Devices, systems, and kits for electroporation, and methods for using them.

Abstract

To provide a method of introducing a composition into a plurality of cells suspended in a flowing liquid.SOLUTION: This method comprises: providing a device comprising a first hollow cylindrical electrode, a second hollow cylindrical electrode, and an electroporation zone therebetween; applying an electrical potential difference between the first and second hollow cylindrical electrodes, thereby generating an electric field in the electroporation zone; and passing the plurality of cells and the composition through the first hollow cylindrical electrode, the electroporation zone and the second hollow cylindrical electrode, thereby enhancing permeability of the plurality of cells and introducing the composition into the plurality of cells.SELECTED DRAWING: None

Description

[Technical Field] 【0001】 Statement on federally funded research This invention was developed with government support under Phase I SBIR Grant No. 1747096 and Phase II SBIR Grant No. 1853194 from the National Science Foundation (NSF). The government has certain rights in this invention. [Background technology] 【0002】 Immunotherapy is currently at the forefront of both basic scientific research and clinical applications driven by pharmaceutical perspectives. This trend is partly due to recent advances in targeted gene modification and the expanding use of CRISPR / Cas complex editing for therapeutic development. To identify gene modifications of therapeutic interest, research organizations often have to screen thousands of gene variants, which may include modifications of endogenous genes or insertions of manipulated genes. This drug discovery process is labor-intensive, requires a great deal of manual work in the laboratory, and creates an industry-wide bottleneck due to a lack of sufficient high-throughput technologies. 【0003】 Biotechnology and pharmaceutical research and development activities are shifting towards automating almost every step of the process. Workflows include liquid handling robots equipped with advanced laboratory management software to enable high-throughput discovery. However, the transfection step remains limited to low-throughput, low-efficiency technologies and user-intensive systems that cannot be automated. Automated platforms for transfection not only have the potential to substantially reduce process costs but also reduce discovery time, coupled with increased cell viability and the quantity of normally operated cells, which is critical in the competitive immunotherapy space. 【0004】 Electroporation's unique strength lies in RNA delivery. While existing viral methods for DNA delivery are comparable to electroporation, they lack GMP-grade non-retroviral RNA viruses. Therefore, companies with electroporation platforms are targeted for collaboration and acquisition for the purpose of delivering mRNA into cells. 【0005】 Current high-throughput gene transfer methods typically require the use of viral delivery (e.g., lentiviral vectors) where viral particles infect cells and transduce the genetic modification of interest. While viral methodologies can be applied to high-throughput automated systems, production has limitations that extend the timeline for research efforts, namely, the cloning of viral vectors, transfection into virus-producing strains, and subsequent purification of viral particles. This process can take research organizations several months, significantly impacting the platform development timeline and potentially increasing the cost of drug discovery. In addition, the use of viral transduction for gene transfer is not suitable for genetic modification of all cell types, as some cells (such as specific immune cell subsets) are resistant to viral infection. Therefore, there is an unmet need within the biotechnology industry for high-throughput automated systems for gene transfer that do not rely on viral delivery mechanisms. [Overview of the Initiative] 【0006】 A device for electroperforating a plurality of cells suspended in a liquid (e.g., a liquid flowing through the device), the device comprising a first electrode and a second electrode and an electroperforation zone. The first electrode comprises a first inlet, a first outlet, and a first lumen including a minimum cross-sectional dimension, and the second electrode comprises a second inlet, a second outlet, and a second lumen including a minimum cross-sectional dimension. The electroporation zone is located between the first outlet and the second inlet and has a minimum cross-sectional dimension greater than approximately 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm, e.g., approximately 0.5 mm, approximately 1.0 mm, approximately 1.5 mm, approximately 2 mm, approximately 5 mm, approximately 10 mm, approximately 15 mm, approximately 25 mm, or approximately 50 mm), and the electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluid communication. 【0007】 In some embodiments, the cross-section of the electroporation zone is a shape selected from the group consisting of circular, disc-shaped, elliptical, regular polygon, non-regular polygon, curved shape, star-shaped, parallelogram, trapezoidal, and irregular shapes (e.g., shapes with protrusions, e.g., protruding slots or grooves, non-regular polygons, and / or curved shapes). In some embodiments, the cross-section of the electroporation zone varies along the length of the electroporation zone (i.e., along the longitudinal axis or flow direction). In some embodiments, the shape is consistent along the length but its position changes with respect to the central longitudinal axis along the length of the electroporation zone (e.g., the cross-sectional shape rotates around the central axis from one end of the electroporation zone to the other, such as a helical shape). In certain embodiments, the electroporation zone has a substantially circular cross-section. In some embodiments, the electroporation zone is approximately 7,850 μm 2 ~approximately 2,000 mm 2 (For example, approximately 8,000 μm) 2 ~about 1mm 2 , about 8,000μm 2 ~about 10mm 2 , about 8,000μm 2 ~approximately 100mm 2Approximately 9,000 μm 2 ~5mm 2 Approximately 1mm 2 ~approximately 10mm 2 Approximately 1mm 2 ~approximately 100mm 2 Approximately 3mm 2 ~approximately 20mm 2 Approximately 10mm 2 ~approximately 50mm 2 Approximately 25mm 2 ~approximately 75mm 2 Approximately 50mm 2 ~approximately 100mm 2 Approximately 75mm 2 ~approximately 200mm 2 Approximately 100mm 2 ~approximately 350mm 2 Approximately 150mm 2 ~approximately 500mm 2 Approximately 300mm 2 ~approximately 750mm 2 Approximately 500mm 2 ~approximately 1,000 mm 2 Approximately 750mm 2 ~approximately 1,500mm 2 、またはabout 950mm 2 ~approximately 2,000mm 2 , example, about 8,000μm 2 Approximately 9,000 μm 2 Approximately 1mm 2 Approximately 5mm 2 Approximately 10mm 2 Approximately 15mm 2 Approximately 20mm 2 Approximately 25mm 2 Approximately 50mm 2 Approximately 60mm 2 Approximately 75mm 2 Approximately 80mm 2 Approximately 100mm 2 Approximately 150mm 2 Approximately 200mm 2 Approximately 250mm 2 Approximately 300mm 2 Approximately 350mm 2 Approximately 400mm 2 Approximately 450mm 2 Approximately 500mm 2 Approximately 600mm 2 Approximately 700mm2 Approximately 800mm 2 Approximately 900mm 2 Approximately 1,000 mm 2 Approximately 1,100 mm 2 , approx. 1,200mm 2 , approx. 1,300mm 2 Approximately 1,400 mm 2 Approximately 1,500 mm 2 Approximately 1,600 mm 2 , approx. 1,700mm 2 Approximately 1,800 mm 2 Approximately 1,900 mm 2 , or approximately 2,000 mm 2 It has a cross-sectional area of ​​). 【0008】 In some embodiments, the electroporation zone is 0.005mm to 50mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.005mm to 25mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.1mm to 50mm, 0.5mm to 5mm, 0.5mm to 25mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 50mm) Having lengths of mm, 15mm-25mm, 20mm-30mm, 25mm-40mm, or 30mm-50mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 15mm, approximately 20mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm). In some embodiments, the electroporation zone is 0.005mm to 25mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.5mm to 5mm, 0.5mm to 10mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 10mm, 7mm to 15mm, 1 Having a length of 0mm to 20mm, or 15mm to 25mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 10mm, approximately 12mm, approximately 15mm, approximately 18mm, approximately 20mm, approximately 23mm, or approximately 25mm). 【0009】 In some embodiments, the lumen of either the first electrode and / or the second electrode is 0.01 mm to 500 mm (for example, 0.01 mm to 0.1 mm, 0.01 mm to 0.5 mm, 0.01 mm to 10 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 0.5 mm to 50 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 15 mm, 3 mm to 50 mm, 10 mm to 20 mm, 10 mm to 100 mm, 15 mm to 30 mm, 20 mm to 40 mm, 20 mm to 200 mm, 30 mm to 50 mm, 30 mm to 300 mm, 45 mm to 60 mm, 50 mm to 100 mm, 50 mm to 500 mm, 75 mm to 150 mm, 75 mm to 300 mm, 100 mm) m~200mm, 100mm~500mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7m It has a minimum cross-sectional dimension of approximately 8mm, 10mm, 15mm, 25mm, 30mm, 35mm, 40mm, 45mm, 50mm, 60mm, 70mm, 80mm, 90mm, 100mm, 150mm, 200mm, 250mm, 300mm, 350mm, 400mm, 450mm, or 500mm. 