suspension comprises a purified population of cells. In some embodiments, the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or erythrocytes. In some embodiments, the immune cells are lymphocytes, T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. In some embodiments, the cells are cells from a mouse, dog, cat, horse, rat, goat, or rabbit. In some embodiments, the cells are human cells.

[0018] In some embodiments, which can be combined with previous embodiments, the compound comprises a nucleic acid. In some embodiments, the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. In some embodiments, the compound is a peptide nucleic acid. In some embodiments, the compound comprises a transposon. In some embodiments, the compound is a plasmid. In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound comprises a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid encoding a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound comprises a nuclease, TALEN protein, zinc finger nuclease, meganuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase. In some embodiments, the compound is an antibody. In some embodiments, the compound is a transcription factor. In some embodiments, the compound is a small molecule. In some embodiments, the compound is a nanoparticle. In some embodiments, the compound is a chimeric antigen receptor. In some embodiments, the compound is a fluorescently tagged molecule. In some embodiments, the compound is a liposome. In some embodiments, the compound is a virus.

[0019] In some embodiments that can be combined with previous embodiments, the cell suspension comes into contact with the compound either simultaneously with or after passing through the pores. In some embodiments, the delivered compound is coated on the surface. In some embodiments, the device is between 0°C and 45°C. In some embodiments, the cells pass through the pores by positive or negative pressure. In some embodiments, the cells pass through the pores by constant or variable pressure. In some embodiments, the pressure is applied using a syringe. In some embodiments, the pressure is applied using a pump. In some embodiments, the pressure is applied using decompression. In some embodiments, the cells pass through the pores by capillary pressure. In some embodiments, the cells pass through the pores by blood pressure. In some embodiments, the cells pass through the pores by g-force. In some embodiments, the cells pass through the pores under pressures ranging from about 0.05 psi to about 500 psi. In some embodiments, the cells pass through the pores under pressures of about 2 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 2.5 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 3 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 5 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 10 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 20 psi. In some embodiments, the cells pass through the pores by fluid flow. In some embodiments, the fluid flow is turbulent before the cells pass through the pores. In some embodiments, the fluid flow through the pores is laminar. In some embodiments, the fluid flow is turbulent after the cells have passed through the pores. In some embodiments, the cells pass through the pores at a uniform cell velocity. In some embodiments, the cells pass through the pores at a fluctuating cell velocity. In some embodiments, the cells pass through the pores at a speed ranging from about 0.1 mm / second to about 20 m / second.In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe.

[0020] In some embodiments, which can be combined with previous embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization. In some embodiments, the agents that affect actin polymerization are latruncrin A, cytochalasin, and / or colchicine. In some embodiments, the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. In some embodiments, the viscosity of the cell suspension is about 8.9 × 10⁻⁶ -4 From Pa·seconds to approximately 4.0 × 10 -3 The range is in the Pa·second range. In some embodiments, the device includes a plurality of surfaces. In some embodiments, the surfaces are transwells. In some embodiments, at least one electrode is located in close proximity to the surface and generates an electric field.

[0021] Certain embodiments of this disclosure include cells containing perturbations, which are generated by passing the cells through a surface containing pores, causing the pores to deform the cells, thereby generating perturbations that allow compounds to enter the cells. In some embodiments, the surface is a membrane. In some embodiments, the surface is a filter. In some embodiments, the surface is the surface of a meandering pathway. In some embodiments, the surface includes a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, and ceramic. In some embodiments, the entrance to the pores is wider than the pores, narrower than the pores, or the same width as the pores. In some embodiments, the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, microporous punching, polymer sponge, direct foam molding, extrusion, and hot embossing.

[0022] In some embodiments, which can be combined with previous embodiments, the pore size correlates with the cell diameter. In some embodiments, the pore size is about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cell diameter. In some embodiments, the width of the surface cross-section is in the range of about 1 mm to about 1 m. In some embodiments, the width of the pore cross-section is in the range of about 0.01 μm to about 300 μm. In some embodiments, the width of the pore cross-section is in the range of about 0.01 to about 35 μm. In some embodiments, the pore size is about 0.4 μm, 4 μm, 5 μm, 8 μm, 10 μm, 12 μm, or about 14 μm. In some embodiments, the pore size is about 200 μm. In some embodiments, the pore size is heterogeneous. In some embodiments, the heterogeneous pore size varies within a range of 10 to 20%. In some embodiments, the pore size is homogeneous.

[0023] In some embodiments that can be combined with previous embodiments, the pores have the same inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the shape of the cross-section of the pores is selected from annular, circular, quadrilateral, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal shapes. In some embodiments, the shape of the cross-section of the pores is selected from cylindrical or conical shapes. In some embodiments, the edges of the pores are smooth. In some embodiments, the edges of the pores are sharp. In some embodiments, the passages of the pores are straight. In some embodiments, the passages of the pores are curved. In some embodiments, the pores constitute about 10 to 80% of the total surface area. In some embodiments, the surface totals about 1.0 × 10 5 From approximately 1.0 x 10 30 It contains 1 pore. In some embodiments, the surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 It contains 100 μm of pores. In some embodiments, the pores are distributed in parallel. In some embodiments, the surfaces having pores are stacked on top of each other. In some embodiments, the surfaces are distributed in series. In some embodiments, the distribution of the pores is regular. In some embodiments, the distribution of the pores is random. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is not uniform. In some embodiments, the thickness of the surface is from about 0.01 μm to about 5 μm. In some embodiments, the thickness of the surface is about 10 μm.

[0024] In some embodiments, which can be combined with previous embodiments, the surface is coated with a material. In some embodiments, the material is Teflon®. In some embodiments, the material includes an adhesive coating that binds to cells. In some embodiments, the material includes a surfactant. In some embodiments, the material includes an anticoagulant. In some embodiments, the material includes a protein. In some embodiments, the material includes an adhesion molecule. In some embodiments, the material includes an antibody. In some embodiments, the material includes a factor that modulates cellular function. In some embodiments, the material includes a nucleic acid. In some embodiments, the material includes a lipid. In some embodiments, the material includes a carbohydrate. In some embodiments, the material includes a complex. In some embodiments, the complex is a lipid-carbohydrate complex. In some embodiments, the material includes a transmembrane protein. In some embodiments, the material is covalently bonded to the surface. In some embodiments, the material is non-covalently bonded to the surface. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged.

[0025] In some embodiments, which can be combined with previous embodiments, the cells are mammalian cells. In some embodiments, the cells are immune cells, cell line cells, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or erythrocytes. In some embodiments, the immune cells are lymphocytes, T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. In some embodiments, the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells. In some embodiments, the cells are human cells.

[0026] In some embodiments that can be combined with previous embodiments, the compound comprises a nucleic acid. In some embodiments, the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. In some embodiments, the compound is a peptide nucleic acid. In some embodiments, the compound comprises a transposon. In some embodiments, the compound is a plasmid. In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound comprises a Cas protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid encoding a Cas protein and guide RNA or donor DNA. In some embodiments, the compound comprises a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid encoding a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, a FLP recombinase, a R recombinase, an integrase, or a transposase. In some embodiments, the compound is an antibody. In some embodiments, the compound is a transcription factor. In some embodiments, the compound is a small molecule. In some embodiments, the compound is a nanoparticle. In some embodiments, the compound is a chimeric antigen receptor. In some embodiments, the compound is a fluorescently tagged molecule. In some embodiments, the compound is a liposome. In some embodiments, the compound is a virus. In some embodiments, the cell suspension includes cells with cell walls. In some embodiments, the cells are plant, yeast, fungal, algal, or bacterial cells.

[0027] In some embodiments that can be combined with previous embodiments, the cells come into contact with the compound before, simultaneously with, or after passing through the pores. In some embodiments, the delivered compound is coated on the surface. In some embodiments, the cells pass through the pores at a temperature between 0°C and 45°C. In some embodiments, the cells pass through the pores by positive or negative pressure. In some embodiments, the cells pass through the pores by constant or variable pressure. In some embodiments, the pressure is applied using a syringe. In some embodiments, the pressure is applied using a pump. In some embodiments, the pressure is applied using decompression. In some embodiments, the cells pass through the pores by capillary pressure. In some embodiments, the cells pass through the pores by blood pressure. In some embodiments, the cells pass through the pores by g-force. In some embodiments, the cells pass through the pores under pressures ranging from about 0.05 psi to about 500 psi. In some embodiments, the cells pass through the pores under pressures of about 2 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 2.5 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 3 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 5 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 10 psi. In some embodiments, the cells pass through the pores under a pressure of approximately 20 psi. In some embodiments, the cells pass through the pores by fluid flow. In some embodiments, the fluid flow is turbulent before the cells pass through the pores. In some embodiments, the fluid flow through the pores is laminar. In some embodiments, the fluid flow is turbulent after the cells have passed through the pores. In some embodiments, the cells pass through the pores at a speed ranging from approximately 0.1 mm / second to approximately 20 m / second. In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe.