【0010】 In some embodiments, the ratio of the minimum cross-sectional dimension of the lumen of either the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is 1:10 to 10:1 (e.g., 1:10 to 1:5, 1:10 to 1:2, 1:10 to 1:1, 1:10 to 2:1, 1:10 to 5:1, 1:5 to 1:2, 1:5 to 1:1, 1:5 to 2:1, 1:5 to 5:1, 1:2 to 2:3, 1:2 to 1:1, 1:2 to 2:1, 1:2 to 6:1, 2:3 to 2:1, 2:3 to 4:1, 1:1 to 2:1, 1:1 to 3:1, 1 :1~10:1, 3:2~3:1, 3:2~6:1, 2:1~3:1, 2:1~5:1, 5:2~5:1, 3:1~4:1, 7:2~5:1, 7:2~10:1, 4:1~8:1, 5:1~10:1, or 7:1~10:1, for example, approximately 1:10, approximately 1:9, approximately 1:8, approximately 1:7, approximately 1:6, approximately 1:5, approximately 1:2, approximately 2:3, approximately 1:1, approximately 3:2, approximately 2:1, approximately 5:2, approximately 3:1, approximately 7:2, approximately 4:1, approximately 9:2, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1). 【0011】 In some embodiments, the ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is 1:100~100:1 (for example, 1:100~1:50, 1:100~1:25, 1:100~1:10, 1:100~1:1, 1:50~1:5, 1:50~1:2, 1:50~2:1, 1:25~1:10, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:2~1:1, 1:2~2:1, 1:1~2:1, 1:1~5:1, 1:1~10:1) , 1:1~50:1, 1:1~100:1, 2:1~5:1, 2:1~20:1, 3:1~10:1, 4:1~25:1, 5:1~50:1, 10:1~50:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50 (approximately 1:25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). 【0012】 In some embodiments of any of the preceding devices, the ratio of the cross-sectional area of ​​the lumen of either the first electrode and / or the second electrode to the cross-sectional area of ​​the electroporation zone is 1:100 to 100:1 (e.g., 1:100 to 1:50, 1:100 to 1:25, 1:100 to 1:10, 1:100 to 1:1, 1:50 to 1:5, 1:50 to 1:2, 1:50 to 2:1, 1:25 to 1:10, 1:25 to 1:5, 1:25 to 1:1, 1:25 to 1:1, 1:25 to 10:1, 1:10 to 1:1, 1:10 to 2:1, 1:10 to 5:1, 1:5 to 1:2, 1:5 to 1:1, 1:5 to 2:1, 1:2 to 1:1, 1:2 to 2:1, 1:1 to 2 :1, 1:1~5:1, 1:1~10:1, 1:1~50:1, 1:1~100:1, 2:1~5:1, 2:1~20:1, 3:1~10:1, 4:1~25:1, 5:1~50:1, 10:1~50:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1 (approximately 75, 1:50, 1:25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). 【0013】 In some embodiments, the device further includes a first reservoir (e.g., a sample bag) in fluid communication with a first inlet, and / or a second reservoir (e.g., a collection bag, e.g., a recovery bag) in fluid communication with a second outlet. In addition, the device may include a third reservoir in fluid communication with the first or second lumen. The third reservoir may contain one or more reagents for transfection, e.g., a gene composition to be delivered to cells. In some embodiments, either the first or second electrode has an additional inlet or outlet for fluid communication with the third reservoir. 【0014】 In some embodiments, either the first electrode or the second electrode may be porous or a conductive fluid (e.g., a conductive liquid). 【0015】 One of the devices in the preceding embodiments may include a delivery source that is in fluid communication with a first inlet. The delivery source may be configured to deliver a plurality of cells in a liquid and / or suspension through a first lumen to a second outlet. The delivery source may also be configured to deliver other components, such as genetic material to be introduced into cells (for example, as a transfection reagent reservoir). 【0016】 In some embodiments, the device further includes one or more additional electroporation zones (e.g., one, two, three, four, six, eight, ten, eleven, twelve, twenty-four, twenty-seven, thirty-six, forty-eight, sixty-four, ninety-six, thirty-eight, fifteen-thirty, or more) which may be arranged in parallel, in series, or in combination thereof. Each of the one or more additional electroporation zones may have a substantially uniform cross-sectional area. 【0017】 In some embodiments of the embodiments described above, the device may further include a housing configured to enclose a first electrode, a second electrode, and an electroporation zone. The housing may include a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the housing may further include a thermal controller configured to raise the temperature of the liquid in which the device and / or a plurality of cells are suspended, wherein the thermal controller is a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing may further include a thermal controller configured to lower the temperature of the liquid in which the device and / or a plurality of cells are suspended, wherein the thermal controller is a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier device. The housing may be integrated into the device or may be detachably connected. 【0018】 In another embodiment, the present invention includes a device for electroperforating a plurality of cells suspended in a liquid, the device comprising: a first electrode comprising a first lumen comprising a first inlet, a first outlet, and a minimum cross-sectional dimension; a second electrode comprising a second lumen comprising a second inlet, a second outlet, and a minimum cross-sectional dimension; a third inlet and a third outlet, the third inlet and third outlet being in fluid communication with the first lumen and the third inlet and third outlet intersecting the first electrode between the first inlet and the first outlet; and a fourth inlet and fourth outlet, the fourth inlet and fourth outlet being in fluid communication with the second lumen and the fourth inlet and fourth outlet being in fluid communication with the second inlet and second outlet The first outlet and the second outlet intersect with the second electrode between them, and the electroporation zone disposed between the first outlet and the second outlet, the electroporation zone having a minimum cross-sectional dimension greater than about 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm, e.g., about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), and the electroporation zone having a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second outlet are in fluid communication. The cross-section of the electroporation zone is a shape selected from the group consisting of circular, disc-shaped, elliptical, regular polygon, non-regular polygon, curved shape, star-shaped, parallelogram, trapezoidal, and irregular shapes (e.g., shapes with protrusions, e.g., protruding slots or grooves, non-regular polygons, and / or curved shapes). In some embodiments, the cross-section of the electroporation zone varies along the length of the electroporation zone (i.e., along the longitudinal axis or flow direction). In some embodiments, the shape is consistent along the length but its position changes with respect to the central longitudinal axis along the length of the electroporation zone (e.g., the cross-sectional shape rotates around the central axis from one end of the electroporation zone to the other, such as a helical shape). In certain embodiments, the electroporation zone has a substantially circular cross-section. In some embodiments, the electroporation zone is approximately 7850 μm 2 ~about 2000mm 2(Example: about 8,000μm 2 ~approximately 1mm 2 Approximately 8,000 μm 2 ~approximately 10mm 2 Approximately 8,000 μm 2 ~approximately 100mm 2 Approximately 9,000 μm 2 ~5mm 2 Approximately 1mm 2 ~approximately 10mm 2 Approximately 1mm 2 ~approximately 100mm 2 Approximately 3mm 2 ~approximately 20mm 2 Approximately 10mm 2 ~approximately 50mm 2 Approximately 25mm 2 ~approximately 75mm 2 Approximately 50mm 2 ~approximately 100mm 2 Approximately 75mm 2 ~approximately 200mm 2 Approximately 100mm 2 ~approximately 350mm 2 Approximately 150mm 2 ~approximately 500mm 2 Approximately 300mm 2 ~approximately 750mm 2 Approximately 500mm 2 ~approximately 1,000 mm 2 Approximately 750mm 2 ~approximately 1,500mm 2 、またはabout 950mm 2 ~approximately 2,000mm 2 , example, about 8,000μm 2 Approximately 9,000 μm 2 Approximately 1mm 2 Approximately 5mm 2 Approximately 10mm 2 Approximately 15mm 2 Approximately 20mm 2 Approximately 25mm 2 Approximately 50mm 2 Approximately 60mm 2 Approximately 75mm 2 Approximately 80mm 2 Approximately 100mm 2 Approximately 150mm 2 Approximately 200mm 2 Approximately 250mm 2, about 300mm 2 , about 350mm 2 Approximately 400mm 2 Approximately 450mm 2 Approximately 500mm 2 Approximately 600mm 2 , about 700mm 2 Approximately 800mm 2 Approximately 900mm 2 Approximately 1,000 mm 2 Approximately 1,100 mm 2 , approx. 1,200mm 2 , approx. 1,300mm 2 Approximately 1,400 mm 2 Approximately 1,500 mm 2 Approximately 1,600 mm 2 , approx. 1,700mm 2 Approximately 1,800 mm 2 Approximately 1,900 mm 2 , or approximately 2,000 mm 2 It has a cross-sectional area of ​​). 【0019】 In some embodiments, the electroporation zone is 0.1mm to 50mm (for example, 0.1mm to 0.5mm, 0.1mm to 1mm, 0.1mm to 5mm, 0.1mm to 10mm, 0.5mm to 5mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 100mm, 15mm to 30mm, 20mm to 40mm, 20mm to 200mm, 30mm to 50mm, 45mm to 60mm, 50mm to 1 It has a minimum cross-sectional dimension of 00mm, 75mm~150mm, 100mm~200mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 10mm, approximately 15mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm. 【0020】 In some embodiments, the electroporation zone is 0.005mm to 50mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.005mm to 25mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.1mm to 50mm, 0.5mm to 5mm, 0.5mm to 25mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 50mm) Having lengths of mm, 15mm-25mm, 20mm-30mm, 25mm-40mm, or 30mm-50mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 15mm, approximately 20mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm). In some embodiments, the electroporation zone is 0.005mm to 25mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.5mm to 5mm, 0.5mm to 10mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 10mm, 7mm to 15mm, 1 Having a length of 0mm to 20mm, or 15mm to 25mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 10mm, approximately 12mm, approximately 15mm, approximately 18mm, approximately 20mm, approximately 23mm, or approximately 25mm). 【0021】 In some embodiments, the lumen of either the first electrode and / or the second electrode is 0.01 mm to 500 mm (for example, 0.01 mm to 0.1 mm, 0.01 mm to 0.5 mm, 0.01 mm to 10 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 0.5 mm to 50 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 15 mm, 3 mm to 50 mm, 10 mm to 20 mm, 10 mm to 100 mm, 15 mm to 30 mm, 20 mm to 40 mm, 20 mm to 200 mm, 30 mm to 50 mm, 30 mm to 300 mm, 45 mm to 60 mm, 50 mm to 100 mm, 50 mm to 500 mm, 75 mm to 150 mm, 75 mm to 300 mm, 100 mm) m~200mm, 100mm~500mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7m It has a minimum cross-sectional dimension of approximately 8mm, 10mm, 15mm, 25mm, 30mm, 35mm, 40mm, 45mm, 50mm, 60mm, 70mm, 80mm, 90mm, 100mm, 150mm, 200mm, 250mm, 300mm, 350mm, 400mm, 450mm, or 500mm.In some embodiments, the ratio of the minimum cross-sectional dimension of the lumen of either the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is 1:10 to 10:1 (e.g., 1:10 to 1:5, 1:10 to 1:2, 1:10 to 1:1, 1:10 to 2:1, 1:10 to 5:1, 1:5 to 1:2, 1:5 to 1:1, 1:5 to 2:1, 1:5 to 5:1, 1:2 to 2:3, 1:2 to 1:1, 1:2 to 2:1, 1:2 to 6:1, 2:3 to 2:1, 2:3 to 4:1, 1:1 to 2:1). The ratios are 1:1-3:1, 1:1-10:1, 3:2-3:1, 3:2-6:1, 2:1-3:1, 2:1-5:1, 5:2-5:1, 3:1-4:1, 7:2-5:1, 7:2-10:1, 4:1-8:1, 5:1-10:1, or 7:1-10:1 (for example, approximately 1:10, approximately 1:5, approximately 1:2, approximately 2:3, approximately 1:1, approximately 3:2, approximately 2:1, approximately 5:2, approximately 3:1, approximately 7:2, approximately 4:1, approximately 9:2, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1). 【0022】 In some embodiments, the ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is 1:100~100:1 (for example, 1:100~1:50, 1:100~1:25, 1:100~1:10, 1:100~1:1, 1:50~1:5, 1:50~1:2, 1:50~2:1, 1:25~1:10, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:2~1:1, 1:2~2:1, 1:1~2:1, 1:1~5:1, 1:1~10:1) , 1:1~50:1, 1:1~100:1, 2:1~5:1, 2:1~20:1, 3:1~10:1, 4:1~25:1, 5:1~50:1, 10:1~50:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50 (approximately 1:25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1).In some embodiments, the ratio of the cross-sectional area of ​​the lumen of either the first electrode and / or the second electrode to the cross-sectional area of ​​the electroporation zone is 1:100~100:1 (e.g., 1:100~1:50, 1:100~1:25, 1:100~1:10, 1:100~1:1, 1:50~1:5, 1:50~1:2, 1:50~2:1, 1:25~1:10, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:2~1:1, 1:2~2:1, 1:1~2:1, 1:1~5: 1, 1:1~10:1, 1:1~50:1, 1:1~100:1, 2:1~5:1, 2:1~20:1, 3:1~10:1, 4:1~25:1, 5:1~50:1, 10:1~50:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, The ratios are approximately 1:50, 1:25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). Either the first electrode or the second electrode, or both, may be porous or conductive fluid (e.g., liquid). 【0023】 In some embodiments, the device further includes a first reservoir that fluid-communicates with a first inlet. In some embodiments, the device further includes a second reservoir that fluid-communicates with a second outlet. In some embodiments, the device further includes a third reservoir that fluid-communicates with a third inlet and a third outlet. In some embodiments, the device further includes a fourth reservoir that fluid-communicates with a fourth inlet and a fourth outlet. In some embodiments, the device further includes a fifth reservoir that fluid-communicates with the lumen of either the first electrode or the second electrode, and either the first electrode or the second electrode has at least one additional inlet for fluid-communicating with the fifth reservoir. In some embodiments, the device further includes a fluid delivery source that fluid-communicates with the first inlet, and the fluid delivery source is configured to deliver a plurality of cells in liquid and / or suspension through the first lumen to the second outlet. In some embodiments, the device further includes a plurality of electroporation zones (arranged, for example, in series, in parallel, or in a combination thereof). Each of the multiple electroporation zones may have a substantially uniform cross-sectional area. 【0024】 In some embodiments, the device further includes a housing configured to enclose a first electrode, a second electrode, and at least one electroporation zone of the device. The housing may include a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the housing further includes a thermal controller configured to raise the temperature of the liquid in which the device and / or a plurality of cells are suspended, the thermal controller being a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to lower the temperature of the liquid in which the device and / or a plurality of cells are suspended, the thermal controller being a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier device. In some embodiments, the housing is either integrated into the device or releasably connected to the device. 【0025】 In another embodiment, the present invention includes a system for electroperforating a plurality of cells suspended in a liquid, the system comprising any of the aforementioned embodiments of the device. 【0026】 In another embodiment, the present invention includes a system for electroperforating a plurality of cells suspended in a liquid, comprising a cell perforation device and a potential source. The cell perforation device comprises a first electrode, a second electrode, and an electroperforation zone. The first electrode comprises a first lumen including a first inlet, a first outlet, and a minimum cross-sectional dimension, and the second electrode comprises a second lumen including a second inlet, a second outlet, and a minimum cross-sectional dimension. The electroporation zone is located between the first outlet and the second inlet and has a minimum cross-sectional dimension greater than approximately 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm, e.g., approximately 0.5 mm, approximately 1.0 mm, approximately 1.5 mm, approximately 2 mm, approximately 5 mm, approximately 10 mm, approximately 15 mm, approximately 25 mm, or approximately 50 mm). The electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluid communication. The system further includes a potential source, and the first and second electrodes of the device are in openable, operable contact with the potential source. In some embodiments, the device further includes a first reservoir in fluid communication with a first inlet, and / or a second reservoir in fluid communication with a second outlet. 【0027】 In some embodiments of the system, the cross-section of the electroporation zone is a shape selected from the group consisting of circular, disc-shaped, elliptical, regular polygon, irregular polygon, curved shape, star-shaped, parallelogram, trapezoid, and irregular. In some embodiments, the electroporation zone has a substantially circular cross-section. In some embodiments, the electroporation zone is 0.1 mm to 50 mm (e.g., 0.1 mm to 0.5 mm, 0.1 mm to 1 mm, 0.1 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 15 mm, 3 mm to 50 mm, 10 mm to 20 mm, 10 mm to 100 mm, 15 mm to 30 mm, 20 mm to 40 mm, 20 mm to 200 mm, 30 mm to 50 mm, 45 mm to 60 mm, 50 mm to 1 It has a minimum cross-sectional dimension of 00mm, 75mm~150mm, 100mm~200mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 10mm, approximately 15mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm. 【0028】 In some embodiments of the system of the present invention, the electroporation zone is approximately 7,850 μm 2 ~approximately 2,000 mm 2 (For example, approximately 8,000 μm) 2 ~about 1mm 2 , about 8,000μm 2 ~about 10mm 2 , about 8,000μm 2 ~approximately 100mm 2 , about 9,000μm 2 ~5mm 2 , about 1mm 2 ~about 10mm 2 , about 1mm 2 ~approximately 100mm 2 , about 3mm 2 ~about 20mm 2 , about 10mm 2 ~approximately 50mm 2 , about 25mm 2 ~about 75mm2 Approximately 50mm 2 ~approximately 100mm 2 , about 75mm 2 ~about 200mm 2 Approximately 100mm 2 ~about 350mm 2 , about 150mm 2 ~approximately 500mm 2 , about 300mm 2 ~about 750mm 2 Approximately 500mm 2 ~approximately 1,000 mm 2 , about 750mm 2 ~approximately 1,500mm 2 , or approximately 950mm 2 ~approximately 2,000 mm 2 For example, approximately 8,000 μm 2 , about 9,000μm 2 , about 1mm 2 , about 5mm 2 , about 10mm 2 , about 15mm 2 , about 20mm 2 , about 25mm 2 Approximately 50mm 2 , about 60mm 2 , about 75mm 2 , about 80mm 2 Approximately 100mm 2 , about 150mm 2 , about 200mm 2 , about 250mm 2 , about 300mm 2 , about 350mm 2 Approximately 400mm 2 Approximately 450mm 2 Approximately 500mm 2 Approximately 600mm 2 , about 700mm 2 Approximately 800mm 2 Approximately 900mm 2 Approximately 1,000 mm 2 Approximately 1,100 mm 2 , approx. 1,200mm 2 , approx. 1,300mm 2 Approximately 1,400 mm 2 Approximately 1,500 mm 2 Approximately 1,600 mm 2 , approx. 1,700mm2 Approximately 1,800 mm 2 Approximately 1,900 mm 2 , or approximately 2,000 mm 2 It has a cross-sectional area of ​​). 【0029】 In some embodiments, the electroporation zone is 0.005mm to 50mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.005mm to 25mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.1mm to 50mm, 0.5mm to 5mm, 0.5mm to 25mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 50mm) Having lengths of mm, 15mm-25mm, 20mm-30mm, 25mm-40mm, or 30mm-50mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 15mm, approximately 20mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm). In some embodiments of the system, the length of the electroporation zone is 0.005mm to 25mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.5mm to 5mm, 0.5mm to 10mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 10mm, 7mm to 25mm, 3mm to 10mm, 7mm to 25mm). These ranges from 15mm, 10mm to 20mm, or 15mm to 25mm (for example, approximately 0.005mm, 0.01mm, 0.05mm, 0.1mm, 0.5mm, 1.0mm, 1.5mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 10mm, 12mm, 15mm, 18mm, 20mm, 23mm, or 25mm). 