[0028] In some embodiments, which can be combined with previous embodiments, the cells are in a cell suspension containing an aqueous solution. In some embodiments, the aqueous solution contains a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization. In some embodiments, the agents that affect actin polymerization are latruncrin A, cytochalasin, and / or colchicine. In some embodiments, the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. In some embodiments, the cells are further passed through an electric field generated by at least one electrode adjacent to the surface. In certain embodiments, for example, the following are provided: (Item 1) A method for delivering a compound into a cell, comprising the step of passing a cell suspension through a surface containing pores, wherein the pores deform the cell, thereby causing a perturbation of the cell that allows the compound to enter the cell, and the cell suspension comes into contact with the compound. (Item 2) The method according to item 1, wherein the surface is a film. (Item 3) The method according to item 1, wherein the surface is a filter. (Item 4) The method according to any one of items 1 to 3, wherein the surface is the surface of a meandering path. (Item 5) The method according to any one of items 1 to 4, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic. (Item 6) The method according to any one of items 1 to 5, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore. (Item 7) The method according to any one of items 1 to 6, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, micro-perforation punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing. (Item 8) The method according to any one of items 1 to 7, wherein the size of the pores correlates with the cell diameter. (Item 9) The method according to any one of items 1 to 8, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. (Item 10) The method according to any one of items 1 to 9, wherein the width of the cross-section of the surface is in the range of approximately 1 mm to approximately 1 m. (Item 11) The method according to any one of items 1 to 10, wherein the width of the cross-section of the pore is in the range of about 0.01 μm to about 300 μm. (Item 12) The method according to any one of items 1 to 11, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm. (Item 13) The method according to any one of items 1 to 12, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm. (Item 14) The method according to any one of items 1 to 11, wherein the width of the cross-section of the pore is approximately 200 μm. (Item 15) The method according to any one of items 1 to 14, wherein the size of the pores is heterogeneous. (Item 16) The method according to any one of items 1 to 15, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies within a range of 10 to 20%. (Item 17) The method according to any one of items 1 to 14, wherein the size of the pores is homogeneous. (Item 18) The method according to any one of items 1 to 17, wherein the pores have the same inlet and outlet angles. (Item 19) The method according to any one of items 1 to 17, wherein the pore has different inlet and outlet angles. (Item 20) The method according to any one of items 1 to 19, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. (Item 21) The method according to any one of items 1 to 19, wherein the shape of the cross-section of the pore is selected from cylindrical or conical. (Item 22) The method according to any one of items 1 to 21, wherein the edges of the pores are smooth. (Item 23) The method according to any one of items 1 to 21, wherein the edges of the pores are sharp. (Item 24) The method according to any one of items 1 to 23, wherein the passage of the pore is straight. (Item 25) The method according to any one of items 1 to 23, wherein the passage of the pore is curved. (Item 26) The method according to any one of items 1 to 25, wherein the pores constitute approximately 10 to 80% of the total surface area. (Item 27) The aforementioned surface is approximately 1.0 × 10 in total. 5 From approximately 1.0 x 10 30 The method according to any one of items 1 to 26, comprising a number of pores. (Item 28) The aforementioned surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 The method according to any one of items 1 to 27, comprising a number of pores. (Item 29) The method according to any one of items 1 to 28, wherein the pores are distributed in parallel. (Item 30) The method according to any one of items 1 to 29, wherein multiple surfaces are distributed in series. (Item 31) The method according to any one of items 1 to 30, wherein the distribution of the pores is regular. (Item 32) The method according to any one of items 1 to 30, wherein the distribution of the pores is random. (Item 33) The method according to any one of items 1 to 32, wherein the thickness of the surface is uniform. (Item 34) The method according to any one of items 1 to 32, wherein the thickness of the surface is not uniform. (Item 35) The method according to any one of items 1 to 34, wherein the surface has a thickness of about 0.01 μm to about 5 m. (Item 36) The method according to any one of items 1 to 35, wherein the surface has a thickness of approximately 10 μm. (Item 37) The method according to any one of items 1 to 36, wherein the surface is covered with a material. (Item 38) The method according to item 37, wherein the material is Teflon®. (Item 39) The method according to item 37, wherein the material includes an adhesive coating that binds to cells. (Item 40) The method according to item 37, wherein the material includes a surfactant. (Item 41) The method according to item 37, wherein the material includes an anticoagulant. (Item 42) The method according to item 37, wherein the material comprises a polypeptide. (Item 43) The method according to item 37, wherein the material comprises adhesive molecules. (Item 44) The method according to item 37, wherein the material includes an antibody. (Item 45) The method according to item 37, wherein the material contains a factor that regulates cellular function. (Item 46) The method according to item 37, wherein the material comprises nucleic acid. (Item 47) The method according to item 37, wherein the material includes lipids. (Item 48) The method according to item 37, wherein the material contains carbohydrates. (Item 49) The method according to item 37, wherein the material includes a composite. (Item 50) The method according to item 49, wherein the complex is a lipid-carbohydrate complex. (Item 51) The method according to item 37, wherein the material comprises a transmembrane protein. (Item 52) The method according to any one of items 37 to 51, wherein the material is covalently bonded to the surface. (Item 53) The method according to any one of items 37 to 51, wherein the material is non-covalently bonded to the surface. (Item 54) The method according to any one of items 1 to 53, wherein the surface is hydrophilic. (Item 55) The method according to any one of items 1 to 53, wherein the surface is hydrophobic. (Item 56) The method according to any one of items 1 to 55, wherein the surface is charged. (Item 57) The method according to any one of items 1 to 56, wherein the cell suspension comprises mammalian cells. (Item 58) The cell suspension includes a mixed population of cells as described in any one of items 1 to 57. method. (Item 59) The method according to any one of items 1 to 58, wherein the cell suspension is whole blood. (Item 60) The method according to any one of items 1 to 58, wherein the cell suspension is lymph. (Item 61) The method according to any one of items 1 to 58, wherein the cell suspension comprises peripheral blood mononuclear cells. (Item 62) The method according to any one of items 1 to 57, wherein the cell suspension comprises a purified cell population. (Item 63) The method according to any one of items 1 to 58 or 62, wherein the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or red blood cells. (Item 64) The method according to item 63, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. (Item 65) The method according to any one of items 1 to 64, wherein the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells. (Item 66) The method according to any one of items 1 to 64, wherein the cells are human cells. (Item 67) The method according to any one of items 1 to 66, wherein the compound comprises a nucleic acid. (Item 68) The method according to any one of items 1 to 67, wherein the compound comprises nucleic acids encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. (Item 69) The method according to any one of items 1 to 68, wherein the compound is a plasmid. (Item 70) The method according to any one of items 1 to 66, wherein the compound comprises a polypeptide-nucleic acid complex. (Item 71) The method according to any one of items 1 to 66 or 70, wherein the compound comprises a Cas9 protein and guide RNA or donor DNA. (Item 72) The method according to any one of items 1 to 67, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA. (Item 73) The method according to any one of items 1 to 66, wherein the compound comprises a protein or a peptide. (Item 74) The method according to any one of items 1 to 66 or 73, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase. (Item 75) The method according to any one of items 1 to 66 or 73, wherein the compound is an antibody. (Item 76) The method according to any one of items 1 to 66 or 73, wherein the compound is a transcription factor. (Item 77) The method according to any one of items 1 to 66, wherein the compound is a small molecule. (Item 78) The method according to any one of items 1 to 66, wherein the compound is a nanoparticle. (Item 79) The method according to any one of items 1 to 66, wherein the compound is a chimeric antigen receptor. (Item 80) The method according to any one of items 1 to 79, wherein the compound is a molecule tagged by fluorescence. (Item 81) The method according to any one of items 1 to 66, wherein the compound is a liposome. (Item 82) The method according to any one of items 1 to 81, wherein the cell suspension comes into contact with the compound before, simultaneously with, or after passing through the pores. (Item 83) The method according to any one of items 1 to 81, wherein the delivered compound is coated on the surface. (Item 84) The method described in any one of items 1 to 83, performed between 0°C and 45°C. (Item 85) The method according to any one of items 1 to 84, wherein the cells pass through the pores due to positive or negative pressure. (Item 86) The method according to any one of items 1 to 85, wherein the cells pass through the pores under constant or variable pressure. (Item 87) The method described in any one of items 1 through 86, wherein pressure is applied using a syringe. (Item 88) Pressure is applied using a pump, as described in any one of items 1 through 86. (Item 89) The method according to any one of items 1 to 86, wherein pressure is applied using depressurization. (Item 90) The method according to any one of items 1 to 86, wherein the cells pass through the pores by capillary pressure. (Item 91) The method according to any one of items 1 to 86, wherein the cells pass through the pores due to blood pressure. (Item 92) The aforementioned cells pass through the pores by g-force, any one of items 1 to 86 The method described in section [section number]. (Item 93) The method according to any one of items 1 to 92, wherein the cells pass through the pores under a pressure ranging from about 0.05 psi to about 500 psi. (Item 94) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of about 2 psi. (Item 95) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of approximately 2.5 psi. (Item 96) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of about 3 psi. (Item 97) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of approximately 5 psi. (Item 98) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of about 10 psi. (Item 99) The method according to any one of items 1 to 93, wherein the cells pass through the pores under a pressure of approximately 20 psi. (Item 100) The method according to any one of items 1 to 99, wherein the cells are passed through the pores by the flow of a fluid. (Item 101) The method according to item 100, wherein the fluid flow is turbulent before the cells pass through the pores. (Item 102) The method according to item 100, wherein the flow of the fluid through the pores is laminar. (Item 103) The method according to item 100, wherein after the cells have passed through the pores, the fluid flow is turbulent. (Item 104) The method according to any one of items 1 to 103, wherein the cells pass through the pores at a uniform cellular velocity. (Item 105) The method according to any one of items 1 to 103, wherein the cells pass through the pores at a fluctuating cellular velocity. (Item 106) The method according to any one of items 1 to 105, wherein the cells pass through the pores at a speed ranging from about 0.1 mm / second to about 20 m / second. (Item 107) The method according to any one of items 1 to 106, wherein the surface is contained within a larger module. (Item 108) The method according to any one of items 1 to 107, wherein the surface is contained within a syringe. (Item 109) The method according to any one of items 1 to 108, wherein the cell suspension comprises an aqueous solution. (Item 110) The method according to item 109, wherein the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization. (Item 111) The method according to item 110, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine. (Item 112) The method according to item 110, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. (Item 113) The viscosity of the cell suspension is approximately 8.9 × 10⁻⁶ -4 From Pa·seconds to approximately 4.0 × 10 -3 The method described in any one of items 1 to 112, in the range of Pa·seconds. (Item 114) The method according to any one of items 1 to 113, further comprising the step of passing the cells through an electric field generated by at least one electrode adjacent to the surface. (Item 115) A device for delivering a compound into a cell, comprising a surface containing pores, wherein the pores are configured to allow cells suspended in a solution to pass through, and the pores cause perturbation of the cells, thereby deforming them so that the compound enters them. (Item 116) The device according to item 115, wherein the surface is a film. (Item 117) The device according to item 115, wherein the aforementioned surface is a filter. (Item 118) The device according to any one of items 115 to 117, wherein the surface is the surface of a meandering path. (Item 119) The device according to any one of items 115 to 118, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic. (Item 120) The device according to any one of items 115 to 119, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore. (Item 121) The device according to any one of items 115 to 120, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, micro-perforation punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing. (Item 122) The device according to any one of items 115 to 121, wherein the size of the pores correlates with the cell diameter. (Item 123) The device according to any one of items 115 to 122, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. (Item 124) A device as described in any one of items 115 to 123, wherein the width of the cross-section of the surface is in the range of approximately 1 mm to approximately 1 m. (Item 125) The device according to any one of items 115 to 124, wherein the width of the cross-section of the pores is in the range of about 0.01 μm to about 300 μm. (Item 126) The device according to any one of items 115 to 125, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm. (Item 127) The device according to any one of items 115 to 126, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm. (Item 128) The device according to any one of items 115 to 125, wherein the width of the cross-section of the pore is approximately 200 μm. (Item 129) The device according to any one of items 115 to 128, wherein the size of the pores is heterogeneous. (Item 130) A device according to any one of items 115 to 129, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies within a range of 10 to 20%. (Item 131) The device according to any one of items 115 to 128, wherein the size of the pores is homogeneous. (Item 132) The device according to any one of items 115 to 131, wherein the pores have the same inlet and outlet angles. (Item 133) The device according to any one of items 115 to 131, wherein the pores have different inlet and outlet angles. (Item 134) The device according to any one of items 115 to 133, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. (Item 135) The device according to any one of items 115 to 133, wherein the shape of the pore is selected from cylindrical or conical. (Item 136) The device according to any one of items 115 to 135, wherein the edges of the pores are smooth. (Item 137) The device according to any one of items 115 to 135, wherein the edges of the pores are sharp. (Item 138) The device according to any one of items 115 to 137, wherein the passage of the pore is straight. (Item 139) The device according to any one of items 115 to 137, wherein the passage of the pore is curved. (Item 140) The device according to any one of items 115 to 139, wherein the pores constitute approximately 10 to 80% of the total surface area. (Item 141) The aforementioned surface is approximately 1.0 × 10 in total. 5 From approximately 1.0 x 10 30 A device according to any one of items 115 to 140, comprising a number of pores. (Item 142) The aforementioned surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 A device according to any one of items 115 to 141, comprising a number of pores. (Item 143) The device according to any one of items 115 to 142, wherein the pores are distributed in parallel. (Item 144) A device according to any one of items 115 to 143, wherein multiple surfaces are distributed in series. (Item 145) The device according to any one of items 115 to 144, wherein the distribution of the pores is regular. (Item 146) The device according to any one of items 115 to 144, wherein the distribution of the pores is random. (Item 147) The device according to any one of items 115 to 146, wherein the thickness of the surface is uniform. (Item 148) The device according to any one of items 115 to 146, wherein the thickness of the surface is not uniform. (Item 149) The device according to any one of items 115 to 148, wherein the surface has a thickness of approximately 0.01 μm to approximately 5 m. (Item 150) The device according to any one of items 115 to 149, wherein the surface has a thickness of approximately 10 μm. (Item 151) The device according to any one of items 115 to 150, wherein the aforementioned surface is coated with a material. (Item 152) The device according to item 151, wherein the material is Teflon®. (Item 153) The device according to item 151, wherein the material includes an adhesive coating that binds to cells. (Item 154) The device according to item 151, wherein the material comprises a surfactant. (Item 155) The device according to item 151, wherein the material comprises an anticoagulant. (Item 156) The device according to item 151, wherein the material comprises a polypeptide. (Item 157) The device according to item 151, wherein the material includes adhesive molecules. (Item 158) The device according to item 151, wherein the material includes an antibody. (Item 159) The device according to item 151, wherein the material contains a factor that modulates cellular function. (Item 160) The device according to item 151, wherein the material comprises nucleic acid. (Item 161) The device according to item 151, wherein the material includes lipids. (Item 162) The device according to item 151, wherein the material includes carbohydrates. (Item 163) The device according to item 151, wherein the material includes a composite. (Item 164) The device according to item 163, wherein the complex is a lipid-carbohydrate complex. (Item 165) The device according to item 151, wherein the material comprises a transmembrane protein. (Item 166) The device according to any one of items 151 to 165, wherein the material is covalently bonded to the surface. (Item 167) The device according to any one of items 151 to 165, wherein the material is non-covalently bonded to the surface. (Item 168) The device according to any one of items 115 to 167, wherein the surface is hydrophilic. (Item 169) The device according to any one of items 115 to 167, wherein the surface is hydrophobic. (Item 170) The device according to any one of items 115 to 169, wherein the surface is charged. (Item 171) The device according to any one of items 115 to 170, wherein the cell suspension comprises mammalian cells. (Item 172) The device according to any one of items 115 to 171, wherein the cell suspension comprises a hybrid population of cells. (Item 173) The device according to any one of items 115 to 172, wherein the cell suspension is whole blood. (Item 174) The device according to any one of items 115 to 172, wherein the cell suspension is lymph. (Item 175) The device according to any one of items 115 to 172, wherein the cell suspension comprises peripheral blood mononuclear cells. (Item 176) The device according to any one of items 115 to 171, wherein the cell suspension comprises a purified cell population. (Item 177) The device according to any one of items 115 to 172 or 176, wherein the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or red blood cells. (Item 178) The device according to item 177, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. (Item 179) The device according to any one of items 115 to 178, wherein the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells. (Item 180) The device according to any one of items 115 to 178, wherein the cells are human cells. (Item 181) The device according to any one of items 115 to 180, wherein the compound comprises nucleic acid. (Item 182) The device according to any one of items 115 to 181, wherein the compound comprises nucleic acids encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. (Item 183) The device according to any one of items 115 to 182, wherein the compound is a plasmid. (Item 184) The device according to any one of items 115 to 180, wherein the compound comprises a polypeptide-nucleic acid complex. (Item 185) The device according to any one of items 115 to 180 or 184, wherein the compound comprises a Cas9 protein and guide RNA or donor DNA. (Item 186) The device according to any one of items 115 to 181, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA. (Item 187) The device according to any one of items 115 to 180, wherein the compound comprises a protein or peptide. (Item 188) The device according to any one of items 115 to 180 or 187, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase. (Item 189) The device according to any one of items 115 to 180 or 187, wherein the compound is an antibody. (Item 190) The device according to any one of items 115 to 180 or 187, wherein the compound is a transcription factor. (Item 191) The device according to any one of items 115 to 180, wherein the compound is a small molecule. (Item 192) The device according to any one of items 115 to 180, wherein the compound is a nanoparticle. (Item 193) The device according to any one of items 115 to 180, wherein the compound is a chimeric antigen receptor. (Item 194) The device according to any one of items 115 to 193, wherein the compound is a molecule tagged by fluorescence. (Item 195) The device according to any one of items 115 to 180, wherein the compound is a liposome. (Item 196) The device according to any one of items 115 to 195, wherein the cell suspension comes into contact with the compound before, simultaneously with, or after passing through the pores. (Item 197) The device according to any one of items 115 to 195, wherein the delivered compound is coated on the surface. (Item 198) The device is one of the devices described in any one of items 115 to 197, wherein the temperature range is between 0°C and 45°C. (Item 199) The device according to any one of items 115 to 198, wherein the cells pass through the pores by positive or negative pressure. (Item 200) The device according to any one of items 115 to 199, wherein the cells pass through the pores at constant or variable pressure. (Item 201) A device as described in any one of items 115 to 200, in which pressure is applied using a syringe. (Item 202) A device as described in any one of items 115 to 200, in which pressure is applied using a pump. (Item 203) A device according to any one of items 115 to 200, in which pressure is applied using depressurization. (Item 204) The device according to any one of items 115 to 200, wherein the cells pass through the pores by capillary pressure. (Item 205) The device according to any one of items 115 to 200, wherein the cells pass through the pores due to blood pressure. (Item 206) The device according to any one of items 115 to 200, wherein the cells pass through the pores by g-force. (Item 207) The device according to any one of items 115 to 206, wherein the cells pass through the pores under a pressure ranging from about 0.05 psi to about 500 psi. (Item 208) The device according to any one of items 115 to 207, wherein the cells pass through the pores under a pressure of approximately 2 psi. (Item 209) The device according to any one of items 115 to 207, wherein the cells pass through the pores under a pressure of approximately 2.5 psi. (Item 210) The device according to any one of items 115 to 207, wherein the cells pass through the pores under a pressure of approximately 3 psi. (Item 211) The cells pass through the pores under a pressure of approximately 5 psi, items 115 to 20 A device as described in any one of item 7. (Item 212) The device according to any one of items 115 to 207, wherein the cells pass through the pores under a pressure of approximately 10 psi. (Item 213) The device according to any one of items 115 to 207, wherein the cells pass through the pores under a pressure of approximately 20 psi. (Item 214) A device according to any one of items 115 to 213, which allows the cells to pass through the pores by the flow of a fluid. (Item 215) The device according to item 214, wherein the fluid flow is turbulent before the cells pass through the pores. (Item 216) The device according to item 214, wherein the fluid flow through the pores is laminar. (Item 217) The device according to item 214, wherein after the cells have passed through the pores, the fluid flow is turbulent. (Item 218) The device according to any one of items 115 to 217, wherein the cells pass through the pores at a uniform cellular velocity. (Item 219) The device according to any one of items 115 to 217, wherein the cells pass through the pores at a fluctuating cellular velocity. (Item 220) The device according to any one of items 115 to 219, wherein the cells pass through the pores at a speed ranging from about 0.1 mm / second to about 20 m / second. (Item 221) The device according to any one of items 115 to 220, wherein the aforementioned surface is contained within a larger module. (Item 222) The device according to any one of items 115 to 221, wherein the aforementioned surface is contained within a syringe. (Item 223) The device according to any one of items 115 to 222, wherein the cell suspension comprises an aqueous solution. (Item 224) The device according to item 223, wherein the aqueous solution comprises cell culture medium, PBS, salt, sugar, growth factor, animal-derived product, filler, surfactant, lubricant, vitamin, protein, chelating agent, and / or agent affecting actin polymerization. (Item 225) The device according to item 224, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine. (Item 226) The device according to item 224, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. (Item 227) The viscosity of the cell suspension is approximately 8.9 × 10⁻⁶ -4 From Pa·seconds to approximately 4.0 × 10 -3 A device as described in any one of items 115 to 226, with a range of Pa·seconds. (Item 228) A device having multiple surfaces, as described in any one of items 115 to 227. (Item 229) The device according to any one of items 115 to 228, wherein the aforementioned surface is a transwell. (Item 230) The device according to any one of items 115 to 229, wherein at least one electrode is in close proximity to the surface and generates an electric field. (Item 231) A cell containing perturbation, wherein the cell is generated by passing the cell through a surface containing pores, and the pores deform the cell, thereby causing a perturbation that allows a compound to enter the cell. (Item 232) The cell described in item 231, wherein the surface is a membrane. (Item 233) The cell described in item 231, wherein the aforementioned surface is a filter. (Item 234) The cell according to any one of items 231 to 233, wherein the surface is the surface of a meandering pathway. (Item 235) The cell according to any one of items 231 to 234, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic. (Item 236) A cell according to any one of items 231 to 235, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore. (Item 237) The cells according to any one of items 231 to 236, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, microporous punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing. (Item 238) A cell according to any one of items 231 to 237, wherein the width of the cross-section of the pore correlates with the cell diameter. (Item 239) A cell according to any one of items 231 to 238, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. (Item 240) A cell as described in any one of items 231 to 239, wherein the width of the cross-section of the surface is in the range of approximately 1 mm to approximately 1 m. (Item 241) A cell according to any one of items 231 to 240, wherein the width of the cross-section of the pores is in the range of approximately 0.01 μm to approximately 300 μm. (Item 242) A cell according to any one of items 231 to 241, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm. (Item 243) The cells described in any one of items 231 to 242, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm. (Item 244) A cell according to any one of items 231 to 241, wherein the width of the cross-section of the pore is approximately 200 μm. (Item 245) The cells described in any one of items 231 to 244, wherein the size of the pores is heterogeneous. (Item 246) Cells as described in any one of items 231 to 245, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies within a range of 10 to 20%. (Item 247) The cells described in any one of items 231 to 244, wherein the size of the pores is homogeneous. (Item 248) The cell according to any one of items 231 to 247, wherein the pore has the same entrance angle and exit angle. (Item 249) The cell according to any one of items 231 to 247, wherein the pores have different inlet and outlet angles. (Item 250) The cell according to any one of items 231 to 249, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. (Item 251) The cell according to any one of items 231 to 249, wherein the shape of the pores is selected from cylindrical or conical. (Item 252) The cells described in any one of items 231 to 251, wherein the edges of the pores are smooth. (Item 253) The cells described in any one of items 231 to 251, wherein the edges of the pores are sharp. (Item 254) The cell according to any one of items 231 to 253, wherein the passage of the pore is straight. (Item 255) The cell according to any one of items 231 to 253, wherein the passage of the pore is curved. (Item 256) A cell according to any one of items 231 to 255, wherein the pores constitute approximately 10 to 80% of the total surface area. (Item 257) The aforementioned surface is approximately 1.0 × 10 in total. 5 From approximately 1.0 x 10 30 A cell containing a number of pores, as described in any one of items 231 to 256. (Item 258) The aforementioned surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 A cell containing a number of pores, as described in any one of items 231 to 257. (Item 259) A cell according to any one of items 231 to 258, wherein the pores are distributed in parallel. (Item 260) A cell as described in any one of items 231 to 259, wherein multiple surfaces are distributed in series. (Item 261) A cell as described in any one of items 231 to 260, wherein the distribution of the pores is regular. (Item 262) A cell according to any one of items 231 to 260, wherein the distribution of the pores is random. (Item 263) The cells described in any one of items 231 to 262, wherein the thickness of the surface is uniform. (Item 264) The cells described in any one of items 231 to 262, wherein the thickness of the surface is not uniform. (Item 265) A cell according to any one of items 231 to 264, wherein the surface has a thickness of approximately 0.01 μm to approximately 5 m. (Item 266) The cell according to any one of items 231 to 265, wherein the surface has a thickness of about 10 μm. (Item 267) The cell according to any one of items 231 to 266, wherein the surface is coated with a material. (Item 268) The cell according to item 267, wherein the material is Teflon (registered trademark). (Item 269) The cell according to item 267, wherein the material contains an adhesive coating that binds to the cell. (Item 270) The cell according to item 267, wherein the material contains a surfactant. (Item 271) The cell according to item 267, wherein the material contains an anticoagulant. (Item 272) The cell according to item 267, wherein the material contains a polypeptide. (Item 273) The cell according to item 267, wherein the material contains an adhesion molecule. (Item 274) The cell according to item 267, wherein the material contains an antibody. (Item 275) The cell according to item 267, wherein the material contains a factor that regulates cell function. (Item 276) The cell according to item 2, wherein the material contains nucleic acid. (Item 277) The cell according to item 267, wherein the material contains a lipid. (Item 278) The cell according to item 267, wherein the material contains a carbohydrate. (Item 279) The cell according to item 267, wherein the material contains a complex. (Item 280) The cell according to item 279, wherein the complex is a lipid-carbohydrate complex. (Item 281) The cell according to item 267, wherein the material contains a transmembrane protein. (Item 282) The cell according to any one of items 267 to 281, wherein the material is covalently bonded to the surface. (Item 283) The cell according to any one of items 267 to 281, wherein the material is non-covalently bonded to the surface. (Item 284) The cell according to any one of items 231 to 283, wherein the surface is hydrophilic. (Item 285) The cell according to any one of items 231 to 283, wherein the surface is hydrophobic. (Item 286) The cell according to any one of items 231 to 285, wherein the surface is charged. (Item 287) The cells described in any one of items 231 to 286, wherein the cells are mammalian cells. (Item 288) The cells described in any one of items 231 to 287, wherein the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or red blood cells. (Item 289) The cells described in item 288, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils. (Item 290) The cells described in any one of items 231 to 289, wherein the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells. (Item 291) The cells described in any one of items 231 to 289, wherein the cells are human cells. (Item 292) The cell according to any one of items 231 to 291, wherein the compound comprises nucleic acid. (Item 293) The cells described in any one of items 231 to 292, wherein the compound comprises nucleic acids encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. (Item 294) A cell according to any one of items 231 to 293, wherein the compound is a plasmid. (Item 295) The cell according to any one of items 231 to 291, wherein the compound comprises a polypeptide-nucleic acid complex. (Item 296) The cells described in any one of items 231 to 291 or 295, wherein the compound comprises the Cas9 protein and guide RNA or donor DNA. (Item 297) The cell according to any one of items 231 to 292, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA. (Item 298) The cell according to any one of items 231 to 291, wherein the compound comprises a protein or peptide. (Item 299) The cells described in any one of items 231 to 291 or 298, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase. (Item 300) The cells described in any one of items 231 to 291 or 298, wherein the compound is an antibody. (Item 301) The cell according to any one of items 231 to 291 or 298, wherein the compound is a transcription factor. (Item 302) A cell according to any one of items 231 to 291, wherein the compound is a small molecule. (Item 303) The cell according to any one of items 231 to 291, wherein the compound is a nanoparticle. (Item 304) The cell according to any one of items 231 to 291, wherein the compound is a chimeric antigen receptor. (Item 305) The cell according to any one of items 231 to 304, wherein the compound is a fluorescently tagged molecule. (Item 306) The cell according to any one of items 231 to 291, wherein the compound is a liposome. (Item 307) The cell according to any one of items 231 to 306, wherein the cell contacts the compound before, simultaneously with, or after passing through the pore. (Item 308) The cell according to any one of items 231 to 306, wherein the compound to be delivered is coated on the surface. (Item 309) The cell according to any one of items 231 to 308, wherein the cell passes through the pore between 0°C and 45°C. (Item 310) The cell according to any one of items 231 to 309, wherein the cell passes through the pore by positive or negative pressure. (Item 311) The cell according to any one of items 231 to 310, wherein the cell passes through the pore by constant or variable pressure. (Item 312) The cell according to any one of items 231 to 311, wherein the pressure is applied using a syringe. ​​​​​​​​​​The cells described in any one of items 231 to 311, wherein the cells pass through the pores by capillary pressure. (Item 316) The cells described in any one of items 231 to 311, wherein the cells pass through the pores due to blood pressure. (Item 317) The cell described in any one of items 231 to 311, wherein the cell passes through the pore by g-force. (Item 318) The cells described in any one of items 231 to 317, wherein the cells pass through the pores under a pressure ranging from about 0.05 psi to about 500 psi. (Item 319) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 2 psi. (Item 320) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 2.5 psi. (Item 321) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 3 psi. (Item 322) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 5 psi. (Item 323) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 10 psi. (Item 324) The cells described in any one of items 231 to 318, wherein the cells pass through the pores under a pressure of approximately 20 psi. (Item 325) A cell according to any one of items 231 to 324, wherein the cell is passed through the pore by the flow of a fluid. (Item 326) The cell according to item 325, wherein the fluid flow is turbulent before the cell passes through the pore. (Item 327) The cell according to item 325, wherein the fluid flow through the pores is laminar. (Item 328) The cell according to item 325, wherein after the cell has passed through the pore, the fluid flow is turbulent. (Item 329) The cells described in any one of items 231 to 328, wherein the cells pass through the pores at a speed ranging from approximately 0.1 mm / second to approximately 20 m / second. (Item 330) The cell according to any one of items 231 to 329, wherein the aforementioned surface is contained within a larger module. (Item 331) The cell according to any one of items 231 to 330, wherein the aforementioned surface is contained in a syringe. (Item 332) The aforementioned cells are in a cell suspension containing an aqueous solution, one of items 231 to 331 The cells described in the section. (Item 333) The cells according to item 332, wherein the aqueous solution comprises cell culture medium, PBS, salt, sugar, growth factor, animal-derived product, filler, surfactant, lubricant, vitamin, protein, chelating agent, and / or agent affecting actin polymerization. (Item 334) The cells described in item 333, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine. (Item 335) The cells according to item 333, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. (Item 336) The cells according to any one of items 231 to 335, wherein the cells further pass through an electric field generated by at least one electrode adjacent to the surface. [Brief explanation of the drawing]

[0029] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure can be better understood by referring to one or more of these drawings together with the detailed descriptions of the specific embodiments presented herein.

[0030] [Figure 1] Figure 1A shows a photograph of an exemplary polycarbonate filter for use in the described embodiment. Figure 1B shows a photograph of the pores of an exemplary polycarbonate filter.

[0031] [Figure 2A-B] Figures 2A–G show exemplary FACS plots demonstrating the delivery of 3kDa PacBlue and 10kDa Alexa488 dextran particles to HeLa cells (2 × 10⁶ cells / mL) at low pressure (5 psi, 10 psi, or 20 psi) through pores of 8 μm, 10 μm, 12 μm, or 14 μm filters. Figures 2A–C represent cells passing through pores of 14 μm (Figure 2A), 12 μm (Figure 2B), and 10 μm (Figure 2C) filters at a pressure of 5 psi. Figures 2E and G represent cells passing through 12 μm filter pores at pressures of 10 psi (Figure 2E) and 20 psi (Figure 2G). Figures 2D and F represent plots of endocytosis control (Figure 2D) and negative control (Figure 2F). [Figure 2C-D] Figures 2A–G show exemplary FACS plots demonstrating the delivery of 3kDa PacBlue and 10kDa Alexa488 dextran particles to HeLa cells (2 × 10⁶ cells / mL) at low pressure (5 psi, 10 psi, or 20 psi) through pores of 8 μm, 10 μm, 12 μm, or 14 μm filters. Figures 2A–C represent cells passing through pores of 14 μm (Figure 2A), 12 μm (Figure 2B), and 10 μm (Figure 2C) filters at a pressure of 5 psi. Figures 2E and G represent cells passing through 12 μm filter pores at pressures of 10 psi (Figure 2E) and 20 psi (Figure 2G). Figures 2D and F represent plots of endocytosis control (Figure 2D) and negative control (Figure 2F). [Figure 2E-F] Figures 2A–G show exemplary FACS plots demonstrating the delivery of 3kDa PacBlue and 10kDa Alexa488 dextran particles to HeLa cells (2 × 10⁶ cells / mL) at low pressure (5 psi, 10 psi, or 20 psi) through pores of 8 μm, 10 μm, 12 μm, or 14 μm filters. Figures 2A–C represent cells passing through pores of 14 μm (Figure 2A), 12 μm (Figure 2B), and 10 μm (Figure 2C) filters at a pressure of 5 psi. Figures 2E and G represent cells passing through 12 μm filter pores at pressures of 10 psi (Figure 2E) and 20 psi (Figure 2G). Figures 2D and F represent plots of endocytosis control (Figure 2D) and negative control (Figure 2F). [Figure 2G] Figures 2A–G show exemplary FACS plots demonstrating the delivery of 3kDa PacBlue and 10kDa Alexa488 dextran particles to HeLa cells (2 × 10⁶ cells / mL) at low pressure (5 psi, 10 psi, or 20 psi) through pores of 8 μm, 10 μm, 12 μm, or 14 μm filters. Figures 2A–C represent cells passing through pores of 14 μm (Figure 2A), 12 μm (Figure 2B), and 10 μm (Figure 2C) filters at a pressure of 5 psi. Figures 2E and G represent cells passing through 12 μm filter pores at pressures of 10 psi (Figure 2E) and 20 psi (Figure 2G). Figures 2D and F represent plots of endocytosis control (Figure 2D) and negative control (Figure 2F).

[0032] [Figure 3] Figures 3A and 3B show FACS plots demonstrating the delivery of 3kDa PacBlue and 10kDa Alexa488 dextran particles to newly isolated human T cells (4 × 10⁶ cells / mL) through the pores of a 5μm-sized filter, either manually by syringe (Figure 3A) or under constant pressure of 5 psi (Figure 3B). Cell viability is shown.

[0033] [Figure 4]Figure 4 shows the delivery efficiency and cell viability after delivery of 3kDa PacBlue dextran particles to HeLa cells mediated by commercially available (COTS) filters or custom-made syringe filters.

[0034] [Figure 5A-B] Figures 5A and 5B show representative flow cytometry histogram plots demonstrating the mean fluorescence intensity (MFI) values ​​of delivery of 3kDa PacBlue dextran particles to HeLa cells mediated by COTS (Figure 5A) or a custom syringe filter (Figure 5B) at 3 psi. [Figure 5C] Figure 5C shows the mean relative mean fluorescence intensity (rMFI) values ​​for delivery of 3kDa PacBlue dextran particles to HeLa cells mediated by COTS or custom syringe filters at 2 psi and 3 psi.

[0035] [Figure 6] Figure 6 shows the delivery efficiency, cell viability, and rMFI values ​​after delivery of 3kDa PacBlue dextran particles and EGFP mRNA to HeLa cells mediated by COTS filters at 2 psi, 2.5 psi, and 3 psi.

[0036] [Figure 7] Figures 7A and 7B show exemplary flow cytometry histogram plots representing fluorescence after compression-mediated delivery (SQZ) of IgG antibody (Figure 7A) and dextran particles (Figure 7B) to human RBCs compared with endocytosis controls.

[0037] [Figure 8] Figure 8A shows the delivery efficiency of IgG antibody and dextran particles after compression-mediated delivery to human RBCs. Figure 8B shows the estimated cell viability after compression-mediated delivery of IgG antibody and dextran particles to human RBCs.

[0038] [Figure 9A]Figure 9A shows an exemplary flow cytometry histogram plot representing fluorescence after compression-mediated delivery of IgG antibody to mouse RBCs. [Figure 9B-C] Figure 9B shows the estimated cell viability after compression-mediated delivery of IgG antibody to mouse RBCs. Figure 9C shows the delivery efficiency after compression-mediated delivery of IgG antibody to mouse RBCs.

[0039] [Figure 10A-C] Figures 10A–I show exemplary FACS plots demonstrating the delivery of dextran particles to mouse RBCs under 2 psi (Figure 10D), 4 psi (Figure 10E), 6 psi (Figure 10F), 10 psi (Figure 10G), and 20 psi (Figure 10H), or using manual syringe pressurization (Figure 10I), compared to endocytosis (Figure 10A), negative control (Figure 10B), and uninfected control (Figure 10C). [Figure 10D-F] Figures 10A–I show exemplary FACS plots demonstrating the delivery of dextran particles to mouse RBCs under 2 psi (Figure 10D), 4 psi (Figure 10E), 6 psi (Figure 10F), 10 psi (Figure 10G), and 20 psi (Figure 10H), or using manual syringe pressurization (Figure 10I), compared to endocytosis (Figure 10A), negative control (Figure 10B), and uninfected control (Figure 10C). [Figure 10G-I] Figures 10A–I show exemplary FACS plots demonstrating the delivery of dextran particles to mouse RBCs under 2 psi (Figure 10D), 4 psi (Figure 10E), 6 psi (Figure 10F), 10 psi (Figure 10G), and 20 psi (Figure 10H), or using manual syringe pressurization (Figure 10I), compared to endocytosis (Figure 10A), negative control (Figure 10B), and uninfected control (Figure 10C).

[0040] [Figure 11A] Figure 11A shows the estimated cell viability after compression-mediated delivery of dextran particles to mouse RBCs. [Figure 11B-C]Figure 11B shows the delivery efficiency of IgG antibody or dextran particles after compression-mediated delivery to mouse RBCs. Figure 11C shows the geometric mean fluorescence after compression-mediated delivery of IgG antibody or dextran particles to mouse RBCs.

[0041] [Figure 12A-B] Figure 12A shows an exemplary flow cytometry histogram plot representing fluorescence after compression-mediated delivery of dextran particles to mouse RBCs. Figure 12B shows estimated cell viability after compression-mediated delivery of dextran particles to mouse RBCs. [Figure 12C-D] Figure 12C shows the delivery efficiency of dextran particles after compression-mediated delivery to mouse RBCs. Figure 12D shows the geometric mean fluorescence after compression-mediated delivery of dextran particles to mouse RBCs.

[0042] [Figure 13A-B] Figure 13A shows cell viability after microsieve-mediated delivery of dextran particles to HeLa cells. Figure 13B shows the delivery efficiency after microsieve-mediated delivery of dextran particles to HeLa cells. [Figure 13C-D] Figures 13C and 13D show exemplary flow cytometry histogram plots representing fluorescence after compression-mediated delivery of dextran particles to HeLa cells using AQUAMARIJN microsieves (Figure 13C) or STERLITECH® microsieves (Figure 13D).

[0043] [Figure 14A] Figure 14A shows cell viability and delivery efficiency after microsieve-mediated delivery of dextran particles to T cells. [Figure 14B] Figure 14B shows an exemplary flow cytometry histogram plot representing fluorescence after compression-mediated delivery of dextran particles to T cells using an AQUAMARIJN microsieve. [Modes for carrying out the invention]

[0044] Detailed explanation Certain aspects of the disclosures described herein are based on the remarkable discovery that intracellular delivery of compounds can be achieved by passing a cell suspension through a pore-containing surface. Certain aspects of the disclosure relate to a method for delivering a compound into cells, comprising the step of passing a cell suspension through a pore-containing surface, wherein the pores deform the cells, thereby causing cellular perturbation to allow the compound to enter the cells, and the cell suspension comes into contact with the compound. Certain aspects of the disclosure relate to a device for delivering a compound into cells, comprising a pore-containing surface, wherein the pores are configured to allow cells suspended in a solution to pass through, and the pores deform the cells, thereby causing cellular perturbation to allow the compound to enter the cells. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the surface is a microsieve.

[0045] I. General techniques The techniques and procedures described or referenced herein include, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (DOI: 10.1002 / 0471142727); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (EA Greenfield, ed., 2013); Culture of Animal Cells. :A Manual of Basic Technique and Specialized Applications(R.I. Freshney, 6th Edition, J. Wiley and Sons, 2010); Oligonucleotides and Analogues (F. Eckstein, ed., 1992); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Handbook (JE Celis, ed., Academic Press, 2005); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, Springer, 2013); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., J. Wiley and Sons, 1993-98); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (DOI: 10.1002 / 0471142735); Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Janeway's Immunobiology (K. Murphy and C. Weaver, Garland Science, 2016); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D.Methodologies described in *Lane, Cold Spring Harbor Laboratory Press, 1999*; *Making and Using Antibodies: A Practical Handbook* (edited by GC Howard and MR Kaser, CRC Press, 2013); *The Antibodies*, Volumes 1-7 (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995-2007); and *Cancer: Principles and Practice of Oncology* (edited by VT DeVita et al., JB Lippincott Company, 2011) are generally well understood by those skilled in the art and are commonly used using conventional methodologies.

[0046] II. Definition For the purposes of describing this specification, the following definitions apply, and wherever appropriate, a term used in the singular form also includes its plural form, and vice versa. In the event of any conflict between any definition below and any document incorporated herein by reference, the definition given below shall prevail.

[0047] As used herein, the singular forms “a,” “an,” and “the” include multiple references unless otherwise indicated.

[0048] The aspects and embodiments of the disclosure described herein are understood to include “including,” “consisting of,” and “essentially consisting of.”

[0049] All compositions described herein, and all methods using the compositions described herein, may contain or "be essentially composed of" the listed components or steps. Where a composition is described as "be essentially composed of" the listed components, the composition contains the listed components and may contain other components that do not substantially affect the disclosed method, but does not contain any other components other than those explicitly listed that substantially affect the disclosed method; or, if a composition certainly contains extra components other than those explicitly listed that substantially affect the disclosed method, the composition does not contain extra components in a concentration or quantity sufficient to substantially affect the disclosed method. Where a method is described as "be essentially composed of" the listed steps, the method contains the listed steps and may contain other steps that do not substantially affect the disclosed method, but does not contain any other steps other than those explicitly listed that substantially affect the disclosed method. As a non-limiting example, where it is stated that a composition "essentially consists of" components, the composition may further contain any amount of pharmaceutically acceptable carriers, vehicles, or diluents and other components that do not substantially affect the disclosed method.

[0050] As used herein, the term “about” refers to the normal range of error for each value as readily understood by those skilled in the art. References to “about” values ​​or parameters herein include (and describe) embodiments directed toward that value or parameter itself.

[0051] As used herein, the term “pore” refers to an opening in a material, including but not limited to holes, cracks, cavities, gaps, fissures, voids, or perforations. In some examples, (where indicated) the term refers to pores on the surface of the disclosure. In other examples, (where indicated) the term “pore” may refer to pores in a cell membrane.

[0052] As used herein, the term “membrane” refers to a selective barrier or sheet containing pores. This term includes flexible, sheet-like structures that function as boundaries or backings. In some examples, the term refers to a surface or filter containing pores. This term is distinctly different from the term “cell membrane.”

[0053] As used herein, the term "filter" refers to a porous article that allows selective passage through its pores. In some instances, the term refers to a surface or membrane containing pores.

[0054] As used herein, the term “heterogeneous” refers to a mixture or non-uniform material in its structure or composition. In some cases, the term refers to pores that have varied sizes, shapes, or distributions within a given surface.

[0055] As used herein, the term “homogeneous” refers to an object whose structure or composition is consistent or uniform throughout. In some examples, the term refers to pores that have a consistent size, shape, or distribution within a given surface.

[0056] As used herein, the term “heterogeneous” refers to molecules originating from different organisms. In some examples, this term refers to nucleic acids or proteins that are not typically found or expressed within a given organism.

[0057] As used herein, the term “homologous” refers to molecules originating from the same organism. In some examples, this term refers to nucleic acids or proteins that are commonly found or expressed within a given organism.

[0058] As used herein, the terms “polynucleotide” or “nucleic acid” refer to any polymeric form of nucleotides of any length, whether ribonucleotide or deoxyribonucleotide. Therefore, the term includes, but is not limited to, single, double, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The polynucleotide backbone may contain sugars and phosphate groups (typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the polynucleotide backbone may contain polymers of synthetic subunits such as phosphoramic acids, and thus may be oligodeoxynucleoside phosphoramic acids (P-NH2) or mixed phosphoramic acid-phosphate diester oligomers. Furthermore, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing a complementary strand and annealing the strand under appropriate conditions, or by using DNA polymerase with appropriate primers to newly synthesize a complementary strand.

[0059] The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues, not limited to the minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and their fragments are included by definition. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this invention, “polypeptide” refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conserved in nature) to its native sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0060] Any structural or functional features described herein are known in the art, and the methods for determining these features are also known.

[0061] surface having pores. In certain embodiments, the present disclosure relates to a method for delivering a compound into cells, comprising the step of passing a cell suspension through a surface containing pores, wherein the pores deform the cells and cause cellular perturbation, and the cell suspension is brought into contact with the compound, for example, before the cells in the suspension pass through the pores, or before, so that the compound enters the cells. The surfaces disclosed herein are made of any one of several materials and can take any one of several forms. In some embodiments, the surface is a membrane. In some embodiments, the surface is a filter. Filters and membranes are typically used to separate substances from solutions and thereby obtain the retained substance. In embodiments where the surface is a filter or membrane, the filter or membrane is used alternatively to deliver the compound into cells by passing a cell suspension through the filter or membrane pores, thereby causing cellular perturbation so that the compound enters the cells. In some embodiments, the surface is a STERLITECH® polycarbonate filter (PCT8013100). In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface is a sponge or sponge-like matrix. In some embodiments, the surface is a matrix. In some embodiments, the surface is a microsieve. In some embodiments, the surface is not a mesh.

[0062] In some embodiments, the surface is the surface of a meandering path. In some embodiments, the surface of the meandering path contains cellulose acetate. In some embodiments, the surface contains, but is not limited to, materials selected from synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, nitrocellulose, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, graphite, and ceramics.

[0063] The surfaces of the disclosed material may be manufactured using any technique known in the art, including but not limited to etching, track etching, lithography, laser ablation, injection molding, stamping, micro-perforation punching, polymer sponge, direct foam molding, extrusion, and hot embossing. Etching involves the process of cutting a metal surface in a desired pattern using a chemical substance such as a strong acid. Track-etched films are formed by emitting particles onto a solid film to form tracks of damaged material within the film. The film is then exposed to a chemical agent to selectively etch the damaged tracks, creating perforations in the film. The width of the pore cross-section can be controlled by the incubation time of the etching solution on the film. Lithography can include microlithography, nanolithography, and X-ray lithography. In lithography, a lithography apparatus places a desired pattern onto a target portion of a substrate. For example, in X-ray lithography, X-rays are used to transfer the pattern from a mask to a photosensitive chemical photoresist on the desired substrate. In photolithography, light is used to transfer a desired pattern from a photomask to a photosensitive photoresist on a substrate. Subsequent chemical coating is used to engrave the pattern into the substrate material directly beneath the photoresist. Laser ablation is the process of removing material from a solid surface by irradiation with a laser beam, while injection molding involves injecting material into a desired mold, where the material then cools and hardens into the desired structure. Stamping and micro-perforation punching methods use tools to cut or imprint a desired shape into a substrate material. To produce ceramic filter films, the polymer sponge method involves saturating a polymer sponge with a ceramic slurry and then burning it to leave a porous ceramic. In the direct foam molding method, a chemical mixture containing the desired ceramic component and organic material is processed to release gas. Bubbles are then generated in the material and it is foamed. The resulting porous chimeric material is then dried and fired.To create honeycomb or cellular structures, a plastic forming method called extrusion is used, in which a mixture of ceramic powder plus additives is extruded through a molding die. Hot embossing involves stamping patterns into a polymer that has been softened by raising the polymer's temperature to just above its glass transition temperature.

[0064] In some embodiments, the surface is a nanostructured film. In some embodiments, a phase transition is used to fabricate the nanostructured film. Phase transition methods include, but are not limited to, sedimentation from the gas phase, wet-dry phase inversion, and thermal induction phase separation. In the sedimentation from the gas phase method, a cast polymer solution consisting of polymer and solvent is introduced into a non-solvent vapor environment saturated with solvent vapor. The saturated solvent vapor inhibits the evaporation of the solvent from the film. Non-solvent molecules then diffuse into the film, resulting in the curing of the polymer. In the wet-dry phase inversion method, a polymer solution consisting of polymer and solvent is prepared. The solution is cast onto a suitable surface, and after partial evaporation of the solvent, the cast film is immersed in gelatin. The non-solvent then diffuses into the polymer solution film through a thin solid layer, while the solvent diffuses outward, creating a porous film. In the thermal induction phase separation method, the polymer is mixed with the solvent at high temperature, and the polymer solution is cast onto the film. As the solution cools, it enters an immiscible region due to the loss of its solubility.

[0065] In some embodiments, the surface is a block copolymer. A block copolymer consists of two or more blocks of different polymerized monomers linked by covalent bonds. The block copolymer components can undergo microphase separation to form periodic nanostructures. In this process, the block copolymer undergoes phase separation due to incompatibility between blocks, creating nanometer-sized structures.

[0066] In some embodiments, the surface is a microsieve. In some examples, the microsieve is used in cell separation, CTC isolation, or droplet emulsification techniques. In some embodiments, the pores of the microsieve are fabricated by photolithography on a ceramic substrate with a silicon support. In some embodiments, the microsieve is made of polycarbonate. Microsieves are available from Aquamarijn or Sterlitech. In some embodiments, the microsieve has a pore size ranging from about 4 μm to about 10 μm. In some embodiments, the microsieve has a porosity greater than any of the following: about 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or greater than about 50%.

[0067] The surfaces disclosed herein may have any shape known in the art, for example, a three-dimensional shape. The two-dimensional shape of the surface may be, but is not limited to, annular, elliptical, circular, quadrilateral, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments, the surface has a circular shape. In some embodiments, the three-dimensional shape of the surface is cylindrical, conical, or cubic.

[0068] The terms “pore size” and “pore cross-sectional width” are used interchangeably and, as used herein, refer to the minimum cross-sectional width across the pore. In some embodiments, the pore is annular or nearly annular, and the pore size or pore cross-sectional width is the minimum width of the polygon. In some embodiments, the pore is polygonal (e.g., quadrilateral, rectangular, pentagonal, hexagonal, etc.), and the pore size or pore cross-sectional width is the minimum width of the polygon. Those skilled in the art will understand that a triangular pore may not have a width, but rather is described in terms of its base and height. In some embodiments, the pore size or pore cross-sectional width of a triangular pore is the minimum height of the triangle (the minimum distance between the base and its opposite angle).

[0069] The surface can have various cross-sectional widths and thicknesses. In some embodiments, the width of the surface cross-section is between approximately 1 mm and approximately 3 m, or any cross-sectional width or range between these. In some embodiments, the width of the surface cross-section is between approximately 1 mm and approximately 750 mm, approximately 1 mm and approximately 500 mm, approximately 1 mm and approximately 250 mm, approximately 1 mm and approximately 100 mm, approximately 1 mm and approximately 50 mm, approximately 1 mm and approximately 25 mm, approximately 1 mm and approximately 10 mm, approximately 1 mm and approximately 5 mm, or approximately 1 mm and approximately 2.5 mm. In some embodiments, the width of the surface cross-section is between approximately 5 mm and approximately 1 m, approximately 10 mm and approximately 1 m, approximately 25 mm and approximately 1 m, approximately 50 mm and approximately 1 m, approximately 100 mm and approximately 1 m, approximately 250 mm and approximately 1 m, approximately 500 mm and approximately 1 m, or approximately 750 mm and approximately 1 m.

[0070] In some embodiments, the surface has a predetermined thickness. In some embodiments, the surface thickness is uniform. In some embodiments, the surface thickness is variable. For example, in some embodiments, one part of the surface is thicker or thinner than the other parts of the surface. In some embodiments, the surface thickness varies by about 1 to 90% or any percentage or range between them. In some embodiments, the surface thickness varies by one of the following: about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, or about 1% to about 5%. In some embodiments, the surface thickness varies from approximately 5% to approximately 90%, approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, or approximately 80% to approximately 90%. In some embodiments, the surface thickness is between approximately 0.01 μm and approximately 5 mm, or any thickness or range between these. In some embodiments, the surface is approximately 0.01 μm to approximately 5 mm, approximately 0.01 μm to approximately 2.5 mm, approximately 0.01 μm to approximately 1 mm, approximately 0.01 μm to approximately 750 μm, approximately 0.01 μm to approximately 500 μm, approximately 0.01 μm to approximately 250 μm, approximately 0.01 μm to approximately 100 μm, approximately 0.01 μm to approximately 90 μm, approximately 0.01 μm to approximately 80 μm, approximately 0.01 μm to approximately 70 μm, and approximately 0.01 The thickness is between μm and approximately 60 μm, approximately 0.01 μm and approximately 50 μm, approximately 0.01 μm and approximately 40 μm, approximately 0.01 μm and approximately 30 μm, approximately 0.01 μm and approximately 20 μm, approximately 0.01 μm and approximately 10 μm, approximately 0.01 μm and approximately 5 μm, approximately 0.01 μm and approximately 1 μm, approximately 0.01 μm and approximately 0.5 μm, approximately 0.01 μm and approximately 0.1 μm, or approximately 0.01 μm and approximately 0.05 μm.In some embodiments, the surface thickness is between approximately 0.01 μm and 5 mm, approximately 0.05 μm and 5 mm, approximately 0.1 μm and 5 mm, approximately 0.5 μm and 5 mm, approximately 1 μm and 5 mm, approximately 5 μm and 5 mm, approximately 10 μm and 5 mm, approximately 20 μm and 5 mm, approximately 30 μm and 5 mm, approximately 40 μm and 5 mm, approximately 50 μm and 5 mm, approximately 60 μm and 5 mm, approximately 70 μm and 5 mm, approximately 80 μm and 5 mm, approximately 90 μm and 5 mm, approximately 100 μm and 5 mm, approximately 250 μm and 5 mm, approximately 500 μm and 5 mm, approximately 750 μm and 5 mm, approximately 1 mm and 5 mm, or approximately 2.5 mm and 5 mm. In some embodiments, the surface thickness is approximately 10 μm.

[0071] In some embodiments, the flow velocity through the surface is approximately 0.001 mL / cm². 2 From / second to approximately 100 L / cm³ 2 The rate is any rate or range between a period of time per second or between such periods. In some embodiments, the flow rate is about 0.001 mL / cm². 2 From / second to approximately 75 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 50 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 25 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 10 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 7.5 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 5.0 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 2.5 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 1 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 0.1 L / cm³ 2 / sec, approx. 0.001mL / cm 2 From / second to approximately 75 mL / cm³ 2 / sec, approx. 0.001mL / cm 2from about 50 mL / cm per second 2 / second, about 0.001 mL / cm 2 from about 25 mL / cm per second 2 / second, about 0.001 mL / cm 2 from about 10 mL / cm per second 2 / second, about 0.001 mL / cm 2 from about 1 mL / cm per second 2 / second, about 0.001 mL / cm 2 from about 0.1 mL / cm per second 2 / second, or about 0.001 mL / cm 2 from about 0.01 mL / cm per second 2 / second. In some embodiments, the flow rate is about 0.001 mL / cm 2 from about 100 L / cm per second 2 / second, about 0.01 mL / cm 2 from about 100 L / cm per second 2 / second, about 0.1 mL / cm 2 from about 100 L / cm per second 2 / second, about 1 mL / cm 2 from about 100 L / cm per second 2 / second, about 10 mL / cm 2 from about 100 L / cm per second 2 / second, about 50 mL / cm 2 from about 100 L / cm per second 2 / second, about 0.1 L / cm 2 from about 100 L / cm per second 2 / second, about 0.5 L / cm 2 from about 100 L / cm per second 2 / second, about 1 L / cm 2 from about 100 L / cm per second 2 / second, about 2.5 L / cm 2 from about 100 L / cm per second 2 / second, about 5 L / cm 2 from about 100 L / cm per second 2 / second, about 7.5 L / cm 2 from about 100 L / cm per second 2 / second, about 10 L / cm 2 from about 100 L / cm per second 2 / second, about 25 L / cm 2 from about 100 L / cm per second 2 / second, approximately 50 L / cm 2 / second to approximately 100 L / cm 2 / second, or approximately 75 L / cm 2 / second to approximately 100 L / cm 2 / second.

[0072] The width of the pore cross-section is related to the type of cell being treated. In some embodiments, the pore size correlates with the diameter of the cell being treated. In some embodiments, the pore size is the degree to which perturbation is imparted to the cell upon passage through the pore. In some embodiments, the pore size is less than the cell diameter. In some embodiments, the pore size is about 20 to 90% of the cell diameter. In some embodiments, the pore size is one of approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cell diameter, or any value in between. The optimal pore size can vary based on the application and / or cell type. Many cells have a diameter between approximately 5 and 15 μm; for example, dendritic cells have a diameter of 7 to 8 μm. For example, the width of the pore cross-section is less than approximately 4.5, 5, 5.5, 6, or 6.5 μm for single-cell treatment. In another example, the cross-sectional width of the pores for processing human oocytes is between approximately 6.2 μm and 8.4 μm, although larger and smaller pores are possible (the diameter of a human oocyte is approximately 120 μm). In some embodiments, if the clusters of cells are not destroyed when passing through the pores, clusters of cells (e.g., embryos) are processed using pore cross-sectional widths between approximately 12 μm and 17 μm. In some embodiments, the cross-sectional width of the pores is between approximately 0.01 μm and 300 μm or any size or range in between. In some embodiments, the sizes of the cross-sectional width of the pores are in the range of approximately 0.01 μm to 250 μm, approximately 0.01 μm to 200 μm, approximately 0.01 μm to 150 μm, approximately 0.01 μm to 100 μm, approximately 0.01 μm to 50 μm, or approximately 0.01 μm to 25 μm. In some embodiments, the width of the pore cross-section is in the range of approximately 1 μm to approximately 300 μm, approximately 25 μm to approximately 300 μm, approximately 50 μm to approximately 300 μm, approximately 100 μm to approximately 300 μm, approximately 150 μm to approximately 300 μm, approximately 200 μm to approximately 300 μm, or approximately 250 μm to approximately 300 μm. In some embodiments, the width of the pore cross-section is in the range of approximately 0.01 to approximately 35 μm. In some embodiments, the width of the pore cross-section is approximately 0.4 μm, approximately 5 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm, or less than any of these.In some embodiments, the width of the pore cross-section is at least about 1 μm, 2 μm, 3 μm, 4 μm, or 5 μm. In some embodiments, the width of the pore cross-section is about 200 μm or less. In some embodiments, the pores have heterogeneous or homogeneous cross-sectional widths across a given surface. In some embodiments, the heterogeneous pore cross-sectional widths vary by 10 to 20% or any percentage or range between them. In some embodiments, the pores deform the cells to any one of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cell diameter or any value between them. In some embodiments, the cell size is the size of the cell in suspension.