【0030】 In some embodiments, the lumen of either the first electrode and / or the second electrode is 0.01 mm to 500 mm (for example, 0.01 mm to 0.1 mm, 0.01 mm to 0.5 mm, 0.01 mm to 10 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 0.5 mm to 50 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 15 mm, 3 mm to 50 mm, 10 mm to 20 mm, 10 mm to 100 mm, 15 mm to 30 mm, 20 mm to 40 mm, 20 mm to 200 mm, 30 mm to 50 mm, 30 mm to 300 mm, 45 mm to 60 mm, 50 mm to 100 mm, 50 mm to 500 mm, 75 mm to 150 mm, 75 mm to 300 mm, 100 mm) m~200mm, 100mm~500mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7m It has a minimum cross-sectional dimension of approximately 8mm, 10mm, 15mm, 25mm, 30mm, 35mm, 40mm, 45mm, 50mm, 60mm, 70mm, 80mm, 90mm, 100mm, 150mm, 200mm, 250mm, 300mm, 350mm, 400mm, 450mm, or 500mm. 【0031】 In some embodiments of the system of the present invention, the ratio of the minimum cross-sectional dimension of the lumen of either the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is 1:10 to 10:1 (for example, 1:10 to 1:5, 1:10 to 1:2, 1:10 to 1:1, 1:10 to 2:1, 1:10 to 5:1, 1:5 to 1:2, 1:5 to 1:1, 1:5 to 2:1, 1:5 to 5:1, 1:2 to 2:3, 1:2 to 1:1, 1:2 to 2:1, 1:2 to 6:1, 2:3 to 2:1, 2:3 to 4:1, 1:1 to 2:1, 1:1 to 3: 1, 1:1~10:1, 3:2~3:1, 3:2~6:1, 2:1~3:1, 2:1~5:1, 5:2~5:1, 3:1~4:1, 7:2~5:1, 7:2~10:1, 4:1~8:1, 5:1~10:1, or 7:1~10:1 (for example, approximately 1:10, approximately 1:9, approximately 1:8, approximately 1:7, approximately 1:6, approximately 1:5, approximately 1:2, approximately 2:3, approximately 1:1, approximately 3:2, approximately 2:1, approximately 5:2, approximately 3:1, approximately 7:2, approximately 4:1, approximately 9:2, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1). In some embodiments, the ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is 1:100~100:1 (for example, 1:100~1:50, 1:100~1:25, 1:100~1:10, 1:100~1:1, 1:50~1:5, 1:50~1:2, 1:50~2:1, 1:25~1:10, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:2~1:1, 1:2~2:1, 1:1~2:1, 1:1~5:1, 1:1~10:1) , 1:1~50:1, 1:1~100:1, 2:1~5:1, 2:1~20:1, 3:1~10:1, 4:1~25:1, 5:1~50:1, 10:1~50:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50 (approximately 1:25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). 【0032】 In some embodiments, the ratio of the cross-sectional area of ​​the lumen of either the first electrode and / or the second electrode to the cross-sectional area of ​​the electroporation zone is 1:100 to 100:1 (e.g., 1:100 to 1:50, 1:100 to 1:25, 1:100 to 1:10, 1:100 to 1:1, 1:50 to 1:5, 1:50 to 1:2, 1:50 to 2:1, 1:25 to 1:1) 0, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:10~10:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:5~50:1, 1:2~1:1, 1:2~2:1, 1:2~10:1, 1:1~2:1, 1:1~5:1, 1:1~10:1, 1:1~50:1, 1 :1~100:1, 2:1~5:1, 2:1~20:1, 2:1~50:1, 3:1~10:1, 3:1~30:1, 4:1~25:1, 5:1~10:1, 5:1~50:1, 10:1~50:1, 10:1~100:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50, approximately 1 The ratios are approximately 25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). Either the first electrode or the second electrode, or both, may be porous or conductive fluid (e.g., liquid). 【0033】 In some embodiments, the system includes a third reservoir that is in fluid communication with the lumen of either the first or second electrode, and either the first or second electrode has an additional inlet for fluid communication with the third reservoir. In some embodiments, the system further includes a fluid delivery source that is in fluid communication with the first inlet, and the fluid delivery source is configured to deliver a plurality of cells in liquid and / or suspension through the first lumen to a second outlet. 【0034】 In some embodiments, the system of the present invention further includes a controller operably coupled to a potential source to deliver voltage pulses to a first electrode and a second electrode, the voltage pulses generating a potential difference between the first electrode and the second electrode, and thus generating an electric field within the electroporation zone. In some embodiments, the system includes a plurality of electroporation zones (e.g., as part of any multiple embodiments of the device provided herein). Each of the plurality of electroporation zones may have a substantially uniform or non-uniform cross-sectional area. 【0035】 In some embodiments, the system further includes an outer structure comprising a housing configured to enclose a first electrode, a second electrode, and at least one electroporation zone of the device (for example, the outer structure further includes a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode). The housing may include a thermal controller configured to raise the temperature of the liquid in which the device and / or multiple cells are suspended. The thermal controller may be a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In addition, or alternatively, the thermal controller may be configured to lower the temperature of the liquid in which the device and / or multiple cells are suspended, and the thermal controller is a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier device. 【0036】 In some embodiments of the system of the present invention, a potential source is releasably connected to a first electrical input and a second electrical input of an outer structure. The releasable connection between the first or second electrical input and the potential source may be selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. The outer structure may be integrated with or releasably connected to the device. In some embodiments, the housing is configured to energize a plurality of devices in parallel, in series, or in a time-offset manner, and the housing further includes a tray housing a plurality of electroporation devices, the tray is modified with two grid electrodes, the first grid electrode is electrically isolated from the second grid electrode, and the outside of the first electrode of each of the plurality of devices is releasably and operably in contact with one of a first spring-biased electrode, a first mechanically connected electrode, or a first inductively connected electrode, and the outside of the second electrode of each of the plurality of devices is in contact with a second spring-biased electrode, a second Each of the multiple devices is releasably and operably in contact with either a mechanically connected electrode or a second inductively coupled electrode, and each of the multiple devices enters the housing releasably through an opening in the grid electrode, and of each device, either a first spring-biased electrode, a first mechanically connected electrode, or a first inductively coupled electrode is operably in contact with the first grid electrode, and of each device, either a second spring-biased electrode, a second mechanically connected electrode, or a second inductively coupled electrode is operably in contact with the second grid electrode, and the grid electrode is connected to a potential source. 【0037】 In some embodiments of the system, a potential source delivers voltage pulses to grid electrodes, so that a first grid electrode is energized with a specific applied voltage, while a second grid electrode is energized with a specific applied voltage, and each of the multiple devices is energized by the grid electrodes with the same applied voltage pulses such that the magnitude of the electric field generated within each of at least one electroporation zones of each device is substantially the same. In some embodiments, the potential source includes additional circuitry or programming configured to modulate the delivery of voltage pulses to the grid electrodes, so that each of the multiple devices may receive different voltages to the grid electrodes, and the magnitude of the electric field generated within each of at least one electroporation zones of each device is different. 