[0073] In some embodiments, the cross-sectional area of ​​the pore correlates with the cross-sectional area of ​​the cell. In some embodiments, the two-dimensional shape of the pore is annular, elliptical, circular, quadrilateral, rectangular, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal, and the cross-sectional area of ​​the pore correlates with the cross-sectional area of ​​the cell. In some embodiments, the cross-sectional area of ​​the pore is at least about 1 μm 2 , 4μm 2 , 9μm 2 , 16 μm 2 , 25μm 2 , 50 μm 2 , 100 μm 2 , 150 μm 2 , 200 μm 2 , 250 μm 2 , 500μm 2 or 1000 μm 2 In some embodiments, the pores have heterogeneous or homogeneous cross-sectional areas across a given surface. In some embodiments, the heterogeneous pore cross-sectional area varies by 10 to 20% or any percentage or range between them. In some embodiments, the pores deform the cell to one of approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the cell's cross-sectional area or any value between them. In some embodiments, the cell size is the size of the cell in suspension.

[0074] The entrance and exit of the pore passage may have various angles. The pore angle can be selected to minimize clogging of the pore while cells are passing through. For example, the angle of the entrance or exit portion can be between approximately 0 and approximately 90 degrees. In some embodiments, pores have the same entrance and exit angles. In some embodiments, pores have different entrance and exit angles. In some embodiments, the edges of the pores are smooth, e.g., rounded or curved. Smooth pore edges are continuous, flat, and have a smooth surface without any ridges, protrusions, or bumps. In some embodiments, the edges of the pores are sharp. Sharp pore edges have thin edges that are pointed or steeply angled. In some embodiments, the pore passage is straight. Straight pore passages do not contain curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. Curved pore passages are curved or deviate from a straight line. In some embodiments, the pore passage has multiple curves, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves.

[0075] Pores can have any shape known in the art, including two-dimensional or three-dimensional shapes. Pore shapes (e.g., cross-sectional shapes) can be, but are not limited to, annular, elliptical, circular, rectangular, quadrilateral, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-section of the pore is circular. In some embodiments, the three-dimensional shape of the pore is cylindrical or conical. In some embodiments, the pore has grooved inlet and outlet shapes. In some embodiments, the pore shapes are homogeneous (i.e., consistent or regular) among the pores on a given surface. In some embodiments, the pore shapes are heterogeneous (i.e., hybrid or varied) among the pores on a given surface.

[0076] The surfaces described herein may have a certain range of total pore counts. In some embodiments, the pores occupy about 10 to 80% of the total surface area. In some embodiments, the surface is about 1.0 × 10 3 From approximately 1.0 x 1030 It contains all of the pores or any number or range of pores between them. For example, the surface has at least about 1.0 × 10 3 , 1.0 × 10 4 , 1.0 × 10 5 , 1.0 × 10 6 , 1.0 × 10 7 , 1.0 × 10 8 , 1.0 × 10 9 , 1.0 × 10 10 , 1.0 × 10 15 , 1.0 × 10 20 , 1.0 × 10 25 , 1.0 × 10 30 It may contain any or more total pores. In some embodiments, the surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 It includes pores between particles. For example, the surface has a surface area of ​​1 mm². 2 Approximately 1.0 x 10 2 From approximately 1.0 x 10 15 , about 1.0×10 3 From approximately 1.0 x 10 15 , about 1.0×10 5 From approximately 1.0 x 10 15 , about 1.0×10 7 From approximately 1.0 x 10 15 , about 1.0×10 10 From approximately 1.0 x 10 15 , about 1.0×10 12 From approximately 1.0 x 10 15 It may contain a number of pores. In some embodiments, the surface has a surface area of ​​1 mm². 2 Approximately 10 to approximately 1.0 x 10 15 Approximately 10 to approximately 1.0 x 10 12 Approximately 10 to approximately 1.0 x 10 10 Approximately 10 to approximately 1.0 x 10 7 Approximately 10 to approximately 1.0 x 10 5 Approximately 10 to approximately 1.0 x 10 3 , or approximately 10 to approximately 1.0 × 10 2 It may contain 10⁴ pores. In some embodiments, a surface with a radius of 13 mm has approximately 6.0 × 10⁴ pores. 5 It contains a number of pores.

[0077] Pores can be distributed in numerous ways within a given surface. In some embodiments, pores are distributed parallel to each other within a given surface. In some embodiments, surfaces having pores are stacked on top of each other. In one such example, pores are distributed in the same direction and are at the same distance from each other within a given surface. In some embodiments, the pore distribution is regular or homogeneous. In one such example, pores are distributed in a regular, organized pattern or are at the same distance from each other within a given surface. In some embodiments, the pore distribution is random or heterogeneous. In one such example, pores are distributed in an irregular and disordered pattern or are at different distances from each other within a given surface. In some embodiments, pores in a surface are arranged in an irregular pattern. In some embodiments, pores in a surface are heterogeneous in size. In some embodiments, pores in a surface are heterogeneous in shape. In some embodiments, pores in a surface are arranged in an irregular pattern and are heterogeneous in size. In some embodiments, the pores in the surface are arranged in an irregular pattern and have a heterogeneous shape. In some embodiments, the pores in the surface are heterogeneous in size and shape. In some embodiments, the pores in the surface are arranged in an irregular pattern, are heterogeneous in size and shape.

[0078] In some embodiments, multiple surfaces are distributed in series. The multiple surfaces may be homogeneous or heterogeneous in surface size, shape, and / or roughness. The multiple surfaces may further contain pores having homogeneous or heterogeneous pore sizes, shapes, and / or numbers, thereby enabling simultaneous delivery of a certain range of compounds to different cell types. In some embodiments, the multiple surfaces are stacked. In some embodiments, the cell suspension passes through the multiple surfaces.

[0079] In some embodiments, individual pores have a uniform width dimension (i.e., a constant width along the length of the pore passage). In some embodiments, individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore passage). In some embodiments, pores within a given surface have the same individual pore depth. In some embodiments, pores within a given surface have different individual pore depths. In some embodiments, pores are immediately adjacent to each other. In some embodiments, pores are separated from each other by distance. In some embodiments, pores are separated from each other by distances ranging from approximately 0.001 μm to approximately 30 mm or any distance or range between these. For example, the pores may be separated from each other by a distance between approximately 0.001 μm and 30 mm, approximately 0.01 μm and 30 mm, approximately 0.05 μm and 30 mm, approximately 0.1 μm and 30 mm, approximately 0.5 μm and 30 mm, approximately 1 μm and 30 mm, approximately 5 μm and 30 mm, approximately 10 μm and 30 mm, approximately 50 μm and 30 mm, approximately 100 μm and 30 mm, approximately 250 μm and 30 mm, approximately 500 μm and 30 mm, approximately 1 mm and 30 mm, approximately 10 mm and 30 mm, or approximately 20 mm and 30 mm. In some embodiments, the pores may be separated from each other by a distance between approximately 0.001 μm and 0.01 μm, approximately 0.001 μm and 0.05 μm, approximately 0.001 μm and 0.1 μm, approximately 0.001 μm and 0.5 μm, approximately 0.001 μm and 1 μm, approximately 0.001 μm and 5 μm, approximately 0.001 μm and 10 μm, approximately 0.001 μm and 50 μm, approximately 0.001 μm and 100 μm, approximately 0.001 μm and 250 μm, approximately 0.001 μm and 500 μm, approximately 0.001 μm and 1 mm, approximately 0.001 μm and 10 mm, or approximately 0.001 μm and 20 mm.

[0080] In some embodiments, the surface is coated with a material. The material may include, but is not limited to, any material known in the art, such as Teflon®, adhesive coatings, surfactants, anticoagulants such as heparin, EDTA, citrates, and oxalates, proteins, adhesion molecules, antibodies, factors that regulate cell function, nucleic acids, lipids, carbohydrates, lipid-carbohydrate complexes, or transmembrane proteins. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently bonded to the surface. In some embodiments, the material is non-covalently bonded to the surface. In some embodiments, surface molecules are released as cells pass through the pores.

[0081] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface is positively charged in some areas and negatively charged in others. In some embodiments, the surface has a positive or negative charge overall. In some embodiments, the surface can be smooth, electropolished, roughened, or plasma treated. In some embodiments, the surface contains dipole ions or dipole compounds.

[0082] IV. Cell suspension In certain embodiments, this disclosure relates to passing a cell suspension through a surface containing pores. In some embodiments, the cell suspension includes animal cells. In some embodiments, the cell suspension includes frog, chicken, insect, or nematode cells. In some embodiments, the cell suspension includes mammalian cells. In some embodiments, the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells. In some embodiments, the cells are human cells.

[0083] In some embodiments, the cell suspension includes cells with cell walls. In some embodiments, the cells are plant, yeast, fungal, algal, or bacterial cells. In some embodiments, the cells are plant cells. In some embodiments, the plant cells are crop, model, ornamental plant, vegetable, leguminous, coniferous, or grass plant cells. In some embodiments, the cells are yeast cells. In some embodiments, the yeast cells are Candida or Saccharomyces cells. In some embodiments, the cells are fungal cells. In some embodiments, the fungal cells are Aspergillus or Penicillium cells. In some embodiments, the cells are algal cells. In some embodiments, the algal cells are Chlamydomonas, Dunaliella, or Chlorella cells. In some embodiments, the cell suspension includes bacterial cells. In some embodiments, the bacterial cells are Gram-positive bacterial cells. In some embodiments, Gram-positive bacteria have a cell wall containing a thick peptidoglycan layer. In some embodiments, the bacterial cells are Gram-negative bacterial cells. Gram-negative bacteria have a cell wall containing a thin peptidoglycan layer between the inner cytoplasmic cell membrane and the outer membrane. In some embodiments, the bacterial cells are Streptococcus, Escherichia, Enterobacter, Bacillus, Pseudomonas, Klebsiella, or Salmonella cells.

[0084] The cell suspension may be a mixed or purified population of cells. In some embodiments, the cell suspension is a mixed population of cells such as whole blood, lymph, and / or peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell suspension is a purified population of cells. In some embodiments, the cells are primary cells or cells of a cell line. In some embodiments, the cells are blood cells. In some embodiments, the blood cells are immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, or DC2.4 dendritic cells. In some embodiments, the immune cells are primary human T cells. In some embodiments, the blood cells are red blood cells. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are cells of a cancer cell line such as HeLa cells. In some embodiments, the cancer cells are tumor cells. In some embodiments, the cancer cells are circulating tumor cells (CTCs). In some embodiments, the cells are stem cells. Examples of stem cells include, but are not limited to, induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hepatic stem cells, cardiomyocyte stem cells, neural stem cells, and hematopoietic stem cells. In some embodiments, the cells are fibroblasts such as primary fibroblasts or neonatal human foreskin fibroblasts (Nuff cells). In some embodiments, the cells are cells from immortalized cell lines such as HEK293 cells or CHO cells. In some embodiments, the cells are skin cells. In some embodiments, the cells are germ cells such as oocytes, egg cells, or zygotes. In some embodiments, the cells are nerve cells. In some embodiments, the cells are clusters of cells such as embryos, provided that the clusters of cells are not destroyed as they pass through the pores.

[0085] The composition of the cell suspension (e.g., molar osmotic concentration, salt concentration, serum content, cell concentration, pH, etc.) may affect the delivery of compounds. In some embodiments, the suspension contains whole blood. Alternatively, the cell suspension is a mixture of cells in saline or a physiological medium other than blood. In some embodiments, the cell suspension contains an aqueous solution. In some embodiments, the aqueous solution contains cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization. In some embodiments, the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO. Furthermore, the solution buffer may include one or more lubricants (pluronic or other surfactants) that can be designed to reduce or remove surface clogging and improve cell viability. Examples of surfactants include, but are not limited to, poloxamers, polysorbates, sugars such as mannitol, animal-derived serum, and albumin proteins.

[0086] In some configurations using certain types of cells, the cells can be incubated with one or more solutions that help deliver compounds into the cell interior. In some embodiments, the aqueous solution contains an agent that affects actin polymerization. In some embodiments, the agents that affect actin polymerization are lantrunculin A, cytochalasin, and / or colchicine. For example, the cells are incubated with lantrunculin A for 1 hour prior to delivery. The actin cytoskeleton can be depolymerized by incubation with a depolymerization solution (0.1 μg / ml). As an additional example, the microtubule network can be depolymerized by incubation with 10 μM colchicine (Sigma) for 2 hours prior to delivery.

[0087] The viscosity of the cell suspension may also affect the methods disclosed herein. In some embodiments, the viscosity of the cell suspension is approximately 8.9 × 10⁻⁶ -4 From Pa·seconds to approximately 4.0 × 10 -3The viscosity ranges to Pa·seconds or any value or range between them. In some embodiments, the viscosity is approximately 8.9 × 10⁻⁶. -4 From Pa·seconds to approximately 4.0 × 10 -3 Pa·sec, approximately 8.9×10 -4 From Pa·seconds to approximately 3.0 × 10 -3 Pa·sec, approximately 8.9×10 -4 From Pa·seconds to approximately 2.0 × 10 -3 Pa·seconds, or approximately 8.9 × 10⁻⁶ -3 From Pa·seconds to approximately 1.0 × 10 -3 The viscosity is in the range of any one of Pa·seconds. In some embodiments, viscosity is in the range of any one of about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about 0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some embodiments, a shear reduction effect is observed in which the viscosity of the cell suspension decreases under shear strain conditions. Viscosity can be measured by any method known in the art, including but not limited to glass capillary viscometers or rheometers. Viscometers measure viscosity under one flow condition, while rheometers are used to measure viscosity that varies with flow conditions. In some embodiments, viscosity is measured for shear reduction solutions such as blood. In some embodiments, viscosity is measured between about 0°C and about 45°C. For example, viscosity is measured at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), above physiological temperature (e.g., above about 37°C and 45°C or higher), below temperature (e.g., from about 0°C to about 4°C), or at temperatures in between these example temperatures.

[0088] V. Compounds to be delivered In certain embodiments, this disclosure relates to a method for delivering a compound into a cell. In some embodiments, the compound is a single compound. In some embodiments, the compound is a mixture of compounds. In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. Examples of nucleic acids include, but are not limited to, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, shRNA, self-replicating mRNA, and peptide nucleic acids. In some embodiments, the nucleic acid is homologous to intracellular nucleic acids. In some embodiments, the nucleic acid is heterogeneous to intracellular nucleic acids. In some embodiments, the nucleic acid comprises a transposon and, optionally, a sequence encoding a transposase. In some embodiments, the compound is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide. In some embodiments, the nucleic acid encodes a reporter or a selectable marker. Examples of reporter markers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), auquorin, beta-galactosidase, uroporphyrinogen III methyltransferase (UMT), and luciferase. Examples of selectable markers include, but are not limited to, blastosidine, G418 / genethecin, hygromycin B, puromycin, zeosin, adenine phosphoribosyltransferase, and thymidine kinase.

[0089] In some embodiments, the compound comprises a protein-nucleic acid complex. In some embodiments, the compound is a protein-nucleic acid complex. In some embodiments, protein-nucleic acid complexes such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 are used in genome editing applications. These complexes contain a sequence-specific DNA-binding domain in combination with a nonspecific DNA-cleaving nuclease. These complexes enable targeted genome editing, including the addition, disruption, or replacement of sequences of specific genes. In some embodiments, non-functional CRISPR is used to block or induce transcription of a target gene. In some embodiments, the compound comprises a Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA. In some embodiments, the compound comprises a nucleic acid containing a transposase protein and a transposon.

[0090] In some embodiments, the compound comprises a protein or polypeptide. In some embodiments, the compound is a protein or polypeptide. In some embodiments, the protein or polypeptide is a therapeutic protein, antibody, fusion protein, antigen, synthetic protein, reporter marker, or selectable marker. In some embodiments, the protein is a gene-editing protein or a nuclease such as a zinc finger nuclease (ZFN), activator-like effector nuclease (TALEN), meganuclease, CRE recombinase, FLP recombinase, R recombinase, integrase, or transposase. In some embodiments, the fusion protein may include, but is not limited to, a chimeric protein drug such as an antibody-drug conjugate or a recombinant fusion protein such as a protein tagged with GST or streptavidin. In some embodiments, the compound is a transcription factor. Examples of transcription factors include, but are not limited to, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORγt.

[0091] In some embodiments, the compound contains an antigen. In some embodiments, the compound is an antigen. An antigen is a substance that stimulates a specific immune response, such as a cell- or antibody-mediated immune response. Antigens bind to receptors expressed by immune cells, such as T cell receptors (TCRs), which are specific to a particular antigen. Subsequently, antigen-receptor binding triggers intracellular signaling pathways, leading to downstream immune effector pathways such as cell activation, cytokine production, cell migration, cytotoxic factor secretion, and antibody production. In some embodiments, the antigen originates from an exogenous source, such as bacteria, fungi, viruses, or allergens. In some embodiments, the antigen originates from an internal source, such as cancer cells or autoantigens (i.e., autoantigens). Autoantigens are antigens present on the organism's own cells. Autoantigens do not normally stimulate an immune response, but they may stimulate an immune response in situations such as autoimmune diseases, such as type 1 diabetes or rheumatoid arthritis. In some embodiments, the antigen is a novel antigen. A novel antigen is an antigen that is not present in the normal human genome but is produced in oncogenic cells as a result of tumor-specific DNA modifications that form novel protein sequences.

[0092] In some embodiments, a protein or polypeptide is the reporter or selectable marker. Examples of reporter markers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), aequorin, beta-galactosidase, uroporphyrinogen (UMT) III methyltransferase (UMT), and luciferase. Examples of selectable markers include, but are not limited to, blastosidine, G418 / genethecin, hygromycin B, puromycin, zeosin, adenine phosphoribosyltransferase, and thymidine kinase.

[0093] In some embodiments, the compound comprises an antibody. In some embodiments, the compound is an antibody. In some embodiments, the antibody is a full-length antibody or an antibody fragment. Antibodies for use in this disclosure include, but are not limited to, human or humanized antibodies, antibody variants, labeled antibodies, antibody fragments such as Fab or F(ab)2 fragments, single-domain antibodies, single-chain antibodies, multispecific antibodies, antibody fusion proteins, and immunoadhesins. The antibody may also be an isotype known in the art, including IgA, IgG, IgE, IgD, or IgM.

[0094] In some embodiments, the compound comprises small molecules. Examples of small molecules include, but are not limited to, fluorescent kerkers, dyes, pharmaceuticals, metabolites, adjuvants, or radionucleotides. In some embodiments, pharmaceuticals are therapeutic drugs and / or cytotoxic drugs. In some embodiments, adjuvants include CpG, oligodeoxyribonucleotides, R848, lipopolysaccharide (LPS), rhIL-2, anti-CD40 or CD40L, IL-12, cyclic dinucleotides, and interferon gene stimulator (STING) agonists. , but not limited to these.

[0095] In some embodiments, the compound comprises nanoparticles. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendrimers, and liposomes. In some embodiments, the nanoparticles contain therapeutic molecules. In some embodiments, the nanoparticles contain nucleic acids such as mRNA. In some embodiments, the nanoparticles contain labels such as fluorescent or radioactive labels.

[0096] In some embodiments, the compound comprises a chimeric antigen receptor (CAR). In some embodiments, the compound is a chimeric antigen receptor (CAR). In some embodiments, the CAR is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding, the intracellular signaling portion of the CAR can initiate an activation-related response in immune cells, such as the release of cytokines or cytolytic molecules. In some embodiments, the CAR is a chimeric T cell antigen receptor. In some embodiments, the CAR contains an antigen-binding domain specific to a tumor antigen. In some embodiments, the CAR antigen-binding domain is a single-chain antibody variable fragment (scFv).

[0097] In some embodiments, the compound includes a fluorescently tagged molecule. In some embodiments, the compound is a fluorescently tagged molecule, such as a molecule tagged with a fluorescent dye such as Pacific Blue, Alexa288, Cy5, or Cascade Blue. In some embodiments, the compound is a radioactive nucleotide, dextran particles, magnetic beads, or an impermeable dye. In some embodiments, the compound is a 3 kDa dextran particle labeled with PacBlue. In some embodiments, the compound is a 10 kDa dextran particle labeled with Alexa488. In some embodiments, the compound is a small molecule fluorophor-tagged protein. In some embodiments, the compound is a small molecule tagged with Alexa647.

[0098] In some embodiments, the compound comprises a virus or virus-like particles. In some embodiments, the compound is a virus or virus-like particles. In some embodiments, the virus is a retrovirus. In some embodiments, the virus is a therapeutic virus. In some embodiments, the virus is an oncolytic virus. In some embodiments, the virus or virus-like particles contain nucleic acids encoding therapeutic molecules such as therapeutic polypeptides.

[0099] In some embodiments, the delivered compound is purified. In some embodiments, the compound is at least about 60% by weight (dry weight) of the target compound. In some embodiments, the purified compound is at least about 75%, 90%, or 99% of the target compound. In some embodiments, the purified compound is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the target compound. Purity is determined by any known method, including but not limited to column chromatography, thin-layer chromatography, HPLC analysis, NMR, mass spectrometry, or SDS-PAGE. Purified DNA or RNA is defined as DNA or RNA free of exogenous nucleic acids, carbohydrates, and lipids.

[0100] VI. Cell perturbation In certain embodiments, the disclosure relates to passing a cell suspension through a pore-containing surface to deform the cells through the pores and produce cellular perturbations. Deformations can be caused, for example, by pressure induced by mechanical strain and / or shear forces. A cellular perturbation is a cellular tear (e.g., a hole, rupture, cavity, opening, pore, cut, gap, perforation) that allows material from outside the cell to move into the cell. In some embodiments, the perturbation is a perturbation within the cell membrane. In some embodiments, the perturbation is transient. In some embodiments, the cellular perturbation is approximately 1.0 × 10⁻⁶ -9 It lasts from seconds to about 2 hours, or any time in between or for a range of times. In some embodiments, the cellular perturbation is about 1.0 × 10⁻⁶ -9 The duration ranges from a few seconds to about one second, from about one second to about one minute, or from about one minute to about one hour. In some embodiments, the cellular perturbation is approximately 1.0 × 10⁻⁶ -9 From approximately 1.0 x 10 -1 , about 1.0×10 -9 From approximately 1.0 x 10 -2 , about 1.0×10 -9 From approximately 1.0 x 10 -3 , about 1.0×10 -9 From approximately 1.0 x 10 -4 , about 1.0×10 -9 From approximately 1.0 x 10-5 , about 1.0×10 -9 From approximately 1.0 x 10 -6 , about 1.0×10 -9 From approximately 1.0 x 10 -7 , or approximately 1.0 × 10 -9 From approximately 1.0 x 10 -8 It lasts for any one of the following seconds. In some embodiments, the cellular perturbation is approximately 1.0 × 10⁻⁶ -8 From approximately 1.0 x 10 -1 , about 1.0×10 -7 From approximately 1.0 x 10 -1 , about 1.0×10 -6 From approximately 1.0 x 10 -1 , about 1.0×10 -5 From approximately 1.0 x 10 -1 , about 1.0×10 -4 From approximately 1.0 x 10 -1 , about 1.0×10 -3 From approximately 1.0 x 10 -1 , or approximately 1.0 × 10 -2 From approximately 1.0 x 10 -1 It lasts for any one of the seconds. The cellular perturbations (e.g., pores or holes) produced by the methods described herein are not formed as a result of the assembly of protein subunits that form a porous structure of a multimer, such as one produced by complement or bacterial hemolysin.

[0101] As cells pass through surface pores, the deformation transiently imparts damage to the cell membrane, causing perturbative passive diffusion of substances. In some embodiments, cells are deformed for only a short time, approximately 100 μseconds, to minimize the possibility of activating apoptotic pathways through cellular signaling mechanisms, although other durations are possible (e.g., ranging from nanoseconds to several hours). In some embodiments, cells are deformed for approximately 1.0 × 10⁻¹⁶ -9 The cells are deformed for a period of time ranging from seconds to approximately 2 hours, or any time in between. In some embodiments, the cells are approximately 1.0 × 10⁻⁶ -9 The deformation lasts for approximately 1 second to 1 second, 1 second to 1 minute, or 1 minute to 1 hour. In some embodiments, the cells are approximately 1.0 × 10⁻⁶ -9 From approximately 1.0 x 10-1 , about 1.0×10 -9 From approximately 1.0 x 10 -2 , about 1.0×10 -9 From approximately 1.0 x 10 -3 , about 1.0×10 -9 From approximately 1.0 x 10 -4 , about 1.0×10 -9 From approximately 1.0 x 10 -5 , about 1.0×10 -9 From approximately 1.0 x 10 -6 , about 1.0×10 -9 From approximately 1.0 x 10 -7 , or approximately 1.0 × 10 -9 From approximately 1.0 x 10 -8 The deformation occurs for one of the following seconds. In some embodiments, the cell is approximately 1.0 × 10⁻⁶ -8 From approximately 1.0 x 10 -1 , about 1.0×10 -7 From approximately 1.0 x 10 -1 , about 1.0×10 -6 From approximately 1.0 x 10 -1 , about 1.0×10 -5 From approximately 1.0 x 10 -1 , about 1.0×10 -4 From approximately 1.0 x 10 -1 , about 1.0×10 -3 From approximately 1.0 x 10 -1 , or approximately 1.0 × 10 -2 From approximately 1.0 x 10 -1 The cell is deformed for any one of the following seconds. In some embodiments, deforming the cell includes deforming the cell for a time ranging from about 1 microsecond to about 750 milliseconds, for example, for at least about 1 microsecond, 10 microseconds, 50 microseconds, 100 microseconds, 500 microseconds, or 750 microseconds.

[0102] In some embodiments, the passage of the compound into the cell occurs simultaneously with the passage of the cell through pores and / or cellular perturbations. In some embodiments, the passage of the compound into the cell occurs after the cell has passed through the pores. In some embodiments, the passage of the compound into the cell occurs approximately a few minutes after the cell has passed through the surface pores. In some embodiments, the passage of the compound into the cell occurs approximately 1.0 × 10⁻¹⁶ minutes after the cell has passed through the pores.-2 This occurs from seconds to about 30 minutes later. For example, the passage of a compound into a cell takes about 1.0 × 10⁻¹⁰ minutes after the cell passes through the pore. -2 This occurs approximately 1 second to 1 second, approximately 1 second to approximately 1 minute, or approximately 1 minute to approximately 30 minutes later. In some embodiments, the passage of the compound into the cell occurs approximately 1.0 × 10⁻¹⁰ minutes after the compound has passed through the pores of the cell. -2 From seconds to approximately 10 minutes, approximately 1.0 × 10 -2 From seconds to approximately 5 minutes, approximately 1.0 × 10 -2 From seconds to approximately 1 minute, approximately 1.0 × 10 -2 From seconds to approximately 50 seconds, approximately 1.0 × 10 -2 From seconds to approximately 10 seconds, approximately 1.0 × 10 -2 From seconds to approximately 1 second, or approximately 1.0 × 10⁻⁶ -2 This occurs approximately 0.1 seconds after the cell passes through the pore. In some embodiments, the passage of the compound into the cell occurs approximately 1.0 × 10⁻¹⁶ seconds after the cell passes through the pore. -1 This occurs from seconds to about 10 minutes, from about 1 second to about 10 minutes, from about 10 seconds to about 10 minutes, from about 50 seconds to about 10 minutes, from about 1 minute to about 10 minutes, or from about 5 minutes to about 10 minutes. In some embodiments, cellular perturbations after it has passed through the pore are corrected within approximately 5 minutes after the cell has passed through the pore.

[0103] In some embodiments, cell viability after passing through surface pores is about 10 to about 100%. In some embodiments, cell viability after passing through pores is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, cell viability is about 1.0 × 10⁻⁶ after the cell passes through the pore. -2 It is measured from seconds to about 10 days later. For example, cell viability is measured approximately 1.0 × 10⁻¹⁶ days after the cell passes through the pore. -2 Cell viability is measured from seconds to approximately 1 second, from approximately 1 second to approximately 1 minute, from approximately 1 minute to approximately 30 minutes, or from approximately 30 minutes to approximately 2 hours later. In some embodiments, cell viability is measured from approximately 1.0 × 10⁻¹⁶ hours after the cell has passed through the pore. -2 From seconds to approximately 2 hours, approximately 1.0 × 10⁻¹⁰ -2 From seconds to approximately 1 hour, approximately 1.0 × 10⁻¹⁰ -2 From seconds to approximately 30 minutes, approximately 1.0 × 10 -2 From seconds to approximately 1 minute, approximately 1.0 × 10-2 From seconds to approximately 30 seconds, approximately 1.0 × 10 -2 From seconds to approximately 1 second, or approximately 1.0 × 10⁻⁶ -2 Cell viability is measured from about 1 second to about 0.1 seconds after the cell has passed through the pore. In some embodiments, cell viability is measured from about 1.5 hours to about 2 hours, from about 1 hour to about 2 hours, from about 30 minutes to about 2 hours, from about 15 minutes to about 2 hours, from about 1 minute to about 2 hours, from about 30 seconds to about 2 hours, or from about 1 second to about 2 hours after the cell has passed through the pore. In some embodiments, cell viability is measured from about 2 hours to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 10 days after the cell has passed through the pore.

[0104] VII. Delivery Parameters In certain embodiments, the present disclosure relates to a method for delivering a compound into cells, comprising the steps of passing a cell suspension through a surface containing pores, wherein the pores deform the cells and cause cellular perturbations, and contacting the cell suspension with the compound. The cell suspension may be contacted with the compound before, in parallel with, or after passing through the pores. The cells may pass through pores suspended in a solution containing the compound to be delivered, but the compound may be added to the cell suspension after the cells have passed through the pores. In some embodiments, the compound to be delivered is coated on a surface.

[0105] Several parameters can influence the delivery of compounds into cells. For example, pore size, pore entrance angle, pore surface properties (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), operating flow rate (e.g., cell migration time into the pore), cell concentration, concentration of the compound in the cell suspension, and the time it takes for cells to recover or incubate after passing through the pore can all affect the passage of the compound into cells. Further parameters influencing the delivery of compounds into cells may include cell velocity in the pore, shear rate in the pore, velocity component perpendicular to the flow rate, and time within the pore. Such parameters can be designed to control compound delivery. In some embodiments, the cell concentration is approximately 10 to 10 times the number of cells.12 This refers to any concentration or range of concentrations within the range of cells / ml. For example, the cell concentration is approximately 10 to 10 10 cells / ml, approximately 10 to 10 8 cells / ml, approximately 10 to 10 6 cells / ml, approximately 10 to 10 4 cells / ml or approximately 10 to 10 2 It is within the range of cells / ml. In some embodiments, the cell concentration is approximately 10 cells 2 From about 10 12 cells / ml, approximately 10 cells 4 From about 10 12 cells / ml, approximately 10 cells 6 From about 10 12 cells / ml, approximately 10 cells 8 From about 10 12 cells / ml or approximately 10 cells 10 From about 10 12The concentration of the delivered compound is within the range of particles / ml. The concentration of the delivered compound may be within the range of approximately 10 ng / ml to approximately 1 g / ml, or any concentration in between, or within a range of concentrations. For example, the compound concentration may be in the range of approximately 10 ng / ml to approximately 500 mg / ml, approximately 10 ng / ml to approximately 250 mg / ml, approximately 10 ng / ml to approximately 1 mg / ml, approximately 10 ng / ml to approximately 500 μg / ml, approximately 10 ng / ml to approximately 250 μg / ml, approximately 10 ng / ml to approximately 1 μg / ml, approximately 10 ng / ml to approximately 500 ng / ml, approximately 10 ng / ml to approximately 250 ng / ml, or approximately 10 ng / ml to approximately 100 ng / ml. In some embodiments, the compound concentration may be in the range of about 10 ng / ml to about 1 g / ml, about 250 ng / ml to about 1 g / ml, about 500 ng / ml to about 1 g / ml, about 1 μg / ml to about 1 g / ml, about 250 μg / ml to about 1 g / ml, about 500 μg / ml to about 1 g / ml, about 1 mg / ml to about 1 g / ml, about 250 mg / ml to about 1 g / ml, about 500 mg / ml to about 1 g / ml, or about 750 mg / ml to about 1 g / ml. The delivered compound concentration may be in the range of about 1 pM to about 2 M. For example, the compound concentration may be in the range of approximately 1 pM to approximately 1 M, approximately 1 pM to approximately 500 mM, approximately 1 pM to approximately 250 mM, approximately 1 pM to approximately 1 mM, approximately 1 pM to approximately 500 μM, approximately 1 pM to approximately 250 μM, approximately 1 pM to approximately 1 μM, approximately 1 pM to approximately 500 nM, approximately 1 pM to approximately 250 nM, approximately 1 pM to approximately 100 nM, approximately 1 pM to approximately 10 nM, approximately 1 pM to approximately 5 nM, approximately 1 pM to approximately 1 nM, approximately 1 pM to approximately 750 pM, approximately 1 pM to approximately 500 pM, approximately 1 pM to approximately 250 pM, approximately 1 pM to approximately 100 pM, approximately 1 pM to approximately 50 pM, approximately 1 pM to approximately 25 pM, or approximately 1 pM to approximately 10 pM. In some embodiments, the compound concentration may be in the range of about 5 pM to about 2 M, about 10 pM to about 2 M, about 25 pM to about 2 M, about 50 pM to about 2 M, about 100 pM to about 2 M, about 250 pM to about 2 M, about 500 pM to about 2 M, about 750 pM to about 2 M, about 1 nM to about 2 M, about 10 nM to about 2 M, about 100 nM to about 2 M, about 500 nM to about 2 M, about 1 μM to about 2 M, about 500 μM to about 2 M, about 1 mM to about 2 M, or about 500 mM to about 2 M.

[0106] The temperature used in the methods of this disclosure can be adjusted to affect compound delivery and cell viability. In some embodiments, the methods are carried out between approximately -5°C and approximately 45°C. For example, the methods can be carried out at room temperature (e.g., approximately 20°C), physiological temperature (e.g., approximately 37°C), temperatures higher than physiological temperature (e.g., above approximately 37°C and up to 45°C, or higher), or temperatures lower than physiological temperature (e.g., approximately -5°C to approximately 4°C), or temperatures in between these exemplary temperatures.

[0107] Various methods can be used to propel cells through the pores. For example, pressure can be applied at the inlet side by a pump (e.g., a gas cylinder or compressor), decompression can be applied at the outlet side by a decompression pump, capillary action can be applied through a tube, and / or gravity can be supplied to the system. A displacement-based flow system can also be used (e.g., syringe pump, peristaltic pump, manual syringe or pipette, piston, etc.). In some embodiments, cells are passed through the pores by positive or negative pressure. In some embodiments, cells are passed through the pores by constant or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump. In some embodiments, pressure is applied using decompression. In some embodiments, cells are passed through the pores by capillary pressure. In some embodiments, cells are passed through the pores by blood pressure. In some embodiments, cells are passed through the pores by g-force. In some embodiments, cells are passed through the pores under a pressure difference in the range of about 0.05 psi to about 500 psi, or any pressure or pressure range in between. In some embodiments, cells are passed through the pores under a pressure difference in the range of about 0.05 psi to about 500 psi, about 0.05 psi to about 400 psi, about 0.05 psi to about 300 psi, about 0.05 psi to about 200 psi, about 0.05 psi to about 100 psi, about 0.05 psi to about 50 psi, about 0.05 psi to about 25 psi, about 0.05 psi to about 10 psi, about 0.05 psi to about 5 psi, or about 0.05 psi to about 1 psi. In some embodiments, cells are passed through the pores under a pressure difference of at least about 5 psi, at least about 10 psi, or at least about 20 psi, or less than about 5 psi, less than about 10 psi, or less than about 20 psi. In some embodiments, cells are passed through pores under a pressure difference of at least about 2 psi, at least about 2.5 psi, or at least about 3 psi, or less than about 2 psi, less than about 2.5 psi, or less than about 3 psi.

[0108] In some embodiments, the fluid flow causes cells to pass through the pores. In some embodiments, the fluid flow is turbulent before the cells pass through the pores. Turbulent flow is fluid flow in which the velocity at a given time point varies irregularly in magnitude and direction. In some embodiments, the fluid flow through the pores is laminar flow. Laminar flow includes continuous flow in a fluid near a solid boundary where the direction of flow remains constant at any given time. In some embodiments, the fluid flow is turbulent after the cells have passed through the pores.

[0109] The speed at which cells pass through the pores may vary. In some embodiments, cells pass through the pores at a uniform cellular velocity. In some embodiments, cells pass through the pores at a fluctuating cellular velocity. Cells pass through the pores at a speed of at least about 0.1 mm / sec to about 5 m / sec, or any speed or range in between. In some embodiments, cells pass through the pores in the range of about 0.1 mm / sec to about 5 m / sec, about 1 mm / sec to about 5 m / sec, or about 1 m / sec to about 5 m / sec. In some embodiments, cells pass through the pores at speeds ranging from approximately 0.1 mm / sec to approximately 4 m / sec, approximately 0.1 mm / sec to approximately 3 m / sec, approximately 0.1 mm / sec to approximately 2 m / sec, approximately 0.1 mm / sec to approximately 1 m / sec, approximately 0.1 mm / sec to approximately 750 mm / sec, approximately 0.1 mm / sec to approximately 500 mm / sec, approximately 0.1 mm / sec to approximately 250 mm / sec, or approximately 0.1 mm / sec to approximately 1 mm / sec. In some embodiments, cells can pass through the pores at speeds faster than approximately 5 m / sec. Cell throughput ranges from less than approximately 1 cell per pore / second to approximately 10 cells per pore. 20 The rate can vary to more than cells per second. In some embodiments, the surface enables high-throughput cell processing of approximately billions of cells per second or minute.

[0110] In some embodiments, cells pass through pores in one direction. In some embodiments, cells pass through pores in more than one direction. For example, by using a syringe to aspirate cells through pores and then releasing them through pores, cells can be forced to pass through pores in one direction and then forced to pass through pores in another direction. In some embodiments, cells pass through a surface with more than one pore.

[0111] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0112] VIII. Applications In some embodiments, a compound or mixture of compounds is delivered to cells to produce a desired effect. In some embodiments, an antigen and / or immunostimulatory molecule is delivered to cells to produce professional antigen-presenting cells, such as dendritic cells, having improved activity levels compared to conventional stimulation methods. In some embodiments, a mixture of antigens is delivered to cells. In some embodiments, dendritic cells, T cells, or B cells are passed through a pore-containing surface and brought into contact with a solution containing the target antigen. The cells can be brought into contact with the antigen before, during, and / or after passing through the pore-containing surface. In some embodiments, the target antigen is a cancer cell antigen or a mixture of cancer cell antigens. In some embodiments, the antigen is an antigen of an infectious disease. In some embodiments, the antigen is an autoprotein antigen. For example, the delivered antigen may be a commonly expressed protein known to be associated with a particular disease, or a patient-specific antigen obtained from a biopsy. In some embodiments, the antigen-presenting cells produced by the methods disclosed herein contribute to an increase in the level of T and B cell-mediated immunity against the target antigen. Thus, such methods may be used as a means of activating the immune system in response to cancer or infection, or in vaccine development. One embodiment of the present invention involves a system in which dendritic cells, T cells, or B cells isolated from a patient's blood are processed ex vivo by the method of the present disclosure to activate them against a specific antigen and then reintroduce them into the patient's bloodstream. In some embodiments, the cancer antigen is from a patient with a hematological malignancy such as B-cell lymphoma, or a cancer such as melanoma or pancreatic cancer. In some embodiments, the cancer antigen is delivered directly to the DC cytoplasm, thereby utilizing the MHC-I antigen presentation pathway to induce a cytotoxic T cell (CTL) response in the patient. These activated T cells then seek out and destroy any cancerous cells expressing the target antigen. In some embodiments, the method can be performed by minimally trained technicians in a typical hospital laboratory. In some embodiments, a patient-operated treatment system can be used.In some embodiments, the method is performed using an inline blood treatment system in which blood is transferred directly from the patient, passes through a pore-containing surface to deliver the compound to blood cells, and is returned directly to the patient by injection after the procedure.

[0113] The methods of this disclosure may be useful for applications in gene therapy. In some embodiments, the methods of this disclosure are used to treat genetic diseases, replace defective gene products, or provide therapeutic nucleic acids. In some embodiments, gene therapy involves delivering nucleic acids encoding functional, therapeutic genes into cells to replace mutated genes. The genes are any genes recognized as beneficial. Representative examples include, for example, proteins or beneficial RNAs; viral proteins such as herpesthymidine kinase; and genes of mammalian origin encoding bacterial cholera toxin for cytotoxic therapy. In some embodiments, nucleic acids such as DNA or RNA are delivered to cells. Nucleic acids can be delivered into cells that are difficult to deliver to, such as stem cells, primary cells, and immune cells. Nucleic acids can include small nucleic acids or very large nucleic acids, such as plasmids or chromosomes. Quantitative delivery of a known amount of a gene construct into cells for studying the expression level of a gene of interest, and its sensitivity to concentration, can also be easily achieved. In some embodiments, delivery of a known amount of DNA sequence can be used together with a known amount of enzyme that enhances DNA recombination to achieve more efficient and stable delivery, homologous recombination, and site-directed mutation.

[0114] The methods and devices described herein may also be useful for quantitative RNA delivery for more efficient and definitive RNA studies. Some embodiments involve the delivery of small interfering RNA (siRNA) into the cytoplasm of cells. In some embodiments, RNA can be delivered into cells for RNA silencing without the need for liposomes. A known amount of RNA molecule can be delivered together with a known amount of dicer molecule to achieve standardized and efficient RNA levels across multiple cell lines in different states. In some embodiments, mRNA can be delivered into cells to study aspects of gene expression regulated at the post-transcriptional level. In some embodiments, a known amount of labeled RNA delivered to cells can be used to study the half-life of RNA in cells. In some embodiments, self-amplifying RNA (saRNA) is delivered to cells. saRNA expresses a protein of interest by cytoplasmically replicating their sequence without integration into the host genome. In some embodiments, the delivered saRNA encodes a desired antigen(s). In some embodiments, saRNA(s) are used to continuously modify cellular function, for example, through the expression of inhibitory, stimulative, or anti-apoptotic proteins.

[0115] For screening, imaging, or diagnostic purposes, the methods of this disclosure can be used to label cells. In some embodiments, a known amount of tagged protein can be delivered to study protein-protein interactions in the cellular environment. For immunostaining and fluorescence-based Western blotting, delivery of labeled antibodies into living cells can be achieved. Methods for labeling cells are performed by passing the cells through a pore-containing surface and bringing the cells into contact with a solution containing a detectable marker. In some embodiments, the detectable marker includes fluorescent molecules, radionuclides, quantum dots, gold nanoparticles, or magnetic beads. In some embodiments, engineered nanomaterials are delivered for applications in living cell imaging. In some embodiments, the compound is a fluorescently labeled and tagged small molecule for use in fluorescence resonance energy transfer (FRET) assays. FRET assays can be used to measure protein interactions for drug discovery applications.

[0116] In some embodiments, targeted cell differentiation is achieved by introducing proteins, mRNA, DNA, and / or growth factors to induce cell reprogramming and produce iPSCs, transgenic stem cell lines, or transgenic organisms. Methods described herein, such as the passage of stem cells or precursor cells, such as induced pluripotent stem cells (iPSCs), through a pore-containing surface, can be used to deliver differentiation factors into such cells. In some embodiments, after the uptake of the introduced factors, the cells proceed along a differentiation pathway determined by the introduced factors.

[0117] In some embodiments, sugars are delivered into cells to improve the cryopreservation of cells such as oocytes.

[0118] IX. Further Embodiments Any of the above methods can be performed in vitro, ex vivo, or in vivo. For in vivo applications, a device, such as an inline stent in an artery or vein, can be implanted in the vascular lumen. In some embodiments, this method is used as part of a bedside system for ex vivo treatment of patient cells and immediate reintroduction of cells into the patient.

[0119] In other embodiments, a combination treatment is used, for example, the method described herein and subsequently or prior to electroporation (an osmotic transfection of the type in which an electric current is used to create temporary holes in the cell membrane, allowing the entry of nucleic acids or macromolecules). In some embodiments, the cells pass through an electric field generated by at least one electrode located close to the surface. In some embodiments, the electrodes are located on one side of the surface. In some embodiments, the electrodes are located on both sides of the surface. In some embodiments, the electric field is between about 0.1 kV / m and about 100 MV / m, or any number or range in between. In some embodiments, an integrated circuit is used to send an electrical signal to activate the electrodes. In some embodiments, the cells are exposed to the electric field for a pulse width between about 1 nanosecond and about 1 second, and for a period between about 100 nanoseconds and about 10 seconds, or any time or range in between.