【0038】 In another aspect, the present invention provides a system for electroperforating a plurality of cells suspended in a liquid, the system being a cell perforation device comprising: a first electrode including a first inlet, a first outlet, and a first lumen; a second electrode including a second inlet, a second outlet, and a second lumen; a third inlet and a third outlet, the third inlet and third outlet being in fluid communication with the first lumen, the third inlet and third outlet crossing the first electrode between the first inlet and the first outlet; and a fourth inlet and fourth outlet, the fourth inlet and fourth The outlet of the first pipe is in fluid communication with the second lumen, and the fourth inlet and fourth outlet intersect with the second electrode between the second inlet and second outlet. An electroporation zone is disposed between the first outlet and the second inlet, and the electroporation zone is 0.005 mm to 50 mm (for example, 0.005 mm to 0.05 mm, 0.005 mm to 0.5 mm, 0.005 mm to 25 mm, 0.01 mm to 1 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.1 mm to 50 mm, 0.5 mm to 5 mm, 0.5 mm to 25 mm, 1 mm to 5 mm, 1 mm m~10mm, 1mm~25mm, 3mm~15mm, 3mm~50mm, 10mm~20mm, 10mm~50mm, 15mm~25mm, 20mm~30mm, 25mm~40, or 30mm~50mm, such as about 0.005mm, about 0.01mm, about 0.05mm, 0.1mm, 0.5mm, 1.0mm, 1.5mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 15mm, 20mm, 25mm, 30mm, 35mm, 40mm, 45mm, or 50mm) The length is such that the minimum cross-sectional dimension of the electroporation zone is greater than approximately 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm, e.g., approximately 0.5 mm, approximately 1.0 mm, approximately 1.5 mm, approximately 2 mm, approximately 5 mm, approximately 10 mm, approximately 15 mm, approximately 25 mm, or approximately 50 mm), the cross-sectional area of ​​the electroporation zone is substantially uniform, and the ratio of the minimum cross-sectional dimension of the first lumen to the minimum cross-sectional dimension of the electroporation zone is1:10~10:1 (for example, 1:10~1:5, 1:10~1:2, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:5~5:1, 1:2~2:3, 1:2~1:1, 1:2~2:1, 1:2~6:1, 2:3~2:1, 2:3~4:1, 1:1~2:1, 1:1~3:1, 1:1~10:1, 3:2~3:1, 3:2~6:1, 2:1~3:1, 2:1~5:1, 5:2~5:1, 3:1~4:1, 7:2~ The ratio is 5:1, 7:2-10:1, 4:1-8:1, 5:1-10:1, or 7:1-10:1, for example, approximately 1:10, approximately 1:5, approximately 1:2, approximately 2:3, approximately 1:1, approximately 3:2, approximately 2:1, approximately 5:2, approximately 3:1, approximately 7:2, approximately 4:1, approximately 9:2, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1), and the ratio of the minimum cross-sectional dimension of the second lumen to the minimum cross-sectional dimension of the electroporation zone is 1:10-10:1 (for example, 1:10-1:5, 1:10-1:2, 1:10 ~1:1, 1:10~2:1, 1:10~5:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:5~5:1, 1:2~2:3, 1:2~1:1, 1:2~2:1, 1:2~6:1, 2:3~2:1, 2:3~4:1, 1:1~2:1, 1:1~3:1, 1:1~10:1, 3:2~3:1, 3:2~6:1, 2:1~3:1, 2:1~5:1, 5:2~5:1, 3:1~4:1, 7:2~5:1, 7:2~10:1, 4:1~8:1, 5:1~10:1, or 7:1 A cell perforation device including an electroperforation zone, the first outlet, the electroperforation zone, and the second inlet being in fluid communication, and a potential source, the first and second electrodes of the device being in openable, operable contact with the potential source. The cross-section of the electroperforation zone is a closed shape selected from the group consisting of circular, disc-shaped, elliptical, regular polygonal, non-regular polygonal, curved, star-shaped, parallelogram, trapezoidal, and irregular shapes. The electroperforation zone may have a substantially circular cross-section. 【0039】 In some embodiments of the system, the electroporation zone is 0.1mm to 50mm (for example, 0.1mm to 0.5mm, 0.1mm to 1mm, 0.1mm to 5mm, 0.1mm to 10mm, 0.5mm to 5mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 100mm, 15mm to 30mm, 20mm to 40mm, 20mm to 200mm, 30mm to 50mm, 45mm to 60mm, 50mm) It has a minimum cross-sectional dimension of m~100mm, 75mm~150mm, 100mm~200mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 10mm, approximately 15mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm. 【0040】 In some embodiments, the electroporation zone is approximately 7,850 μm. 2 ~approximately 2,000 mm 2 (For example, approximately 8,000 μm) 2 ~about 1mm 2 , about 8,000μm 2 ~about 10mm 2 , about 8,000μm 2 ~approximately 100mm 2 , about 9,000μm 2 ~5mm 2 , about 1mm 2 ~about 10mm 2 , about 1mm 2 ~approximately 100mm 2 , about 3mm 2 ~about 20mm 2 , about 10mm 2 ~approximately 50mm 2 , about 25mm 2 ~about 75mm 2 Approximately 50mm 2 ~approximately 100mm 2 , about 75mm 2 ~about 200mm 2 Approximately 100mm 2 ~about 350mm2 , about 150mm 2 ~approximately 500mm 2 , about 300mm 2 ~about 750mm 2 Approximately 500mm 2 ~approximately 1,000 mm 2 , about 750mm 2 ~approximately 1,500mm 2 , or approximately 950mm 2 ~approximately 2,000 mm 2 For example, approximately 8,000 μm 2 , about 9,000μm 2 , about 1mm 2 , about 5mm 2 , about 10mm 2 , about 15mm 2 , about 20mm 2 , about 25mm 2 Approximately 50mm 2 , about 60mm 2 , about 75mm 2 , about 80mm 2 Approximately 100mm 2 , about 150mm 2 , about 200mm 2 , about 250mm 2 , about 300mm 2 , about 350mm 2 Approximately 400mm 2 Approximately 450mm 2 Approximately 500mm 2 Approximately 600mm 2 , about 700mm 2 Approximately 800mm 2 Approximately 900mm 2 Approximately 1,000 mm 2 Approximately 1,100 mm 2 , approx. 1,200mm 2 , approx. 1,300mm 2 Approximately 1,400 mm 2 Approximately 1,500 mm 2 Approximately 1,600 mm 2 , approx. 1,700mm 2 Approximately 1,800 mm 2 Approximately 1,900 mm 2 , or approximately 2,000 mm 2 It has a cross-sectional area of ​​). 【0041】 In some embodiments, the electroporation zone is 0.005mm to 50mm (for example, 0.005mm to 0.05mm, 0.005mm to 0.5mm, 0.005mm to 25mm, 0.01mm to 1mm, 0.05mm to 5mm, 0.1mm to 10mm, 0.1mm to 50mm, 0.5mm to 5mm, 0.5mm to 25mm, 1mm to 5mm, 1mm to 10mm, 1mm to 25mm, 3mm to 15mm, 3mm to 50mm, 10mm to 20mm, 10mm to 50mm) Having lengths of mm, 15mm-25mm, 20mm-30mm, 25mm-40mm, or 30mm-50mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 15mm, approximately 20mm, approximately 25mm, approximately 30mm, approximately 35mm, approximately 40mm, approximately 45mm, or approximately 50mm). In some embodiments of the system of the present invention, the length of the electroporation zone is 0.005 mm to 25 mm (for example, 0.005 mm to 0.05 mm, 0.005 mm to 0.5 mm, 0.01 mm to 1 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 0.5 mm to 10 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 10 mm, 7 mm) These ranges from m to 15mm, 10mm to 20mm, or 15mm to 25mm (for example, approximately 0.005mm, 0.01mm, 0.05mm, 0.1mm, 0.5mm, 1.0mm, 1.5mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 10mm, 12mm, 15mm, 18mm, 20mm, 23mm, or 25mm). 【0042】 In some embodiments, the lumen of either the first electrode and / or the second electrode is 0.01 mm to 500 mm (for example, 0.01 mm to 0.1 mm, 0.01 mm to 0.5 mm, 0.01 mm to 10 mm, 0.05 mm to 5 mm, 0.1 mm to 10 mm, 0.5 mm to 5 mm, 0.5 mm to 50 mm, 1 mm to 5 mm, 1 mm to 10 mm, 1 mm to 25 mm, 3 mm to 15 mm, 3 mm to 50 mm, 10 mm to 20 mm, 10 mm to 100 mm, 15 mm to 30 mm, 20 mm to 40 mm, 20 mm to 200 mm, 30 mm to 50 mm, 30 mm to 300 mm, 45 mm to 60 mm, 50 mm to 100 mm, 50 mm to 500 mm, 75 mm to 150 mm, 75 mm to 300 mm, 100 mm) m~200mm, 100mm~500mm, 150mm~300mm, 200mm~400mm, 300mm~450mm, or 350mm~500mm, for example, approximately 0.005mm, approximately 0.01mm, approximately 0.05mm, approximately 0.1mm, approximately 0.5mm, approximately 1.0mm, approximately 1.5mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm, approximately 6mm, approximately 7m It has a minimum cross-sectional dimension of approximately 8mm, 10mm, 15mm, 25mm, 30mm, 35mm, 40mm, 45mm, 50mm, 60mm, 70mm, 80mm, 90mm, 100mm, 150mm, 200mm, 250mm, 300mm, 350mm, 400mm, 450mm, or 500mm. 【0043】 In some embodiments, the ratio of the minimum cross-sectional dimension of the lumen of either the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is 1:10 to 10:1 (e.g., 1:10 to 1:5, 1:10 to 1:2, 1:10 to 1:1, 1:10 to 2:1, 1:10 to 5:1, 1:5 to 1:2, 1:5 to 1:1, 1:5 to 2:1, 1:5 to 5:1, 1:2 to 2:3, 1:2 to 1:1, 1:2 to 2:1, 1:2 to 6:1, 2:3 to 2:1, 2:3 to 4:1, 1:1 to 2:1). The ratios are 1:1-3:1, 1:1-10:1, 3:2-3:1, 3:2-6:1, 2:1-3:1, 2:1-5:1, 5:2-5:1, 3:1-4:1, 7:2-5:1, 7:2-10:1, 4:1-8:1, 5:1-10:1, or 7:1-10:1 (for example, approximately 1:10, approximately 1:5, approximately 1:2, approximately 2:3, approximately 1:1, approximately 3:2, approximately 2:1, approximately 5:2, approximately 3:1, approximately 7:2, approximately 4:1, approximately 9:2, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1).In some embodiments, the ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is 1:100 to 100:1 (e.g., 1:100 to 1:50, 1:100 to 1:25, 1:100 to 1:10, 1:100 to 1:1, 1:50 to 1:5, 1:50 to 1:2, 1:50 to 2:1, 1:25 to 1:10, 1:25 to 1:5, 1:25) ~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:10~10:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:5~50:1, 1:2~1:1, 1:2~2:1, 1:2~10:1, 1:1~2:1, 1:1~5:1, 1:1~10:1, 1:1~50:1, 1:1~100:1, 2 :1~5:1, 2:1~20:1, 2:1~50:1, 3:1~10:1, 3:1~30:1, 4:1~25:1, 5:1~10:1, 5:1~50:1, 10:1~50:1, 10:1~100:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50, approximately 1:25, approximately The ratios are approximately 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.In some embodiments, the ratio of the cross-sectional area of ​​the lumen of either the first electrode and / or the second electrode to the cross-sectional area of ​​the electroporation zone is 1:100 to 100:1 (e.g., 1:100 to 1:50, 1:100 to 1:25, 1:100 to 1:10, 1:100 to 1:1, 1:50 to 1:5, 1:50 to 1:2, 1:50 to 2:1, 1:25 to 1:1) 0, 1:25~1:5, 1:25~1:1, 1:25~10:1, 1:10~1:1, 1:10~2:1, 1:10~5:1, 1:10~10:1, 1:5~1:2, 1:5~1:1, 1:5~2:1, 1:5~50:1, 1:2~1:1, 1:2~2:1, 1:2~10:1, 1:1~2:1, 1:1~5:1, 1:1~10:1, 1:1~50:1, 1 :1~100:1, 2:1~5:1, 2:1~20:1, 2:1~50:1, 3:1~10:1, 3:1~30:1, 4:1~25:1, 5:1~10:1, 5:1~50:1, 10:1~50:1, 10:1~100:1, 40:1~80:1, 50:1~100:1, or 75:1~90:1, for example, approximately 1:100, approximately 1:75, approximately 1:50, approximately 1 (approximately 25, 1:10, 1:5, 1:2, 1:1, 3:2, 2:1, 5:2, 3:1, 7:2, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1). 【0044】 In some embodiments, the system further includes a first reservoir fluid-communicating with a first inlet, a second reservoir fluid-communicating with a second outlet, a third reservoir fluid-communicating with a third inlet and a third outlet, a fourth reservoir fluid-communicating with a fourth inlet and a fourth outlet, and / or a fifth reservoir fluid-communicating with the lumen of either the first or second electrode, for example, the first or second electrode having at least one additional inlet for fluid communication with the fifth reservoir. In some embodiments, the system further includes a fluid delivery source fluid-communicating with the first inlet, the fluid delivery source being configured to deliver a plurality of cells in liquid and / or suspension through the first lumen to the second outlet. In some embodiments, the device further includes a plurality of electroporation zones, for example, each of the plurality of electroporation zones having a substantially uniform or non-uniform cross-sectional area. 【0045】 The system may further include a controller operably coupled to a potential source to deliver voltage pulses to a first electrode and a second electrode to generate a potential difference between the first electrode and the second electrode, and thus generate an electric field within the electroporation zone. 【0046】 In some embodiments, the system further includes an outer structure comprising a first electrode, a second electrode, and a housing configured to enclose at least one electroporation zone of the device. The system may further include a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. The housing may further include a thermal controller configured to raise the temperature of the liquid in which the device and / or a plurality of cells are suspended, wherein the thermal controller is a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In addition, or alternatively, the housing may further include a thermal controller configured to lower the temperature of the liquid in which the device and / or a plurality of cells are suspended, wherein the thermal controller is a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier device. In some embodiments, the potential source is releasably connected to the first and second electrical inputs of the outer structure, for example, the releasable connection between the first or second electrical input and the potential source is selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. The outer structure and / or housing may be integrated into or releasably connected to the device. 【0047】 In some embodiments, the system further includes a plurality of cell perforation devices having, for example, a plurality of external structures. In some embodiments, the housing is configured to energize the plurality of devices in parallel, in series, or in a time-offset manner, and the housing further includes a tray for housing the plurality of electroperforation devices, the tray is modified with two grid electrodes, the first grid electrode being electrically isolated from the second grid electrode, the outside of the first electrode of each of the plurality of devices being in releasably and operably in contact with one of a first spring-biased electrode, a first mechanically connected electrode, or a first inductively connected electrode, and the outside of the second electrode of each of the plurality of devices being in contact with a second spring-biased electrode, a second Each of the plurality of devices is releasably and operably in contact with either a mechanically connected electrode or a second inductively coupled electrode, and each of the plurality of devices enters the housing releasably through an opening in the grid electrode, and each of the first spring-biased electrode, the first mechanically connected electrode, or the first inductively connected electrode of each device is operably in contact with the first grid electrode, and each of the second spring-biased electrode, the second mechanically connected electrode, or the second inductively connected electrode of each device is operably in contact with the second grid electrode, and the grid electrode is connected to a potential source. In some embodiments, the potential source delivers voltage pulses to the grid electrode so that the first grid electrode is energized with a specific applied voltage, while the second grid electrode is energized with a specific applied voltage, and each of the plurality of devices is energized by the grid electrode with the same applied voltage pulse such that the magnitude of the electric field generated in each of at least one electroporation zones of each device is substantially the same. In some embodiments, the potential source includes additional circuitry or programming configured to modulate the delivery of voltage pulses to the grid electrodes, so that each of the multiple devices may receive a different voltage from the grid electrodes, and the magnitude of the electric field generated within each of at least one electroporation zones of each device may be different. 【0048】 In another aspect, the present invention provides a method for introducing a composition into a plurality of cells suspended in a fluid using any of the devices or systems of the present invention. In particular, the method of the present invention provides a device comprising: a first electrode comprising a first lumen comprising a first outlet, a first inlet, and a minimum cross-sectional dimension; a second electrode comprising a second lumen comprising a second outlet, a second inlet, and a minimum cross-sectional dimension; and an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone is larger than about 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm) The invention provides an electroporation zone having a substantially uniform cross-sectional area, including a minimum cross-sectional dimension (for example, about 0.5 mm, about 1.0 mm, about 1.5 mm, about 2 mm, about 5 mm, about 10 mm, about 15 mm, about 25 mm, or about 50 mm), with a first outlet, the electroporation zone, and a second inlet being in fluid communication; generating an electric field within the electroporation zone by applying a potential difference between the first electrode and the second electrode; and increasing the permeability of multiple cells and a composition by passing multiple cells and a composition through the electroporation zone. In some embodiments, passing multiple cells involves applying a fluid-driven positive pressure. In some embodiments, neither the first lumen, the second lumen, nor the electroporation zone has a minimum cross-sectional dimension that temporarily compresses the cross-sectional dimension of any of the multiple cells suspended in the fluid. Electroporation may be substantially non-thermally reversible electroporation, substantially non-thermally irreversible electroporation, or substantially thermally irreversible electroporation.In some embodiments, the flow rate of multiple cells in the liquid and / or suspension delivered from the fluid delivery source from the first lumen to the electroporation zone is 0.001 mL / min to 1,000 mL / min (e.g., 0.001 mL / min to 0.05 mL / min, 0.001 mL / min to 0.1 mL / min, 0.001 mL / min to 1 mL / min, 0.05 mL / min to 0.5 mL / min, 0.05 mL / min to 5 mL / min, 0.1 mL / min to 1 mL / min, 0.5 mL / min to 2 mL / min, 1 mL / min to 5 mL / min, 1 mL / min to 10mL / min, 1mL / min~100mL / min, 5mL / min~25mL / min, 5mL / min~150mL / min, 10mL / min~100mL / min, 15mL / min~150mL / min, 25mL / min~100mL / min, 25mL / min~200mL / min, 5 0mL / min~150mL / min, 50mL / min~250mL / min, 75mL / min~200mL / min, 75mL / min~350mL / min, 100mL / min~250mL / min, 100mL / min~400mL / min, 150mL / min~450mL / min, 200 mL / min to 500 mL / min, 250 mL / min to 700 mL / min, 300 mL / min to 1,000 mL / min, 400 mL / min to 750 mL / min, 500 mL / min to 1,000 mL / min, or 750 mL / min to 1,000 mL / min, for example, about 0.001 mL / min, about 0.01 mL / min, about 0.05 mL / min, about 0.1 mL / min, about 0.5 mL / min, about 1 mL / min, about 5 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 30 mL / min, about 40 mL / min, about 50 mL / min, about 6 The fluid delivery source is configured to deliver multiple cells in liquid and / or suspension through a first lumen to a second outlet. (The fluid delivery source is configured to deliver multiple cells in liquid and / or suspension through a first lumen to a second outlet.) 【0049】 In some embodiments, the residence time in any of the electroporation zones of multiple cells suspended in a liquid is 0.5ms to 50ms (e.g., 0.5ms to 5ms, 1ms to 10ms, 1ms to 15ms, 5ms to 15ms, 10ms to 20ms, 15ms to 25ms, 20ms to 30ms, 25ms to 35ms, 30ms to 40ms, 35ms to 45ms, or 40ms to 50ms, for example, about 0.5ms, about 0.6ms, about 0.7ms, about 0.8ms, about 0.9ms, about 1ms, about 1. The residence times are approximately 5ms, 2ms, 2.5ms, 3ms, 3.5ms, 4ms, 4.5ms, 5ms, 5.5ms, 6ms, 6.5ms, 7ms, 7.5ms, 8ms, 8.5ms, 9ms, 9.5ms, 10ms, 10.5ms, 11ms, 11.5ms, 12ms, 12.5ms, 13ms, 13.5ms, 14ms, 14.5ms, 15ms, 20ms, 25ms, 30ms, 35ms, 40ms, 45ms, or 50ms. In some embodiments, the residence time is 5–20ms (e.g., 6–18ms, 8–15ms, or 10–14ms). 【0050】 In some embodiments, upon exiting the device's second exit (e.g., within 48 hours after exiting the second exit, e.g., within 24 hours after exiting the second exit, e.g., 1 minute to 24 hours, 5 minutes to 24 hours, 10 minutes to 24 hours, 30 minutes to 24 hours, 1 hour to 24 hours, or 2 hours to 24 hours after exiting the second exit), the phenotype of multiple cells is 0% to approximately 25% (e.g., approximately 0% to approximately 2.5%) relative to the baseline measurement of cell phenotype. It changes by approximately 1% to 5%, 1% to 10%, 5% to 15%, 10% to 20%, 15% to 25%, or 20% to 25%, for example, approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. 【0051】 In some embodiments, once the cells exit the device's second exit (for example, within 48 hours after exiting the second exit, for example, within 24 hours after exiting the second exit, for example, 1 minute to 24 hours, 5 minutes to 24 hours, 10 minutes to 24 hours, 30 minutes to 24 hours, 1 hour to 24 hours, or 2 hours to 24 hours after exiting the second exit), the phenotypes of multiple cells do not change relative to baseline measurements of cell phenotype. 