[0120] Various pretreatment and post-sorting assay techniques can also be utilized, thus enabling the development of continuous high-throughput assays for drug screening and diagnostic methods. In some embodiments, the methods disclosed herein can be performed in sequence with a fluorescence-activated cell sorting (FACS) module. This can enable real-time delivery and sorting of desired cells in the same system. For example, the methods and devices disclosed herein may also include sorter and / or sensor modules (e.g., optical, electrical, and magnetic). In some embodiments, a strainer or size sorter is placed upstream of the surface to separate cells before they pass through the surface. For example, the surface described herein can be lined up with a leukocyte electrophoresis device for separating leukocytes from blood samples. In further embodiments, the surface disclosed herein can be incorporated into a liquid handler or pipette. In some embodiments, the surface is implemented in a bioreactor format for cell culture and / or biomolecule production. Cells modified by the methods of this disclosure can be used in the bioproduction of drugs or other therapeutic molecules.

[0121] In some embodiments, the surface is formed within a tube through which a cell suspension flows. In some embodiments, the surface formed within the tube contains a gradient of pore sizes along its length, allowing for selective delivery to different cell types as the cell suspension flows.

[0122] Some aspects of the present disclosure include perturbed cells produced by passing a cell through a pore-containing surface, wherein the pores deform the cell, thereby causing a perturbation that allows a compound to enter the cell. In some embodiments, the cell passes through an electric field generated by at least one electrode adjacent to the surface.

[0123] X. Devices and Kits Some aspects of the present disclosure include a device for delivering compounds into cells, comprising a surface containing pores, wherein the pores are configured to allow cells suspended in a solution to pass through, and the pores deform the cells, thereby causing cellular perturbations that allow the compounds to enter the cells. The device may include any embodiment described for the methods disclosed above, including a surface having pores, a cell suspension, cellular perturbations, delivery parameters, and / or applications. In some embodiments, at least one electrode is located in proximity to the surface to generate an electric field.

[0124] In some embodiments, the device is TRANSWELL® or a permeable support. In some embodiments, TRANSWELL® can be used in applications for high-throughput screening. For example, a multi-well TRANSWELL® filter system can be used to screen different candidate drug compounds for specific functional or therapeutic endpoints. In some embodiments, each well contains a different compound to be tested. Cells pass through the TRANSWELL® filter pores, thereby causing cellular perturbation, and then enter the bottom side of the solution containing the compound or mixture of compounds to be delivered. These methods may be used as high-throughput methods for screening potential therapeutic compounds to identify novel treatments or to understand disease mechanisms. For example, a 96-well TRANSWELL® format can be used to perform high-throughput screening through the methods of this disclosure.

[0125] In some embodiments, the device includes a series of surfaces. These surfaces may be uniform or non-uniform in size, shape, and pore dimensions and / or number, thereby enabling the simultaneous delivery of various compounds to different cell types.

[0126] Kits or products for use in the manner disclosed herein are also provided. In some embodiments, the kit comprises the compositions described herein (e.g., pore-containing surfaces, cell suspensions and / or compounds to be delivered) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These products can be further sterilized and / or sealed.

[0127] This disclosure also provides a kit comprising elements of the methods described herein, which may further include instructions for performing the methods. The kits described herein may further include other materials, including other buffers, diluents, filters, needles, syringes, and accompanying documentation including instructions for performing any of the methods described herein.

[0128] XI. Exemplary Embodiments Embodiment 1. A method for delivering a compound into a cell, comprising the step of passing a cell suspension through a surface containing pores, wherein the pores deform the cell, thereby causing a perturbation of the cell that allows the compound to enter the cell, and the cell suspension comes into contact with the compound.

[0129] Embodiment 2. The method according to Embodiment 1, wherein the surface is a film.

[0130] Embodiment 3. The method according to Embodiment 1, wherein the surface is a filter.

[0131] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the surface is the surface of a meandering path.

[0132] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic.

[0133] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore.

[0134] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, micro-perforation punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing.

[0135] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the size of the pores correlates with the cell diameter.

[0136] Embodiment 9. The method according to any one of Embodiments 1 to 8, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.

[0137] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the width of the cross-sectional surface is in the range of about 1 mm to about 1 m.

[0138] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the width of the cross-section of the pore is in the range of about 0.01 μm to about 300 μm.

[0139] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm.

[0140] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm.

[0141] Embodiment 14. The method according to any one of Embodiments 1 to 11, wherein the width of the cross-section of the pore is approximately 200 μm.

[0142] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the size of the pores is heterogeneous.

[0143] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies in the range of 10 to 20%.

[0144] Embodiment 17. The method according to any one of Embodiments 1 to 14, wherein the size of the pores is homogeneous.

[0145] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the pore has the same inlet and outlet angles.

[0146] Embodiment 19. The method according to any one of Embodiments 1 to 17, wherein the pore has different inlet and outlet angles.

[0147] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal.

[0148] Embodiment 21. The method according to any one of Embodiments 1 to 19, wherein the shape of the cross-section of the pore is selected from cylindrical or conical.

[0149] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the edges of the pores are smooth.

[0150] Embodiment 23. The method according to any one of Embodiments 1 to 21, wherein the edges of the pores are sharp.

[0151] Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the passage of the pore is straight.

[0152] Embodiment 25. The method according to any one of Embodiments 1 to 23, wherein the passage of the pore is curved.

[0153] Embodiment 26. The method according to any one of Embodiments 1 to 25, wherein the pores constitute about 10 to 80% of the total surface area.

[0154] Embodiment 27. The method according to any one of Embodiments 1 to 26, wherein the surface contains a total of approximately 1.0 × 10⁵ to approximately 1.0 × 10³ pores.

[0155] Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the surface contains about 10 to about 1.0 × 10¹⁵ pores per 1 mm² of surface area.

[0156] Embodiment 29. The method according to any one of Embodiments 1 to 28, wherein the pores are distributed in parallel.

[0157] Embodiment 30. The method according to any one of Embodiments 1 to 29, wherein a plurality of surfaces are distributed in series.

[0158] Embodiment 31. The method according to any one of Embodiments 1 to 30, wherein the distribution of the pores is regular.

[0159] Embodiment 32. The method according to any one of Embodiments 1 to 30, wherein the distribution of the pores is random.

[0160] Embodiment 33. The method according to any one of Embodiments 1 to 32, wherein the thickness of the surface is uniform.

[0161] Embodiment 34. The method according to any one of Embodiments 1 to 32, wherein the thickness of the surface is not uniform.

[0162] Embodiment 35. The method according to any one of Embodiments 1 to 34, wherein the surface has a thickness of about 0.01 μm to about 5 m.

[0163] Embodiment 36. The method according to any one of Embodiments 1 to 35, wherein the surface has a thickness of approximately 10 μm.

[0164] Embodiment 37. The method according to any one of Embodiments 1 to 36, wherein the surface is covered with a material.

[0165] Embodiment 38. The method according to Embodiment 37, wherein the material is Teflon®.

[0166] Embodiment 39. The method according to Embodiment 37, wherein the material includes an adhesive coating that binds to cells.

[0167] Embodiment 40. The method according to Embodiment 37, wherein the material includes a surfactant.

[0168] Embodiment 41. The method according to Embodiment 37, wherein the material includes an anticoagulant.

[0169] Embodiment 42. The method according to Embodiment 37, wherein the material comprises a polypeptide.

[0170] Embodiment 43. The method according to Embodiment 37, wherein the material includes adhesive molecules.

[0171] Embodiment 44. The method according to Embodiment 37, wherein the material comprises an antibody.

[0172] Embodiment 45. The method according to Embodiment 37, wherein the material includes a factor that modulates cell function.

[0173] Embodiment 46. The method according to Embodiment 37, wherein the material comprises nucleic acid.

[0174] Embodiment 47. The method according to Embodiment 37, wherein the material includes lipids.

[0175] Embodiment 48. The method according to Embodiment 37, wherein the material includes carbohydrates.

[0176] Embodiment 49. The method according to Embodiment 37, wherein the material includes a composite.

[0177] Embodiment 50. The method according to Embodiment 49, wherein the complex is a lipid-carbohydrate complex.

[0178] Embodiment 51. The method according to Embodiment 37, wherein the material comprises a transmembrane protein.

[0179] Embodiment 52. The method according to any one of Embodiments 37 to 51, wherein the material is covalently bonded to the surface.

[0180] Embodiment 53. The method according to any one of Embodiments 37 to 51, wherein the material is non-covalently bonded to the surface.

[0181] Embodiment 54. The method according to any one of Embodiments 1 to 53, wherein the surface is hydrophilic.

[0182] Embodiment 55. The method according to any one of Embodiments 1 to 53, wherein the surface is hydrophobic.

[0183] Embodiment 56. The method according to any one of Embodiments 1 to 55, wherein the surface is charged.

[0184] Embodiment 57. The method according to any one of Embodiments 1 to 56, wherein the cell suspension comprises mammalian cells.

[0185] Embodiment 58. The method according to any one of Embodiments 1 to 57, wherein the cell suspension comprises a hybrid cell population.

[0186] Embodiment 59. The method according to any one of Embodiments 1 to 58, wherein the cell suspension is whole blood.

[0187] Embodiment 60. The method according to any one of Embodiments 1 to 58, wherein the cell suspension is lymph fluid.

[0188] Embodiment 61. The method according to any one of Embodiments 1 to 58, wherein the cell suspension comprises peripheral blood mononuclear cells.

[0189] Embodiment 62. The method according to any one of Embodiments 1 to 57, wherein the cell suspension comprises a purified cell population.

[0190] Embodiment 63. The method according to any one of Embodiments 1 to 58 or 62, wherein the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or erythrocytes.

[0191] Embodiment 64. The method according to Embodiment 63, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils.

[0192] Embodiment 65. The method according to any one of Embodiments 1 to 64, wherein the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells.

[0193] Embodiment 66. The method according to any one of Embodiments 1 to 64, wherein the cells are human cells.

[0194] Embodiment 67. The method according to any one of Embodiments 1 to 66, wherein the compound comprises a nucleic acid.

[0195] Embodiment 68. The embodiment according to any one of Embodiments 1 to 67, wherein the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA. The method.

[0196] Embodiment 69. The method according to any one of Embodiments 1 to 68, wherein the compound is a plasmid.

[0197] Embodiment 70. The method according to any one of Embodiments 1 to 66, wherein the compound comprises a polypeptide-nucleic acid complex.

[0198] Embodiment 71. The method according to any one of Embodiments 1 to 66 or 70, wherein the compound comprises a Cas9 protein and guide RNA or donor DNA.

[0199] Embodiment 72. The method according to any one of Embodiments 1 to 67, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide R NA or donor DNA.

[0200] Embodiment 73. The method according to any one of Embodiments 1 to 66, wherein the compound comprises a protein or a peptide.

[0201] Embodiment 74. The method according to any one of Embodiments 1 to 66 or 73, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase.

[0202] Embodiment 75. The method according to any one of Embodiments 1 to 66 or 73, wherein the compound is an antibody.

[0203] Embodiment 76. The method according to any one of Embodiments 1 to 66 or 73, wherein the compound is a transcription factor.

[0204] Embodiment 77. The method according to any one of Embodiments 1 to 66, wherein the compound is a small molecule.

[0205] Embodiment 78. The method according to any one of Embodiments 1 to 66, wherein the compound is a nanoparticle.

[0206] Embodiment 79. The method according to any one of Embodiments 1 to 66, wherein the compound is a chimeric antigen receptor.

[0207] Embodiment 80. The method according to any one of Embodiments 1 to 79, wherein the compound is a molecule tagged by fluorescence.

[0208] Embodiment 81. The method according to any one of Embodiments 1 to 66, wherein the compound is a liposome.

[0209] Embodiment 82. The method according to any one of Embodiments 1 to 81, wherein the cell suspension comes into contact with the compound before, simultaneously with, or after passing through the pores.

[0210] Embodiment 83. The method according to any one of Embodiments 1 to 81, wherein the delivered compound is coated on the surface.

[0211] Embodiment 84. The method according to any one of Embodiments 1 to 83, performed between 0°C and 45°C.

[0212] Embodiment 85. The method according to any one of Embodiments 1 to 84, wherein the cells pass through the pores due to positive or negative pressure.

[0213] Embodiment 86. The method according to any one of Embodiments 1 to 85, wherein the cells pass through the pores under constant or variable pressure.

[0214] Embodiment 87. The method according to any one of Embodiments 1 to 86, wherein pressure is applied using a syringe.

[0215] Embodiment 88. The method according to any one of Embodiments 1 to 86, wherein pressure is applied using a pump.

[0216] Embodiment 89. The method according to any one of Embodiments 1 to 86, wherein pressure is applied using depressurization.

[0217] Embodiment 90. The method according to any one of Embodiments 1 to 86, wherein the cells pass through the pores by capillary pressure.

[0218] Embodiment 91. The method according to any one of Embodiments 1 to 86, wherein the cells pass through the pores due to blood pressure.

[0219] Embodiment 92. The cell passes through the pore by g-force, as in Embodiment 1. The method described in any one of the above 86.

[0220] Embodiment 93. The method according to any one of Embodiments 1 to 92, wherein the cells pass through the pores under a pressure ranging from about 0.05 psi to about 500 psi.

[0221] Embodiment 94. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of about 2 psi.

[0222] Embodiment 95. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of approximately 2.5 psi.

[0223] Embodiment 96. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of approximately 3 psi.

[0224] Embodiment 97. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of about 5 psi.

[0225] Embodiment 98. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of about 10 psi.

[0226] Embodiment 99. The method according to any one of Embodiments 1 to 93, wherein the cells pass through the pores under a pressure of approximately 20 psi.

[0227] Embodiment 100. The method according to any one of Embodiments 1 to 99, wherein the cells are passed through the pores by the flow of a fluid.

[0228] Embodiment 101. The method according to Embodiment 100, wherein the fluid flow is turbulent before the cells pass through the pores.

[0229] Embodiment 102. The method according to Embodiment 100, wherein the fluid flow through the pores is laminar.

[0230] Embodiment 103. The method according to Embodiment 100, wherein the fluid flow is turbulent after the cells have passed through the pores.

[0231] Embodiment 104. The method according to any one of Embodiments 1 to 103, wherein the cells pass through the pores at a uniform cellular velocity.

[0232] Embodiment 105. The method according to any one of Embodiments 1 to 103, wherein the cells pass through the pores at a fluctuating cell velocity.

[0233] Embodiment 106. The method according to any one of Embodiments 1 to 105, wherein the cells pass through the pores at a speed ranging from about 0.1 mm / second to about 20 m / second.

[0234] Embodiment 107. The method according to any one of Embodiments 1 to 106, wherein the surface is contained within a larger module.

[0235] Embodiment 108. The method according to any one of Embodiments 1 to 107, wherein the surface is contained within a syringe.

[0236] Embodiment 109. The method according to any one of Embodiments 1 to 108, wherein the cell suspension comprises an aqueous solution.

[0237] Embodiment 110. The method according to Embodiment 109, wherein the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization.

[0238] Embodiment 111. The method according to Embodiment 110, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine.

[0239] Embodiment 112. The method according to Embodiment 110, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO.

[0240] Embodiment 113. The method according to any one of Embodiments 1 to 112, wherein the viscosity of the cell suspension is in the range of about 8.9 × 10⁻⁴ Pa·s to about 4.0 × 10⁻³ Pa·s.

[0241] Embodiment 114. The method according to any one of Embodiments 1 to 113, further comprising the step of passing the cells through an electric field generated by at least one electrode adjacent to the surface.

[0242] Embodiment 115. A device for delivering a compound into a cell, comprising a surface containing pores, wherein the pores are configured to allow cells suspended in a solution to pass through, and the pores cause perturbation of the cells, thereby deforming them and allowing the compound to enter them.

[0243] Embodiment 116. The device according to Embodiment 115, wherein the surface is a film.

[0244] Embodiment 117. The device according to Embodiment 115, wherein the surface is a filter.

[0245] Embodiment 118. The device according to any one of Embodiments 115 to 117, wherein the surface is the surface of a meandering path.

[0246] Embodiment 119. The device according to any one of Embodiments 115 to 118, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic.

[0247] Embodiment 120. The device according to any one of Embodiments 115 to 119, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore.

[0248] Embodiment 121. The device according to any one of Embodiments 115 to 120, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, micro-perforation punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing.

[0249] Embodiment 122. The device according to any one of Embodiments 115 to 121, wherein the size of the pores correlates with the cell diameter.

[0250] Embodiment 123. The device according to any one of Embodiments 115 to 122, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.

[0251] Embodiment 124. The device according to any one of Embodiments 115 to 123, wherein the width of the cross-sectional surface is in the range of about 1 mm to about 1 m.

[0252] Embodiment 125. The device according to any one of Embodiments 115 to 124, wherein the width of the cross-section of the pore is in the range of about 0.01 μm to about 300 μm.

[0253] Embodiment 126. The device according to any one of Embodiments 115 to 125, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm.

[0254] Embodiment 127. The device according to any one of Embodiments 115 to 126, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm.

[0255] Embodiment 128. The device according to any one of Embodiments 115 to 125, wherein the width of the cross-section of the pore is approximately 200 μm.

[0256] Embodiment 129. The device according to any one of Embodiments 115 to 128, wherein the size of the pores is heterogeneous.

[0257] Embodiment 130. The device according to any one of Embodiments 115 to 129, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies in the range of 10 to 20%.

[0258] Embodiment 131. The device according to any one of Embodiments 115 to 128, wherein the size of the pores is homogeneous.

[0259] Embodiment 132. The device according to any one of Embodiments 115 to 131, wherein the pores have the same inlet and outlet angles.

[0260] Embodiment 133. The device according to any one of embodiments 115 to 131, wherein the pore has different inlet and outlet angles.

[0261] Embodiment 134. The device according to any one of Embodiments 115 to 133, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal.

[0262] Embodiment 135. The device according to any one of Embodiments 115 to 133, wherein the shape of the pore is selected from cylindrical or conical.

[0263] Embodiment 136. The device according to any one of Embodiments 115 to 135, wherein the edges of the pores are smooth.

[0264] Embodiment 137. The device according to any one of Embodiments 115 to 135, wherein the edges of the pores are sharp.

[0265] Embodiment 138. The device according to any one of Embodiments 115 to 137, wherein the passage of the pore is straight.

[0266] Embodiment 139. The device according to any one of Embodiments 115 to 137, wherein the passage of the pore is curved.

[0267] Embodiment 140. The device according to any one of Embodiments 115 to 139, wherein the pores constitute about 10 to 80% of the total surface area.

[0268] Embodiment 141. The device according to any one of Embodiments 115 to 140, wherein the surface contains a total of approximately 1.0 × 10⁵ to approximately 1.0 × 10³ pores.

[0269] Embodiment 142. The device according to any one of Embodiments 115 to 141, wherein the surface contains approximately 10 to approximately 1.0 × 10¹⁵ pores per 1 mm² of surface area.

[0270] Embodiment 143. The device according to any one of Embodiments 115 to 142, wherein the pores are distributed in parallel.

[0271] Embodiment 144. A device according to any one of Embodiments 115 to 143, wherein multiple surfaces are distributed in series.

[0272] Embodiment 145. The device according to any one of Embodiments 115 to 144, wherein the distribution of the pores is regular.

[0273] Embodiment 146. The device according to any one of Embodiments 115 to 144, wherein the distribution of the pores is random.

[0274] Embodiment 147. The device according to any one of Embodiments 115 to 146, wherein the thickness of the surface is uniform.

[0275] Embodiment 148. The device according to any one of Embodiments 115 to 146, wherein the thickness of the surface is not uniform.

[0276] Embodiment 149. The device according to any one of Embodiments 115 to 148, wherein the surface has a thickness of about 0.01 μm to about 5 m.

[0277] Embodiment 150. The device according to any one of Embodiments 115 to 149, wherein the surface has a thickness of approximately 10 μm.

[0278] Embodiment 151. The device according to any one of Embodiments 115 to 150, wherein the surface is covered with a material.

[0279] Embodiment 152. The device according to Embodiment 151, wherein the material is Teflon®.

[0280] Embodiment 153. The device according to Embodiment 151, wherein the material includes an adhesive coating that binds to cells.

[0281] Embodiment 154. The device according to Embodiment 151, wherein the material comprises a surfactant.

[0282] Embodiment 155. The device according to Embodiment 151, wherein the material includes an anticoagulant.

[0283] Embodiment 156. The device according to Embodiment 151, wherein the material comprises a polypeptide.

[0284] Embodiment 157. The device according to Embodiment 151, wherein the material comprises adhesive molecules.

[0285] Embodiment 158. The device according to Embodiment 151, wherein the material comprises an antibody.

[0286] Embodiment 159. The device according to Embodiment 151, wherein the material contains a factor that modulates cellular function.

[0287] Embodiment 160. The device according to Embodiment 151, wherein the material comprises nucleic acid.

[0288] Embodiment 161. The device according to Embodiment 151, wherein the material includes lipids.

[0289] Embodiment 162. The device according to Embodiment 151, wherein the material comprises carbohydrates.

[0290] Embodiment 163. The device according to Embodiment 151, wherein the material includes a composite.

[0291] Embodiment 164. The device according to Embodiment 163, wherein the complex is a lipid-carbohydrate complex.

[0292] Embodiment 165. The device according to Embodiment 151, wherein the material comprises a transmembrane protein.

[0293] Embodiment 166. The device according to any one of Embodiments 151 to 165, wherein the material is covalently bonded to the surface.

[0294] Embodiment 167. The device according to any one of Embodiments 151 to 165, wherein the material is non-covalently bonded to the surface.

[0295] Embodiment 168. The device according to any one of Embodiments 115 to 167, wherein the surface is hydrophilic.

[0296] Embodiment 169. The device according to any one of Embodiments 115 to 167, wherein the surface is hydrophobic.

[0297] Embodiment 170. The device according to any one of Embodiments 115 to 169, wherein the surface is charged.

[0298] Embodiment 171. The device according to any one of Embodiments 115 to 170, wherein the cell suspension comprises mammalian cells.

[0299] Embodiment 172. The device according to any one of Embodiments 115 to 171, wherein the cell suspension comprises a hybrid cell population.

[0300] Embodiment 173. The device according to any one of Embodiments 115 to 172, wherein the cell suspension is whole blood.

[0301] Embodiment 174. The device according to any one of Embodiments 115 to 172, wherein the cell suspension is lymph fluid.

[0302] Embodiment 175. The device according to any one of Embodiments 115 to 172, wherein the cell suspension comprises peripheral blood mononuclear cells.

[0303] Embodiment 176. The device according to any one of Embodiments 115 to 171, wherein the cell suspension comprises a purified cell population.

[0304] Embodiment 177. The device according to any one of Embodiments 115 to 172 or 176, wherein the cells are immune cells, cells of a cell line, stem cells, tumor cells, fibroblasts, skin cells, nerve cells, or red blood cells.

[0305] Embodiment 178. The device according to Embodiment 177, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils.

[0306] Embodiment 179. The device according to any one of Embodiments 115 to 178, wherein the cells are mouse, dog, cat, horse, rat, goat, or rabbit cells.

[0307] Embodiment 180. The device according to any one of Embodiments 115 to 178, wherein the cells are human cells.

[0308] Embodiment 181. The device according to any one of Embodiments 115 to 180, wherein the compound comprises nucleic acid.

[0309] Embodiment 182. The device according to any one of Embodiments 115 to 181, wherein the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA.

[0310] Embodiment 183. The device according to any one of Embodiments 115 to 182, wherein the compound is a plasmid.

[0311] Embodiment 184. The device according to any one of Embodiments 115 to 180, wherein the compound comprises a polypeptide-nucleic acid complex.

[0312] Embodiment 185. The device according to any one of Embodiments 115 to 180 or 184, wherein the compound comprises a Cas9 protein and guide RNA or donor DNA.

[0313] Embodiment 186. The device according to any one of Embodiments 115 to 181, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA.

[0314] Embodiment 187. The device according to any one of Embodiments 115 to 180, wherein the compound comprises a protein or a peptide.

[0315] Embodiment 188. A device according to any one of Embodiments 115 to 180 or 187, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase.

[0316] Embodiment 189. The device according to any one of Embodiments 115 to 180 or 187, wherein the compound is an antibody.

[0317] Embodiment 190. The device according to any one of Embodiments 115 to 180 or 187, wherein the compound is a transcription factor.

[0318] Embodiment 191. The device according to any one of Embodiments 115 to 180, wherein the compound is a small molecule.

[0319] Embodiment 192. The device according to any one of Embodiments 115 to 180, wherein the compound is a nanoparticle.

[0320] Embodiment 193. The device according to any one of Embodiments 115 to 180, wherein the compound is a chimeric antigen receptor.

[0321] Embodiment 194. The device according to any one of Embodiments 115 to 193, wherein the compound is a molecule tagged by fluorescence.

[0322] Embodiment 195. The device according to any one of Embodiments 115 to 180, wherein the compound is a liposome.

[0323] Embodiment 196. The device according to any one of Embodiments 115 to 195, wherein the cell suspension comes into contact with the compound before, simultaneously with, or after passing through the pores.

[0324] Embodiment 197. The device according to any one of Embodiments 115 to 195, wherein the delivered compound is coated on the surface.

[0325] Embodiment 198. The device is the device according to any one of Embodiments 115 to 197, wherein the temperature range is between 0°C and 45°C.

[0326] Embodiment 199. The device according to any one of Embodiments 115 to 198, wherein the cells pass through the pores by positive or negative pressure.

[0327] Embodiment 200. The device according to any one of Embodiments 115 to 199, wherein the cells pass through the pores under constant or variable pressure.

[0328] Embodiment 201. The device according to any one of Embodiments 115 to 200, wherein pressure is applied using a syringe.

[0329] Embodiment 202. The device according to any one of Embodiments 115 to 200, wherein pressure is applied using a pump.

[0330] Embodiment 203. The device according to any one of Embodiments 115 to 200, wherein pressure is applied using depressurization.

[0331] Embodiment 204. The device according to any one of Embodiments 115 to 200, wherein the cells pass through the pores by capillary pressure.

[0332] Embodiment 205. The device according to any one of Embodiments 115 to 200, wherein the cells pass through the pores due to blood pressure.

[0333] Embodiment 206. The device according to any one of Embodiments 115 to 200, wherein the cells pass through the pores by g-force.

[0334] Embodiment 207. The device according to any one of Embodiments 115 to 206, wherein the cells pass through the pores under a pressure ranging from about 0.05 psi to about 500 psi.

[0335] Embodiment 208. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of about 2 psi.

[0336] Embodiment 209. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of approximately 2.5 psi.

[0337] Embodiment 210. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of about 3 psi.

[0338] Embodiment 211. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of approximately 5 psi.

[0339] Embodiment 212. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of about 10 psi.

[0340] Embodiment 213. The device according to any one of Embodiments 115 to 207, wherein the cells pass through the pores under a pressure of about 20 psi.

[0341] Embodiment 214. A device according to any one of Embodiments 115 to 213, wherein the cells are passed through the pores by the flow of a fluid.

[0342] Embodiment 215. The device according to Embodiment 214, wherein the fluid flow is turbulent before the cells pass through the pores.

[0343] Embodiment 216. The device according to Embodiment 214, wherein the fluid flow through the pores is laminar.

[0344] Embodiment 217. The device according to Embodiment 214, wherein the fluid flow is turbulent after the cells have passed through the pores.

[0345] Embodiment 218. The device according to any one of Embodiments 115 to 217, wherein the cells pass through the pores at a uniform cellular velocity.

[0346] Embodiment 219. The device according to any one of Embodiments 115 to 217, wherein the cells pass through the pores at a fluctuating cell velocity.

[0347] Embodiment 220. The device according to any one of Embodiments 115 to 219, wherein the cells pass through the pores at a speed ranging from about 0.1 mm / second to about 20 m / second.

[0348] Embodiment 221. The device according to any one of Embodiments 115 to 220, wherein the surface is contained within a larger module.

[0349] Embodiment 222. The device according to any one of Embodiments 115 to 221, wherein the surface is contained within a syringe.

[0350] Embodiment 223. The device according to any one of Embodiments 115 to 222, wherein the cell suspension comprises an aqueous solution.

[0351] Embodiment 224. The device according to Embodiment 223, wherein the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization.

[0352] Embodiment 225. The device according to Embodiment 224, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine.

[0353] Embodiment 226. The device according to Embodiment 224, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO.

[0354] Embodiment 227. The device according to any one of Embodiments 115 to 226, wherein the viscosity of the cell suspension is in the range of about 8.9 × 10⁻⁴ Pa·s to about 4.0 × 10⁻³ Pa·s.

[0355] Embodiment 228. A device according to any one of Embodiments 115 to 227, comprising a plurality of surfaces.

[0356] Embodiment 229. The device according to any one of Embodiments 115 to 228, wherein the surface is a transwell.

[0357] Embodiment 230. The device according to any one of embodiments 115 to 229, wherein at least one electrode is in close proximity to the surface and generates an electric field.

[0358] Embodiment 231. A cell comprising a perturbation, wherein the cell is generated by passing the cell through a surface containing pores, and the pores deform the cell, thereby causing a perturbation that allows a compound to enter the cell.

[0359] Embodiment 232. The cell according to Embodiment 231, wherein the surface is a membrane.

[0360] Embodiment 233. The cell according to Embodiment 231, wherein the surface is a filter.

[0361] Embodiment 234. A cell according to any one of Embodiments 231 to 233, wherein the surface is the surface of a meandering pathway.

[0362] Embodiment 235. A cell according to any one of Embodiments 231 to 234, wherein the surface comprises a material selected from one of polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, nylon, polyester, polyethersulfone, polytetrafluoroethylene, graphite, and ceramic.

[0363] Embodiment 236. A cell according to any one of Embodiments 231 to 235, wherein the entrance to the pore is wider than the pore, narrower than the pore, or the same width as the pore.

[0364] Embodiment 237. A cell according to any one of Embodiments 231 to 236, wherein the surface is manufactured using a method selected from etching, track etching, lithography, laser ablation, stamping, microporous punching, polymer sponge, direct foam molding, extrusion molding, and hot embossing.

[0365] Embodiment 238. A cell according to any one of Embodiments 231 to 237, wherein the width of the cross-section of the pore correlates with the cell diameter.

[0366] Embodiment 239. A cell according to any one of Embodiments 231 to 238, wherein the width of the cross-section of the pore is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.

[0367] Embodiment 240. A cell according to any one of Embodiments 231 to 239, wherein the width of the cross-sectional surface is in the range of about 1 mm to about 1 m.

[0368] Embodiment 241. A cell according to any one of Embodiments 231 to 240, wherein the width of the cross-section of the pore is in the range of about 0.01 μm to about 300 μm.

[0369] Embodiment 242. A cell according to any one of Embodiments 231 to 241, wherein the width of the cross-section of the pore is in the range of about 0.01 to about 35 μm.

[0370] Embodiment 243. A cell according to any one of Embodiments 231 to 242, wherein the width of the cross-section of the pore is approximately 0.4 μm, approximately 4 μm, approximately 5 μm, approximately 8 μm, approximately 10 μm, approximately 12 μm, or approximately 14 μm.

[0371] Embodiment 244. A cell according to any one of Embodiments 231 to 241, wherein the width of the cross-section of the pore is approximately 200 μm.

[0372] Embodiment 245. A cell according to any one of Embodiments 231 to 244, wherein the pore size is heterogeneous.

[0373] Embodiment 246. A cell according to any one of Embodiments 231 to 245, wherein the width of the cross-sectional area of ​​the heterogeneous pores varies in the range of 10 to 20%.

[0374] Embodiment 247. A cell according to any one of Embodiments 231 to 244, wherein the pore size is homogeneous.

[0375] Embodiment 248. A cell according to any one of Embodiments 231 to 247, wherein the pores have the same entrance angle and exit angle.

[0376] Embodiment 249. The cell according to any one of Embodiments 231 to 247, wherein the pores have different inlet and outlet angles.

[0377] Embodiment 250. A cell according to any one of Embodiments 231 to 249, wherein the shape of the cross-section of the pore is selected from the shapes of annular, circular, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal.

[0378] Embodiment 251. A cell according to any one of Embodiments 231 to 249, wherein the shape of the pores is selected from cylindrical or conical.

[0379] Embodiment 252. The cell according to any one of Embodiments 231 to 251, wherein the edges of the pores are smooth.

[0380] Embodiment 253. A cell according to any one of Embodiments 231 to 251, wherein the edges of the pores are sharp.

[0381] Embodiment 254. A cell according to any one of Embodiments 231 to 253, wherein the pore passages are straight.

[0382] Embodiment 255. The cell according to any one of Embodiments 231 to 253, wherein the pore passages are curved.

[0383] Embodiment 256. A cell according to any one of Embodiments 231 to 255, wherein the pores constitute about 10 to 80% of the total surface area.

[0384] Embodiment 257. A cell according to any one of Embodiments 231 to 256, wherein the surface contains a total of approximately 1.0 × 10⁵ to approximately 1.0 × 10³ pores.

[0385] Embodiment 258. A cell according to any one of Embodiments 231 to 257, wherein the surface contains about 10 to about 1.0 × 10¹⁵ pores per 1 mm² of surface area.

[0386] Embodiment 259. A cell according to any one of Embodiments 231 to 258, wherein the pores are distributed in parallel.

[0387] Embodiment 260. A cell according to any one of Embodiments 231 to 259, wherein multiple surfaces are distributed in series.

[0388] Embodiment 261. A cell according to any one of Embodiments 231 to 260, wherein the distribution of the pores is regular.

[0389] Embodiment 262. A cell according to any one of Embodiments 231 to 260, wherein the distribution of the pores is random.

[0390] Embodiment 263. A cell according to any one of Embodiments 231 to 262, wherein the thickness of the surface is uniform.

[0391] Embodiment 264. A cell according to any one of Embodiments 231 to 262, wherein the thickness of the surface is not uniform.

[0392] Embodiment 265. A cell according to any one of Embodiments 231 to 264, wherein the surface has a thickness of about 0.01 μm to about 5 m.

[0393] Embodiment 266. A cell according to any one of Embodiments 231 to 265, wherein the surface has a thickness of approximately 10 μm.

[0394] Embodiment 267. A cell according to any one of Embodiments 231 to 266, wherein the surface is coated with a material.

[0395] Embodiment 268. The cell according to Embodiment 267, wherein the material is Teflon®.

[0396] Embodiment 269. The cell according to Embodiment 267, wherein the material includes an adhesive coating that binds to the cell.

[0397] Embodiment 270. The cell according to Embodiment 267, wherein the material comprises a surfactant.

[0398] Embodiment 271. The cell according to Embodiment 267, wherein the material comprises an anticoagulant.

[0399] Embodiment 272. The cell according to Embodiment 267, wherein the material comprises polypeptide.

[0400] Embodiment 273. The cell according to Embodiment 267, wherein the material comprises an adhesion molecule.

[0401] Embodiment 274. The cell according to Embodiment 267, wherein the material comprises an antibody.

[0402] Embodiment 275. The cell according to Embodiment 267, wherein the material contains a factor that modulates cell function.

[0403] Embodiment 276. The cell according to Embodiment 267, wherein the material comprises nucleic acid.

[0404] Embodiment 277. The cell according to Embodiment 267, wherein the material comprises lipids.

[0405] Embodiment 278. The cell according to Embodiment 267, wherein the material comprises carbohydrates.

[0406] Embodiment 279. The cell according to Embodiment 267, wherein the material comprises a complex.

[0407] Embodiment 280. The cell according to Embodiment 279, wherein the complex is a lipid-carbohydrate complex.

[0408] Embodiment 281. The cell according to Embodiment 267, wherein the material comprises a transmembrane protein.

[0409] Embodiment 282. A cell according to any one of Embodiments 267 to 281, wherein the material is covalently bonded to the surface.

[0410] Embodiment 283. A cell according to any one of Embodiments 267 to 281, wherein the material is non-covalently bonded to the surface.

[0411] Embodiment 284. A cell according to any one of Embodiments 231 to 283, wherein the surface is hydrophilic.

[0412] Embodiment 285. The cell according to any one of Embodiments 231 to 283, wherein the surface is hydrophobic.

[0413] Embodiment 286. A cell according to any one of Embodiments 231 to 285, wherein the surface is charged.

[0414] Embodiment 287. The cell according to any one of Embodiments 231 to 286, wherein the cell is a mammalian cell.

[0415] Embodiment 288. The cell according to any one of Embodiments 231 to 287, wherein the cell is an immune cell, a cell line cell, a stem cell, a tumor cell, a fibroblast, a skin cell, a nerve cell, or a red blood cell.

[0416] Embodiment 289. The cells according to Embodiment 288, wherein the immune cells are T cells, B cells, dendritic cells, monocytes, macrophages, eosinophils, basophils, NK cells, NKT cells, mast cells, or neutrophils.

[0417] Embodiment 290. The cell according to any one of Embodiments 231 to 289, wherein the cell is a mouse, dog, cat, horse, rat, goat, or rabbit cell.

[0418] Embodiment 291. The cell according to any one of Embodiments 231 to 289, wherein the cell is a human cell.

[0419] Embodiment 292. A cell according to any one of Embodiments 231 to 291, wherein the compound comprises nucleic acid.

[0420] Embodiment 293. A cell according to any one of Embodiments 231 to 292, wherein the compound comprises a nucleic acid encoding DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, miRNA, lncRNA, tRNA, shRNA, or self-amplified mRNA.

[0421] Embodiment 294. A cell according to any one of Embodiments 231 to 293, wherein the compound is a plasmid.

[0422] Embodiment 295. A cell according to any one of Embodiments 231 to 291, wherein the compound comprises a polypeptide-nucleic acid complex.

[0423] Embodiment 296. A cell according to any one of Embodiments 231 to 291 or 295, wherein the compound comprises a Cas9 protein and guide RNA or donor DNA.

[0424] Embodiment 297. A cell according to any one of Embodiments 231 to 292, wherein the compound comprises a nucleic acid encoding the Cas9 protein and guide RNA or donor DNA.

[0425] Embodiment 298. A cell according to any one of Embodiments 231 to 291, wherein the compound comprises a protein or a peptide.

[0426] Embodiment 299. A cell according to any one of Embodiments 231 to 291 or 298, wherein the compound comprises a nuclease, a TALEN protein, a zinc finger nuclease, a meganuclease, a CRE recombinase, an FLP recombinase, an R recombinase, an integrase, or a transposase.

[0427] Embodiment 300. A cell according to any one of Embodiments 231 to 291 or 298, wherein the compound is an antibody.

[0428] Embodiment 301. A cell according to any one of Embodiments 231 to 291 or 298, wherein the compound is a transcription factor.

[0429] Embodiment 302. The cell according to any one of Embodiments 231 to 291, wherein the compound is a small molecule.

[0430] Embodiment 303. A cell according to any one of Embodiments 231 to 291, wherein the compound is a nanoparticle.

[0431] Embodiment 304. A cell according to any one of Embodiments 231 to 291, wherein the compound is a chimeric antigen receptor.

[0432] Embodiment 305. A cell according to any one of Embodiments 231 to 304, wherein the compound is a molecule tagged by fluorescence.

[0433] Embodiment 306. A cell according to any one of Embodiments 231 to 291, wherein the compound is a liposome.

[0434] Embodiment 307. A cell according to any one of Embodiments 231 to 306, wherein the cell comes into contact with the compound before, simultaneously with, or after passing through the pore.

[0435] Embodiment 308. A cell according to any one of Embodiments 231 to 306, wherein the delivered compound is coated on the surface.

[0436] Embodiment 309. The cell according to any one of Embodiments 231 to 308, wherein the cell passes through the pore between 0°C and 45°C.

[0437] Embodiment 310. The cell according to any one of Embodiments 231 to 309, wherein the cell passes through the pore by positive or negative pressure.

[0438] Embodiment 311. The cell according to any one of Embodiments 231 to 310, wherein the cell passes through the pore at a constant or variable pressure.

[0439] Embodiment 312. The cells according to any one of Embodiments 231 to 311, wherein pressure is applied using a syringe.

[0440] Embodiment 313. A cell according to any one of Embodiments 231 to 311, wherein pressure is applied using a pump.

[0441] Embodiment 314. A cell according to any one of Embodiments 231 to 311, wherein pressure is applied using decompression.

[0442] Embodiment 315. The cell according to any one of Embodiments 231 to 311, wherein the cell passes through the pore by capillary pressure.

[0443] Embodiment 316. The cell according to any one of Embodiments 231 to 311, wherein the cell passes through the pore by blood pressure.

[0444] Embodiment 317. The cell according to any one of Embodiments 231 to 311, wherein the cell passes through the pore by g-force.

[0445] Embodiment 318. The cell according to any one of Embodiments 231 to 317, wherein the cell passes through the pore under a pressure ranging from about 0.05 psi to about 500 psi.

[0446] Embodiment 319. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 2 psi.

[0447] Embodiment 320. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 2.5 psi.

[0448] Embodiment 321. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 3 psi.

[0449] Embodiment 322. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 5 psi.

[0450] Embodiment 323. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 10 psi.

[0451] Embodiment 324. The cell according to any one of Embodiments 231 to 318, wherein the cell passes through the pore under a pressure of about 20 psi.

[0452] Embodiment 325. A cell according to any one of Embodiments 231 to 324, wherein the cell is passed through the pore by the flow of a fluid.

[0453] Embodiment 326. The cell according to Embodiment 325, wherein the fluid flow is turbulent before the cell passes through the pore.

[0454] Embodiment 327. The cell according to Embodiment 325, wherein the fluid flow through the pores is laminar.

[0455] Embodiment 328. The cell according to Embodiment 325, wherein the fluid flow is turbulent after the cell has passed through the pore.

[0456] Embodiment 329. The cell according to any one of Embodiments 231 to 328, wherein the cell passes through the pore at a speed in the range of about 0.1 mm / second to about 5 m / second.

[0457] Embodiment 330. The cell according to any one of embodiments 231 to 329, wherein the surface is contained within a larger module.

[0458] Embodiment 331. The cell according to any one of Embodiments 231 to 330, wherein the surface is contained within a syringe.

[0459] Embodiment 332. The cell according to any one of Embodiments 231 to 331, wherein the cell is in a cell suspension containing an aqueous solution.

[0460] Embodiment 333. The cells according to Embodiment 332, wherein the aqueous solution comprises a cell culture medium, PBS, salts, sugars, growth factors, animal-derived products, fillers, surfactants, lubricants, vitamins, proteins, chelating agents, and / or agents that affect actin polymerization.

[0461] Embodiment 334. The cell according to Embodiment 333, wherein the agent affecting actin polymerization is latruncrin A, cytochalasin, and / or colchicine.

[0462] Embodiment 335. The cells according to Embodiment 333, wherein the cell culture medium is DMEM, OptiMEM, IMDM, RPMI, or X-VIVO.

[0463] Embodiment 336. The cell according to any one of Embodiments 231 to 335, wherein the cell further passes through an electric field generated by at least one electrode adjacent to the surface. [Examples]

[0464] The following embodiments are given to illustrate various embodiments of the Disclosure and are not intended to limit the Disclosure. Those skilled in the art will readily understand that the Disclosure is suitable for achieving the objectives, the outcomes and benefits pointed out, and the objectives, outcomes and benefits inherent in this Spec. Any modifications and other uses therein that fall within the scope of the spirit of the Disclosure as defined by the claims will be conceivable to those skilled in the art.

[0465] (Example 1) Delivery of dextran particles to HeLa cells Introduction To evaluate filter-mediated molecular delivery into cells, HeLa cells mixed with fluorescent dextran particles were passed through a filter containing pores of a specified size, and intracellular particle delivery was assessed by FACS analysis.

[0466] material and method Polycarbonate membrane filters were obtained from STERLITECH® (PCT8013100). These polycarbonate filters are produced by exposing a carbonate film to charged particles in a nuclear reactor to create relatively uniform but randomly distributed pore sizes. The resulting pore diameters ranged from 0.010 μm to 35 μm. The filter pores were approximately 10 μm thick. Exemplary images of the polycarbonate filters and filter pores are shown in Figures 1A and 1B. In the studies described herein, filters with pore sizes of 8 μm, 10 μm, 12 μm, and 14 μm were used. Further materials used in filter delivery experiments are listed in Table 1. [Table 1]

[0467] HeLa cells, 4 × 10 6The cells were suspended in optiMEM medium at a concentration of 4 cells / mL. 100 μl of cells were pipetted for use as a negative control. To wet the membrane filter, the polycarbonate membrane filter was suspended in PBS using forceps. The cap of the plastic filter holder was removed, the filter was placed on the inner surface with the shiny side facing up, and the filter holder was recapped. 200 μl of cells mixed with dextran particles were added to the filter holder. Cell count: 4 × 10⁶ 6 Dextran was suspended at 0.2 mg / mL in 5 mL of cells / mL. A nitrogen gas delivery system with a pressure regulator was used for constant-pressure delivery. Filter flow-through was collected in 24-well plates or FACS tubes. Before FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C and washed to remove extracellular dextran. The following steps were taken to prepare for FACS analysis: First, FACS buffer was prepared (PBS + final concentration: 1% FBS + 2 nM EDTA), and propidium iodide was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Forward scattering, side scattering, and fluorescence channel voltages of flow cytometry were adjusted to visualize all cellular events, and 10,000 events were recorded per sample. Cells that mixed with dextran particles but did not pass through the filter were used as controls for particle delivery by endocytosis. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis control. The negative control sample was not in contact with the dye but was subjected to the same steps as the experimental sample.