【0052】 In some embodiments, the electric field is generated by a voltage pulse, which energizes the first electrode at a specific applied voltage while the second electrode is energized at a specific applied voltage, thereby applying a potential difference between the first electrode and the second electrode, where the voltage pulses are each -3kV to 3kV (e.g., -3kV to 1kV, -3kV to -1.5kV, -2kV to 2kV, -1.5kV to 1.5kV, -1.5kV to 2.5kV, -1kV to 1kV, -1kV to 2kV, -0.5kV to 0.5kV, -0.5kV to 1.5kV, -0.5kV to 3kV). -0.01kV~2kV, 0kV~1kV, 0kV~2kV, 0kV~3kV, 0.01kV~0.1kV, 0.01kV~1kV , 0.02kV~0.2kV, 0.03kV~0.3kV, 0.04kV~0.4kV, 0.05kV~0.5kV, 0.05kV~ 1.5kV, 0.06kV~0.6kV, 0.07kV~0.7kV, 0.08kV~0.8kV, 0.09kV~0.9kV, 0. 1kV~0.7kV, 0.1kV~1kV, 0.1kV~2kV, 0.1kV~3kV, 0.15kV~1.5kV, 0.2~0.6 kV, 0.2kV~2kV, 0.25kV~2.5kV, 0.3kV~3kV, 0.5kV~1kV, 0.5kV~3kV, 0.6kV~1.5kV, 0.7kV~1.8kV, 0.8kV~2kV, 0.9kV~3kV, 1kV~2kV, 1.5kV~2.5kV, or 2kV~3kV, for example, approximately -3kV, approximately -2.5kV, approximately -2kV, approximately -1.5kV, approximately -1kV, approximately -0.5kV, approximately -0.01kV, approximately 0kV, approximately 0.01kV, approximately 0.02kV, approximately 0.03kV, approximately 0.04kV, approximately 0.05kV, approximately 0.06kV, It has an amplitude of approximately 0.07kV, 0.08kV, 0.09kV, 0.1kV, 0.2kV, 0.3kV, 0.4kV, 0.5kV, 0.6kV, 0.7kV, 0.8kV, 0.9kV, 1kV, 1.1kV, 1.2kV, 1.3kV, 1.4kV, 1.5kV, 1.6kV, 1.7kV, 1.8kV, 1.9kV, 2kV, 2.1kV, 2.2kV, 2.3kV, 2.4kV, 2.5kV, 2.6kV, 2.7kV, 2.8kV, 2.9kV, or 3kV.In some embodiments, the first electrode is energized with a specific applied voltage while the second electrode is held to ground (e.g., 0kV), thus applying a potential difference between the first and second electrodes. In some embodiments, the voltage pulses are 0.01ms to 1,000ms (e.g., 0.01ms to 0.1ms, 0.01ms to 1ms, 0.01ms to 10ms, 0.05ms to 0.5ms, 0.05ms to 1ms, 0.1ms to 1ms, 0.1ms to 5ms, 0.1ms to 500ms, 0.5ms to 2ms, 1ms to 5ms, 1ms to 10ms, 1ms~25ms, 1ms~100ms, 1ms~1,000ms, 5ms~25ms, 5ms~150ms, 10ms~100ms, 15ms~150ms, 25ms~1 00ms, 25ms~200ms, 50ms~150ms, 50ms~250ms, 75ms~200ms, 75ms~350ms, 100ms~250ms, 100ms~4 00ms, 150ms~450ms, 200ms~500ms, 250ms~700ms, 300ms~1,000ms, 400ms~750ms, 500ms~1,000ms, or 750ms~1,000ms, for example, approximately 0.01ms, approximately 0.05ms, approximately 0.1ms, approximately 0.5ms, approximately 1ms, approximately 5ms, approximately 10ms, approximately 15ms, approximately 20 It has a duration of approximately ms, 30ms, 40ms, 50ms, 60ms, 70ms, 80ms, 90ms, 100ms, 150ms, 200ms, 250ms, 300ms, 350ms, 400ms, 450ms, 500ms, 600ms, 700ms, 800ms, 900ms, or 1,000ms.In the Large embodiment, the voltage pulses range from 1Hz to 50,000Hz (for example, 1Hz to 10Hz, 1Hz to 100Hz, 1Hz to 1,000Hz, 5Hz to 20Hz, 5Hz to 2,000Hz, 10Hz to 50Hz, 10Hz to 100Hz, 10Hz to 1,000Hz, 10Hz to 10,000Hz, 20Hz to 50Hz, 20Hz to 100Hz, 20Hz to 2,000Hz, 20Hz to 20,000Hz, 50Hz to 500Hz, 50Hz to 1,000Hz, 50Hz~50,000Hz, 100Hz~200Hz, 100Hz~500Hz, 100Hz~1,000Hz, 100Hz~10,000Hz, 100Hz~50,000Hz, 200Hz~400H z, 200Hz~750Hz, 200Hz~2,000Hz, 500Hz~1,000Hz, 750Hz~1,500Hz, 750Hz~10,000Hz, 1,000Hz~2,000Hz, 1,000Hz~5,000 Hz, 1,000Hz~10,000Hz, 1,000Hz~50,000Hz, 5,000Hz~10,000Hz, 5,000Hz~20,000Hz, 5,000Hz~50,000Hz, 10,000Hz~15,000Hz, 10,000Hz~25,000Hz, 10,000Hz~50,000Hz, 20,000Hz~30,000Hz, or 20,000~50,000Hz, for example, approximately 1Hz, approximately 5Hz, approximately 10Hz, approximately A current is applied to the first electrode and the second electrode at frequencies of approximately 20Hz, 50Hz, 75Hz, 100Hz, 150Hz, 200Hz, 300Hz, 400Hz, 500Hz, 600Hz, 700Hz, 800Hz, 900Hz, 1,000Hz, 2,000Hz, 5,000Hz, 10,000Hz, 15,000Hz, 20,000Hz, 30,000Hz, 40,000Hz, or 50,000Hz. 【0053】 In some embodiments, the waveform of the voltage pulse is selected from the group consisting of DC, square, pulsed, bipolar, sinusoidal, ramp, asymmetric bipolar, arbitrary, and superposition or combination of any of these. In some embodiments, the electric field generated from the voltage pulse is 1V / cm to 50,000V / cm (e.g., 1V / cm to 50V / cm, 1V / cm to 500V / cm, 1V / cm to 1,000V / cm, 1V / cm to 20,000V / cm, 5V / cm to 10,000V / cm, 25V / cm to 200V / cm, 50V / cm to 250V / cm, 50V / cm to 500V / cm, 50V / cm to 15,000V / cm, 100V / cm to 1,000V / cm, 3 00V / cm~500V / cm, 500V / cm~10,000V / cm, 1000V / cm~25,000V / cm, 5,000V / cm~25,000V / cm, 10,000V / cm~20,000V / cm, 10,000V / cm~50,000V / cm, for example, approximately 1V / cm, approximately 2V / cm, approximately 3V / cm, approximately 4V / cm, approximately 5V / cm, approximately 6V / cm, approximately 7V / cm, approximately 8V / cm, approximately 9V / cm, approximately 10V / cm, approximately 20V / cm, approximately 30V / cm, approximately 40V / cm, approximately 50V / cm, approximately 60V / cm, approximately 70V / cm, approximately 80V / cm, approximately 90V / cm, approximately 100V / cm, approximately 150V / cm, approximately 200V / cm, approximately 250V / cm, approximately 300V / cm, approximately 350V / c m, approximately 400V / cm, approximately 450V / cm, approximately 500V / cm, approximately 550V / cm, approximately 600V / cm, approximately 650V / cm, approximately 700V / cm, approximately 750V / cm, approximately 800V / cm, approximately 900V / cm, approximately 1,000V / cm, approximately It has a magnitude of approximately 2,000V / cm, 3,000V / cm, 4,000V / cm, 5,000V / cm, 6,000V / cm, 7,000V / cm, 8,000V / cm, 9,000V / cm, 10,000V / cm, 15,000V / cm, 20,000V / cm, 25,000V / cm, 30,000V / cm, 35,000V / cm, 40,000V / cm, 45,000V / cm, or 50,000V / cm. 【0054】 In some embodiments, the duty cycle of the voltage pulse is 0.001% to 100% (e.g., 0.001% to 0.1%, 0.001% to 10%, 0.01% to 1%, 0.01% to 100%, 0.1% to 5%, 0.1% to 99%, 1% to 10%, 1% to 97%, 2.5% to 20%, 5% to 25%, 5% to 40%, 10% to 10%). 25%, 10%~50%, 10%~95%, 15%~60%, 15%~85%, 20%~40%, 30%~50%, 40%~60%, 40%~75%, 50%~85%, 50%~100%, 75%~100%, or 90%~100%, for example, approximately 0.001%, approximately 0.002%, approximately 0.003%, approximately 0.004%, approximately 0.0 0.5%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8% The percentages are approximately 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 【0055】 In some embodiments, the liquid has a viscosity of 0.001 mS / cm to 500 mS / cm (for example, 0.001 mS / cm to 0.05 mS / cm, 0.001 mS / cm to 0.1 mS / cm, 0.001 mS / cm to 1 mS / cm, 0.05 mS / cm to 0.5 mS / cm, 0.05 mS / cm to 5 mS / cm, 0.1 mS / cm to 1 mS / cm, 0.1 mS / cm to 100 mS / cm, 0.5 mS / cm to 2 mS / cm, 1 mS / cm to 5 mS / cm, 1 mS / cm). ~10mS / cm, 1mS / cm~100mS / cm, 1mS / cm~500mS / cm, 5mS / cm~25mS / cm, 5mS / cm~150mS / cm, 10mS / cm~100mS / cm, 10mS / cm~250mS / cm, 15mS / cm~150mS / cm, 25mS / cm~100mS / cm, 25mS / cm~200mS / cm, 50mS / cm~150mS / cm, 50mS / cm~250mS / cm, 50mS / cm~500mS / cm, 75mS / cm~200mS / cm, 75mS / cm~350mS / cm, 100mS / cm~250mS / cm, 100mS / cm~400mS / cm, 100mS / cm~500mS / cm, 150mS / cm~450mS / cm, 200mS / cm~500mS / cm, 300mS / cm~500mS / cm, for example, approximately 0.001mS / cm, approximately 0.01mS / cm, approximately 0.05mS / cm, approximately 0.1mS / cm, approximately 0.5mS / cm, approximately 1 It has an electrical conductivity of approximately mS / cm, 5mS / cm, 10mS / cm, 15mS / cm, 20mS / cm, 30mS / cm, 40mS / cm, 50mS / cm, 60mS / cm, 70mS / cm, 80mS / cm, 90mS / cm, 100mS / cm, 150mS / cm, 200mS / cm, 250mS / cm, 300mS / cm, 350mS / cm, 400mS / cm, 450mS / cm, or 500mS / cm. 【0056】 In some embodiments, the temperature of multiple cells suspended in a liquid is 0°C to 50°C (0°C to 5°C, 2°C to 15°C, 3°C to 30°C, 4°C to 10°C, 4°C to 25°C, 5°C to 30°C, 7°C to 35°C, 10°C to 25°C, 10°C to 40°C, 15°C to 50°C, 20°C to 40°C, 25°C to 50°C, or 35°C to 45°C, for example, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, (Approximately 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C). 【0057】 In some embodiments, the method further includes storing a plurality of cells suspended in a liquid in a recovery buffer after perforation. In some embodiments, the cells are concentrated in a range of 0.1% to 99.9% (e.g., 0.1% to 5%, 1% to 10%, 2.5% to 20%, 5% to 40%, 10% to 30%, 10% to 60%, 10% to 90%, 25% to 40%, 25% to 85%, 30% to 50%, 30% to 80%, 40% to 65%, 50% to 75%, 50% to 99.9%, 60% to 80%, 75% to 99.9%, or 85% to 99.9%, for example, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0. The composition has a survival rate after introduction of approximately 45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9%. 【0058】 In some embodiments, the composition is 0.1% to 99.9% (for example, 0.1% to 5%, 1% to 10%, 2.5% to 20%, 5% to 40%, 10% to 30%, 10% to 60%, 10% to 90%, 25% to 40%, 25% to 85%, 30% to 50%, 30% to 80%, 40% to 65%, 50% to 75%, 50% to 99.9%, 60% to 80%, 75% to 99.9%, or 85% to 99.9%, for example, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0 It is introduced into multiple cells with an efficiency of approximately 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9%). 【0059】 In some embodiments, any of the methods of the present invention is 10 4 cells ~10 12 Cells (for example, 10 4 cells ~10 5 cells, 10 4 cells ~10 6 cells, 10 4 cells ~10 7 cells, 5 x 10 4 cells ~5×10 5 cells, 10 5 cells ~10 6 cells, 10 5 cells ~10 7 cells, 10 5 cells ~10 10 cells, 2.5 x 10 5 cells ~10 6 cells, 5 x 10 5 cells ~5×10 6 cells, 10 6 cells ~10 7 cells, 10 6 cells ~10 8 cells, 10 6 cells ~10 12cells, 5 x 10 6 cells ~5×10 7 cells, 10 7 cells ~10 8 cells, 10 7 cells ~10 9 cells, 10 7 cells ~10 12 cells, 5 x 10 7 cells ~5×10 8 cells, 10 8 cells ~10 9 cells, 10 8 cells ~10 10 cells, 10 8 cells ~10 12 cells, 5 x 10 8 cells ~5×10 9 cells, 10 9 cells ~10 10 cells, 10 9 cells ~10 11 cells, 10 10 cells ~10 11 cells, 10 10 cells ~10 12 cells, or 10 11 cells ~10 12 Cells, for example, about 10 4 cells, approximately 2.5 x 10 4 cells, approximately 5 x 10 4 cells, about 10 5 cells, approximately 2.5 x 10 5 cells, approximately 5 x 10 5 cells, about 10 6 cells, approximately 2.5 x 10 6 cells, approximately 5 x 10 6 cells, about 10 7 cells, approximately 2.5 x 10 7 cells, approximately 5 x 10 7 cells, about 10 8 cells, approximately 2.5 x 10 8 cells, approximately 5 x 10 8 cells, about 10 9 cells, approximately 2.5 x 10 9 cells, approximately 5 x 10 9 cells, about 10 10 cells, approximately 5 x 10 10 cells, about 10 11 Cells, or about 10 12 Generates the number of cells to be harvested. 