[0468] result Delivery efficiency of dextran particles to HeLa cells through pores of 8 μm, 10 μm, 12 μm, and 14 μm sized filters. HeLa cells mixed with 3kDa PacBlue and 10kDa Alexa488 dextran particles were passed through pores of 8μm, 10μm, 12μm, and 14μm filters at low pressure (5psi, 10psi, or 20psi). Exemplary flow cytometry plots representing the cells after delivery are shown in Figures 2A–G. Figures 2A–C represent cells that passed through pores of 14μm (Figure 2A), 12μm (Figure 2B), and 10μm (Figure 2C) filters at 5psi. Figures 2E and G represent cells that passed through pores of a 12μm filter at 10psi (Figure 2E) and 20psi (Figure 2G). Figures 2D and F represent plots of endocytosis control (Figure 2D) and negative control (Figure 2F). Cells containing both 3kDa and 10kDa dextran particles after filter delivery are indicated by double-positive cell staining for PacBlue and Alexa488 (Q2 quadrant). The delivery efficiency, indicated by the percentage of cells positive for both 3kDa and 10kDa dextran particles after filter delivery, is shown in Table 2 below. As expected, the delivery efficiency of dextran particles varied with pore size and pressure fluctuations, with the highest delivery (86%) observed at 5 psi in 8 μm pores. [Table 2]

[0469] Cell viability was measured after filter delivery. Cell viability, expressed as the percentage of viable cells after filter delivery, is shown in Table 3 below. Cell viability of cells that passed through the filter decreased with increasing pressure, with 5 psi yielding the highest cell viability for each pore size. Cell viability was reduced in filtered cells compared to endocytosis controls. Cell viability differed as expected with pore size and pressure variations. [Table 3]

[0470] (Example 2) Delivery of dextran particles to human T cells through the pores of a 5 μm-sized filter. Introduction To evaluate the filter-mediated delivery of molecules into primary immune cells, primary human T cells mixed with fluorescent dextran particles were passed through the pores of a 5 μm-sized filter, and intracellular particle delivery was determined by FACS analysis. Filter-mediated delivery of dextran particles to primary human T cells was compared between manual syringe pressurization and constant pressure.

[0471] material and method Peripheral blood mononuclear cells (PBMCs) were first isolated from fresh human blood via Ficol separation. T cells were then separated from the PBMCs by negative selection using a magnetic column. The T cells were divided into 4 × 10⁶ cells. 6 The cells were suspended in optiMEM medium at a concentration of 4 cells / mL. 100 μl of cells were pipetted for use as a negative control. To wet the membrane filter, the polycarbonate membrane filter was suspended in PBS using forceps. The cap of the plastic filter holder was removed, the filter was placed on the inner surface with the shiny side facing up, and the filter holder was recapped. 200 μl of cells mixed with dextran particles were added to the filter holder. Cell count: 4 × 10⁶ 6Dextran was suspended at 0.2 mg / mL in 5 mL of cells / mL. A nitrogen gas delivery system with a pressure regulator was used for constant-pressure delivery. Filter flow-through was collected in 24-well plates or FACS tubes. Before FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C and washed to remove extracellular dextran. The following steps were taken to prepare for FACS analysis: First, FACS buffer was prepared (PBS + final concentration: 1% FBS + 2 nM EDTA), and propidium iodide was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Flow cytometry forward scatter, side scatter, and fluorescence channel voltages were adjusted to visualize all cellular events, and 10,000 events were recorded per sample. Cells that mixed with dextran particles but did not pass through the filter were used as controls for particle delivery by endocytosis. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis control. The negative control sample did not come into contact with the dye but was subjected to the same steps as the experimental sample.

[0472] result Primary human T cells mixed with 3kDa PacBlue and 10kDa Alexa488 dextran particles were passed through a 5μm-sized filter pore under manual syringe pressurization or a constant pressure of 5 psi. As shown by double-positive cell staining for PacBlue and Alexa488 (Q2 quadrant), 8.60% of cells passed under manual syringe pressurization (Figure 3A) and 18.9% of cells passed under a constant pressure of 5 psi (Figure 3B) contained both 3kDa and 10kDa dextran particles after filter delivery. 85.4% of cells passed under manual syringe pressurization (Figure 3A) and 83.1% of cells passed under a constant pressure of 5 psi (Figure 3B) remained viable after delivery.

[0473] (Example 3) Delivery of dextran particles to HeLa cells at 2 psi and 3 psi Introduction To evaluate filter-mediated molecular delivery into cells, HeLa cells mixed with fluorescent dextran particles were passed through a filter containing pores of a specified size, and intracellular particle delivery was assessed by FACS analysis.

[0474] material and method The polycarbonate membrane filter was obtained from STERLITECH®. In the studies described herein, filters with a pore size of 10 μm were used. HeLa cells were sampled in a cell size of 2.5 × 10⁶ cells. 6 Cells were suspended in OptiMEM medium at a concentration of 1 / mL, and 3kDa dextran particles were added to a final concentration of 0.1 mg / mL. Membrane filters and plastic filter holders were prepared as described in Example 1. 200 μl of cells mixed with dextran particles were added to the filter holder. A nitrogen gas delivery system with a pressure regulator was used for constant-pressure delivery. Filter flow-through was collected in 24-well plates or FACS tubes. Before FACS analysis, all samples were centrifuged three times at 400 Relative Centrifugal Force (rcf) for 4 minutes at 4°C, washed, and removed extracellular dextran. The following steps were taken to prepare for FACS analysis: First, FACS buffer was prepared (PBS + final concentration: 1% FBS + 2nM EDTA), and propidium iodide was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Flow cytometry was adjusted for forward scattering, side scattering, and fluorescence channel voltages to visualize all cellular events, and 10,000 events were recorded per sample. Cells mixed with dextran particles but not filtered were used as controls for particle delivery by endocytosis. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis controls. Negative control samples did not come into contact with the dye but were subjected to the same steps as the experimental samples. Three filters were used at each pressure (2 psi and 3 psi), with three samples flowing through each filter per experiment. The experiment was repeated over three separate days, and a total of 27 runs were performed at each pressure.

[0475] result Delivery efficiency of dextran particles to HeLa cells through pores of a 10 μm size filter at 2 psi and 3 psi. HeLa cells mixed with 3kDa PacBlue dextran particles were passed through 10 μm-sized filter pores at 2 psi and 3 psi. Delivery efficiency, indicated by the percentage of cells positive for 3kDa dextran particles, cell viability, indicated by the percentage of viable cells after filter delivery, and relative mean fluorescence intensity (rMFI) by flow cytometry are shown in Tables 4 (2 psi) and 5 (3 psi) below. Delivery efficiency, viability, and rMFI values ​​were reproducible and significant across the entire experiment. At 2 psi, cell viability and delivery efficiency were approximately 80%. At 3 psi, cell viability was approximately 60-70%, but delivery efficiency was approximately 90%. [Table 4] [Table 5]

[0476] (Example 4) Delivery of dextran particles to HeLa cells mediated by commercially available or custom-made syringe filters. Introduction To evaluate filter-mediated molecular delivery into cells, HeLa cells mixed with fluorescent dextran particles were passed through commercially available or custom-made syringe filters, and intracellular particle delivery was assessed by FACS analysis.

[0477] material and method A polycarbonate membrane filter (COTS filter) with 10 μm pores, using a commercially available filter and holder combination, was obtained from STERLITECH®. A custom-made syringe filter with 10 μm pores was also used.

[0478] HeLa cells 3 × 10 6 Cells were suspended in OptiMEM medium at a concentration of 1 / mL, and 3kDa dextran particles were added to a final concentration of 0.1 mg / mL. Membrane filters and plastic filter holders were prepared as described in Example 1. 200 μl of cells mixed with dextran particles were added to the filter holder. A nitrogen gas delivery system with a pressure regulator was used for constant-pressure delivery. Filter flow-through was collected in 24-well plates or FACS tubes. Before FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C and washed to remove extracellular dextran. The following steps were taken to prepare for FACS analysis: First, FACS buffer was prepared (PBS + final concentration: 1% FBS + 2nM EDTA), and propidium iodide was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Flow cytometry was adjusted to visualize all cellular events, with forward scattering, side scattering, and fluorescence channel voltages adjusted, and 10,000 events recorded per sample. Cells mixed with dextran particles but not filtered were used as controls for particle delivery by endocytosis. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis controls. Negative control samples did not come into contact with the dye but were subjected to the same steps as the experimental samples. One filter was used for each pressure (2 psi and 3 psi), and three samples flowed through each filter per experiment. The experiment was repeated over two separate days, and a total of six runs were performed at each pressure.

[0479] result Delivery efficiency of dextran particles to HeLa cells through commercially available or custom-made filters. HeLa cells mixed with 3kDa PacBlue dextran particles were passed through 10 μm sized filter pores at 2 psi and 3 psi. Results from COTS filters and custom-made filters were compared. Table 4 shows delivery efficiency, indicated by the percentage of cells positive for 3kDa dextran particles, and cell viability, indicated by the percentage of viable cells after filter delivery. Representative flow cytometry histogram plots demonstrating the mean fluorescence intensity (MFI) values ​​for three runs at 3 psi are shown in Figure 5A (COTS filter) and Figure 5B (custom-made syringe filter). The mean relative mean fluorescence intensity (rMFI) value for the entire run is shown in Figure 5C. Removal of the mesh insert between experiments performed on day 2 did not affect either delivery efficiency or cell viability. No advantage was observed when using the custom-made filter compared to the COTS filter, as a result of reduced dead volume. Overall, the COTS filter and the custom-made syringe filter yielded comparable cell viability and delivery efficiency. Subsequent experiments were performed using the COTS filter.

[0480] (Example 5) Filter-mediated delivery of EGFP mRNA to HeLa cells Introduction To evaluate cellular functionality after filter-mediated molecular delivery, HeLa cells mixed with fluorescent dextran particles and EGFP mRNA were passed through a COTS filter with a pore size of 10 μm, and intracellular particle delivery and mRNA expression were evaluated by FACS analysis.

[0481] material and method A polycarbonate membrane filter with a pore size of 10 μm, using a commercially available (COTS) filter and holder combination, was obtained from STERLITECH®. HeLa cells were placed in a 2 × 10⁶ cell range. 6The cells were suspended in OptiMEM medium at a concentration of 1 / mL, and 3kDa dextran particles were added at a final concentration of 0.1 mg / mL, followed by EGFP-encoding mRNA at a final concentration of 0.1 mg / mL. Membrane filters and plastic filter holders were prepared as described in Example 1. 200 μl of cells mixed with dextran particles were added to the filter holder and filtered at 2 psi, 2.5 psi, or 3 psi. A nitrogen gas delivery system with a pressure regulator was used for constant-pressure delivery. Flow-through was collected in 24-well plates or FACS tubes.

[0482] Prior to FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C, washed, and removed extracellular dextran. The following steps were taken to prepare for FACS analysis: First, the FACS buffer was prepared (PBS + final concentration: 1% FBS + 2nM). EDTA was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Flow cytometry forward scatter, side scatter, and fluorescence channel voltages were adjusted to visualize all cellular events, and 10,000 events were recorded per sample. Cells mixed with dextran particles or EGFP mRNA but not filtered were used as controls for endocytosis delivery. These cells came into contact with the dye or EGFP mRNA but did not pass through the filter. All other steps were the same as for the endocytosis controls. Negative control samples did not come into contact with either the dye or EGFP mRNA, but were subjected to the same steps as the experimental samples. Four samples were flowed through the filter at each pressure (2 psi, 2.5 psi, or 3 psi).

[0483] result HeLa cells mixed with 3kDa PacBlue dextran particles and EGFP mRNA were passed through 10 μm-sized filter pores at 2 psi, 2.5 psi, or 3 psi. Delivery efficiency, indicated by the percentage of cells positive for 3kDa dextran particles or GFP-expressing cells, cell viability, indicated by the percentage of viable cells after filter delivery, and relative mean fluorescence intensity (rMFI) by flow cytometry are shown in Figure 6 and Tables 6-8 below. Similar delivery efficiency and cell viability were observed at 2.5 psi and 3 psi, with higher delivery observed compared to 2 psi. As indicated by sustained viability and GFP expression, cells retained function 24 hours after co-delivery of dextran particles and EGFP mRNA. [Table 6] [Table 7] [Table 8]

[0484] (Example 6) Compression-mediated delivery of dextran and IgG antibodies to human RBCs Introduction To evaluate filter-mediated molecular delivery into anucleated cells, fluorescent dextran particles or human RBCs mixed with IgG antibody were passed through syringe filters containing pores of a specified size, and intracellular particle delivery was evaluated by FACS analysis.

[0485] material and method Polycarbonate membrane filters (COTS filters) with a 2 μm pore diameter, using a commercially available filter and holder combination, were obtained from STERLITECH®. Human RBCs were separated from whole blood or leukocyte-reduced color using the Ficoll gradient separation method and resuspended in Optimem at the desired concentration (50–500 M / mL). To wet the membrane filter, the polycarbonate membrane filter was suspended in Optimem using forceps. The cap of the plastic filter holder was removed, the filter was placed on the inner surface with the shiny side facing up, and the filter holder was recapped. Dextran particles and IgG antibody were suspended at 0.1 mg / mL. 1 mL of cells mixed with dextran particles or IgG antibody were added to the filter holder at room temperature. Pushing the syringe with a finger was used to pass the cells through the filter. Filter flow-through was collected in a 15 ml Falcon tube or FACS tube. Prior to FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C, washed, and removed extracellular dextran. The following steps were taken to prepare for FACS analysis: FACS buffer was prepared (PBS + final concentration: 1% FBS + 2 mM EDTA), 400 μL of FACS buffer was added per tube, and cells were resuspended. Flow cytometry forward scatter, side scatter, and fluorescence channel voltages were adjusted to visualize all cellular events, recording ≥10,000 events per sample. Cells mixed with dextran particles or IgG antibody but not filtered were used as controls for endocytosis delivery. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis controls.

[0486] result RBCs mixed with 10kDa Alexa Fluor® 647 dextran particles or 150kDa IgG antibody conjugated with Alexa Fluor® 647 were passed through a 2μm filter pore. Exemplary flow cytometry histogram plots showing fluorescence after compression-mediated delivery (SQZ) against an endocytosis control are shown in Figures 7A and 7B. The delivery efficiency of IgG antibody after filter delivery, indicated by the percentage of cells positive for dextran particles, is shown in Figure 8A. The delivery efficiency of IgG antibody after filter delivery was 88.6%, and the delivery efficiency of dextran particles was 95.6%.

[0487] Cell viability was measured after filter delivery. Figure 8B shows the estimated cell viability, expressed as the percentage of cells present in the FSC and SSC gates after filter delivery. The estimated viability of cells delivered with IgG antibody after filter delivery was 68.8%, while the estimated viability of cells delivered with dextran particles was 63.3%.

[0488] Compression-mediated delivery of dextran particles and IgG antibodies into human RBCs was achieved.

[0489] (Example 7) Compression-mediated delivery of dextran and IgG antibodies to mouse RBCs Introduction To evaluate filter-mediated molecular delivery into anucleated cells, fluorescent dextran particles or mouse RBCs mixed with IgG antibody were passed through syringe filters containing pores of a specified size, and intracellular particle delivery was evaluated by FACS analysis.

[0490] material and method Polycarbonate membrane filters (COTS filters) with pore diameters of 1 μm and 2 μm, using commercially available filter and holder combinations, were obtained from STERLITECH®. Whole blood was centrifuged and cells were resuspended in Optimem at the desired concentration (100–500 M / ml). To wet the membrane filter, the polycarbonate membrane filter was suspended in Optimem using forceps. The cap of the plastic filter holder was removed, the filter was placed on the inner surface with the shiny side facing up, and the filter holder was recapped. Dextran particles and IgG antibody were suspended at 0.1 mg / mL. 1 mL of cells mixed with dextran particles or IgG antibody was added to the filter holder at room temperature. Pushing the syringe with a finger was used to pass the cells through the filter. Alternatively, a controlled pressure system was used to pass 200 μL of cells mixed with dextran particles or IgG antibody through the filter. The filter flow-through was collected in a 15 ml Falcon tube or FACS tube. Prior to FACS analysis, all samples were centrifuged three times at 1000 rcf for 10 minutes at 24°C, washed, and removed extracellular dextran. The following steps were taken to prepare for FACS analysis: FACS buffer was prepared (PBS + final concentration: 1% FBS + 2 mM). Cells were resuspended in EDTA, with 400 μL of FACS buffer per tube. Flow cytometry forward scatter, side scatter, and fluorescence channel voltages were adjusted to visualize all cellular events, and ≥10,000 events were recorded per sample. Cells mixed with dextran particles or IgG antibody but not filtered were used as controls for endocytosis delivery. These cells came into contact with the dye but did not pass through the filter. All other steps were the same as for the endocytosis controls. NC (non-contact) samples did not come into contact with the dye but were subjected to the same steps as the experimental samples but did not pass through the filter.

[0491] result Mouse RBCs mixed with IgG antibody (150 kDa) were passed through 1 μm and 2 μm sized filter pores using manual syringe pressurization. Exemplary flow cytometry histogram plots showing fluorescence after compression-mediated delivery (SQZ) for endocytosis (Endo), negative (NC), and no-material controls are shown in Figure 9A. The "no-material" controls did not come into contact with the IgG antibody but were subjected to the same steps as the experimental samples and passed through the filters.

[0492] Cell viability was measured after filter delivery. Figure 9B shows the estimated cell viability, indicated by the percentage of cells present in the FSC and SSC gates after filter delivery of IgG antibody using manual syringe pressurization. Figure 9C shows the delivery efficiency of IgG antibody, indicated by the percentage of cells positive for IgG antibody after filter delivery using manual syringe pressurization.

[0493] Using a controlled pressure system below 2 psi, 4 psi, 6 psi, 10 psi, and 20 psi, or by manual syringe pressurization, RBCs mixed with 70 kDa Alexa Fluor® 488 dextran particles or IgG antibody (150 kDa) were passed through a 2 μm-sized filter pore. Exemplary flow cytometry histogram plots representing fluorescence after compression-mediated delivery (SQZ) for endocytosis (Endo), negative (NC), and no-material controls are shown in Figures 10A-I. Cell viability was measured after filter delivery. Estimated cell viability, indicated by the percentage of cells present in the FSC and SSC gates after filter delivery of dextran particles, is shown in Figure 11A. Delivery efficiency, indicated by the percentage of cells positive for dextran particles or IgG antibody after filter delivery, is shown in Figure 11B. Geometric mean fluorescence after filter delivery of dextran particles or IgG antibody is shown in Figure 11C.

[0494] RBCs mixed with 70 kDa Alexa Fluor® 488 dextran particles were passed through 2 μm-sized filter pores using controlled pressure systems below 10 psi, 12 psi, 14 psi, 16 psi, or 18 psi. Exemplary flow cytometry histogram plots representing fluorescence after compression-mediated delivery (SQZ) for endocytosis (Endo) and NC (Negative) controls are shown in Figure 12A. Cell viability was measured after filter delivery. Estimated cell viability, indicated by the percentage of cells present in the FSC and SSC gates after filter delivery of dextran particles, is shown in Figure 12B. Delivery efficiency, indicated by the percentage of cells positive for dextran particles after filter delivery, is shown in Figure 12C. Geometric mean fluorescence after filter delivery of dextran particles is shown in Figure 12D.

[0495] Compression-mediated delivery of dextran particles and IgG antibodies into mouse RBCs was achieved.

[0496] (Example 8) Microsieve delivery to HeLa cells and T cells To evaluate the microsieve-mediated delivery of molecules, HeLa cells or T cells mixed with fluorescent dextran particles were passed through microsieves, and intracellular particle delivery was assessed by FACS analysis.

[0497] material and method Polycarbonate microsieves with a pore size of 10 μm (porosity 8%) from STERLITECH® or silicon / ceramic microsieves with a pore size of 10 μm (porosity 36.3%) from AQUAMARIJN were used for HeLa cells. HeLa cells were divided into 5 × 10⁶ cells. 6 The cells were suspended in OptiMEM medium at a concentration of cells / mL, and 3kDa dextran particles were added to a final concentration of 1 mg / mL. 200 μl of the cells mixed with the dextran particles were added to a microsieve and passed through the microsieve at 3-7 psi at room temperature. Collection was performed at 37°C.

[0498] Naive T cells 10x10 6 Cells were suspended in OptiMEM medium at a concentration of cells / mL, and 3kDa dextran particles were added to a final concentration of 1 mg / mL. 200 μl of the cells mixed with the dextran particles were passed through a 4 μm filter or 4 μm microsieve at 6–8 psi on ice. Collection was performed at room temperature.

[0499] Prior to FACS analysis, all samples were centrifuged three times at 400 rcf for 4 minutes at 4°C, washed, and removed extracellular dextran. The following steps were taken to prepare for FACS analysis: First, the FACS buffer was prepared (PBS + final concentration: 1% FBS + 2nM). EDTA was added, and propidium iodide was added immediately before use (1:100 dilution). 400 μL of FACS buffer was added per tube, and the cells were resuspended. Flow cytometry forward scatter, side scatter, and fluorescence channel voltages were adjusted to visualize all cellular events, and 10,000 events were recorded per sample. Cells mixed with dextran particles but not filtered were used as controls for endocytosis delivery. These cells came into contact with the dye or EGFP mRNA but did not pass through the filter. All other steps were the same as for the endocytosis controls. Negative control samples did not come into contact with either the dye or EGFP mRNA, but were subjected to the same steps as the experimental samples.

[0500] result The results of microsieve-mediated delivery of dextran to HeLa cells are shown in Figures 13A-13D. Cell viability for STERLITECH® and AQUAMARIJN microsieves is shown in Figure 13A, and dextran delivery is shown in Figure 13B. Representative histograms for AQUAMARIJN and STERLITECH® microsieves are shown in Figures 13C and 13D, respectively.

[0501] The results of microsieve-mediated delivery of dextran to T cells are shown in Figures 14A and 14B. Cell viability and dextran delivery for AQUAMARIJN microsieves are shown in Figure 14A. A representative flow cytometry histogram for AQUAMARIJN microsieves is shown in Figure 14B. The results were comparable to those of filter-mediated delivery.

[0502] The results demonstrate excellent delivery and minimal decrease in viability after microsieve delivery of dextran to HeLa and T cells.

[0503] All compositions and / or methods disclosed and claimed herein can be prepared and performed without undue experimentation in consideration of this disclosure. Although the compositions and methods of this disclosure are described in terms of embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions and / or methods, and to the steps or order of steps of the methods described herein, without departing from the concept, spirit and scope of this disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to fall within the spirit, scope and concept of this disclosure as defined by the appended claims.

Claims

[Claim 1] The invention as shown in the drawings.

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