【0060】 In some embodiments, the method is 0.1%~100% (for example, 0.1%~5%, 1%~10%, 2.5%~20%, 5%~40%, 10%~30%, 10%~60%, 10%~90%, 25%~40%, 25%~85%, 30%~50%, 30%~80%, 40%~65%, 50%~75%, 50%~100%, 60%~80%, 75%~100%, 85%~100%, for example, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about It produces cell recovery rates of approximately 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.In some embodiments, the method is 0.1%~500% (for example, 0.1%~5%, 1%~10%, 2.5%~20%, 5%~40%, 10%~30%, 10%~60%, 10%~90%, 25%~40%, 25%~85%, 30%~50%, 30%~80%, 40%~65%, 50%~75%, 50%~100%, 60%~80%, 60%~150%, 75%~100%, 75%~200%, 85%~ 150%, 90%~250%, 100%~200%, 100%~400%, 150%~300%, 200%~500%, or 300%~500%, for example, approximately 0.1%, approximately 0.15%, approximately 0.2%, approximately 0.25%, approximately 0.3%, approximately 0.35%, approximately 0.4%, approximately 0.45%, approximately 0.5%, approximately 0.55%, approximately 0.6%, approximately 0.65%, approximately 0.7%, approximately 0.75%, approximately 0.8%, approximately 0.85%, approximately 0.9%, Approximately 0.95%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, approximately 100%, approximately 150%, approximately 200%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately It produces a live-operated cell yield (e.g., recovery yield) of approximately 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%. 【0061】 In some embodiments, the composition comprises at least one compound selected from the group consisting of therapeutic agents, vitamins, nanoparticles, charged molecules, uncharged molecules, manipulated nucleases, DNA, RNA, CRISPR-Cas complexes, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing nucleases, meganucleases (mn), megaTALs, enzymes, transposons, peptides, proteins, viruses, polymers, ribonucleoproteins (RNPs), and polysaccharides. In some embodiments, the composition ranges from 0.0001 μg / mL to 1,000 μg / mL (for example, approximately 0.0001 μg / mL to approximately 0.001 μg / mL, approximately 0.001 μg / mL to approximately 0.01 μg / mL, approximately 0.001 μg / mL to approximately 5 μg / mL, approximately 0.005 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 1 μg / mL). , about 0.1μg / mL to about 5μg / mL, about 1μg / mL to about 10μg / mL, about 1μg / mL to about 50μg / mL, about 1μg / mL to about 100μg / mL, about 2.5μg / mL to about 15μg / mL, about 5μg / mL mL ~ approx. 25 μg / mL, approx. 5 μg / mL ~ approx. 50 μg / mL, approx. 5 μg / mL ~ approx. 500 μg / mL, approx. 7.5 μg / mL ~ approx. 75 μg / mL, approx. 10 μg / mL ~ approx. 100 μg / mL, approx. 10 μg / mL ~ approx. 1 ,000μg / mL, about 25μg / mL to about 50μg / mL, about 25μg / mL to about 250μg / mL, about 25μg / mL to about 500μg / mL, about 50μg / mL to about 100μg / mL, about 50μg / mL to about 250μg / mL, approximately 50μg / mL to approximately 750μg / mL, approximately 100μg / mL to approximately 300μg / mL, approximately 100μg / mL to approximately 1,000μg / mL, approximately 200μg / mL to approximately 400μg / mL, approximately 250μg / mL~approximately 500μg / mL, approximately 350μg / mL~approximately 500μg / mL, approximately 400μg / mL~approximately 1,000μg / mL, approximately 500μg / mL~approximately 750μg / mL, approximately 650μg / mL~approximately 1,000μg / mL, or approximately 800μg / mL~approximately 1,000μg / mL, for example, approximately 0.0001μg / mL, approximately 0.0005μg / mL, approximately 0.001μg / mL, approximately 0.005μg / mL, approximately 0.01μg / mL, approximately 0.01μg / mL, approximately 0.02μg / mL, approximately 0.03μg / mL, approximately 0.04μg / mL, approximately 0.05μg / mL, approximately 0.06μg / mL, approximately 0.07μg / mL, approximately 0.08μg / mL, approximately 0.09μg / mL, approximately 0 .1μg / mL, approximately 0.2μg / mL, approximately 0.3μg / mL, approximately 0.4μg / mL, approximately 0.5μg / mL, approximately 0.6μg / mL, approximately 0.7μg / mL, approximately 0.8μg / mL, approximately 0.9μg / mL , about 1μg / mL, about 1.5μg / mL, about 2μg / mL, about 2.5μg / mL, about 3μg / mL, about 3.5μg / mL, about 4μg / mL, about 4.5μg / mL, about 5μg / mL, about 5.5μg / mL, approximately 6μg / mL, approximately 6.5μg / mL, approximately 7μg / mL, approximately 7.5μg / mL, approximately 8μg / mL, approximately 8.5μg / mL, approximately 9μg / mL, approximately 9.5μg / mL, approximately 10μg / mL, approximately 1 5μg / mL, approximately 20μg / mL, approximately 25μg / mL, approximately 30μg / mL, approximately 35μg / mL, approximately 40μg / mL, approximately 45μg / mL, approximately 50μg / mL, approximately 55μg / mL, approximately 60μg / mL , about 65μg / mL, about 70μg / mL, about 75μg / mL, about 80μg / mL, about 85μg / mL, about 90μg / mL, about 95μg / mL, about 100μg / mL, about 200μg / mL, about 25 It has concentrations in liquids of 0 μg / mL, approximately 300 μg / mL, approximately 350 μg / mL, approximately 400 μg / mL, approximately 450 μg / mL, approximately 500 μg / mL, approximately 550 μg / mL, approximately 600 μg / mL, approximately 650 μg / mL, approximately 700 μg / mL, approximately 750 μg / mL, approximately 800 μg / mL, approximately 850 μg / mL, approximately 900 μg / mL, approximately 950 μg / mL, or approximately 1,000 μg / mL. 【0062】 In some embodiments, the plurality of cells suspended in liquid include eukaryotic cells (e.g., animal cells, e.g., human cells), prokaryotic cells (e.g., bacterial cells), plant cells, and / or synthetic cells. The cells may be primary cells (e.g., primary human cells), cell line-derived cells (e.g., human cell lines), cells in suspension, adherent cells, stem cells, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), and / or immune cells (e.g., leukocytes (e.g., innate immune cells or adaptive immune cells)). In some embodiments, the cells (e.g., immune cells, e.g., T cells, B cells, natural killer cells, macrophages, monocytes, or antigen-presenting cells) are unstimulated cells, stimulated cells, or activated cells. In some embodiments, the cells are adaptive immune cells and / or innate immune cells. In some embodiments, the plurality of cells are antigen-presenting cells (APCs). The cells include monocytes, T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, Jurkat cells, THP-1 cells, human embryonic kidney (HEK-293) cells, Chinese hamster ovary (e.g., CHO-K1) cells, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). In some embodiments, the cells may be primary human T cells, primary human macrophages, primary human monocytes, primary human NK cells, or primary human induced pluripotent stem cells (iPSCs). In some embodiments of any of the methods described herein, the method further includes storing a plurality of cells suspended in a liquid in a recovery buffer after perforation. 【0063】 In another embodiment, the present invention provides a kit comprising any of the devices or systems described herein. For example, in one embodiment, the present invention provides a kit for electroperforating a plurality of cells suspended in a liquid, the kit comprising a plurality of cell perforation devices, each of which comprises a first electrode comprising a first lumen comprising a first outlet, a first inlet, and a minimum cross-sectional dimension, a second electrode comprising a second lumen comprising a second outlet, a second inlet, and a minimum cross-sectional dimension, and an electroperforation zone disposed between the first outlet and the second inlet, wherein the electroperforation zone is larger than about 100 μm (e.g., 100 μm to 10 mm, 150 μm to 15 mm, 200 μm to 10 mm, 250 μm to 5 mm, 500 μm to 10 mm, 1 mm to 10 mm, 1 mm to 50 mm, 5 mm to 25 mm, or 20 mm to 50 mm, e.g., about 0.5 mm, about 1 A plurality of cell perforation devices, each comprising an electroperforation zone having a substantially uniform cross-sectional area and an electroperforation zone having a minimum cross-sectional dimension of 0.0 mm, approximately 1.5 mm, approximately 2 mm, approximately 5 mm, approximately 7 mm, approximately 10 mm, approximately 15 mm, approximately 25 mm, or approximately 50 mm, wherein the application of a potential difference to a first electrode and a second electrode generates an electric field within the electroperforation zone; and a plurality of outer structures configured to enclose the plurality of cell perforation devices, each of which comprises a housing configured to enclose a first electrode, a second electrode, and an electroperforation zone of at least one cell perforation device, and a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the plurality of outer structures are integrated into the plurality of cell perforation devices. In some embodiments, the plurality of outer structures are releasably connected to the plurality of cell perforation devices. In some embodiments, the housing further includes a thermal controller configured to raise the temperature of at least one cell piercing device, the thermal controller being a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater.In some embodiments, the housing further includes a thermal controller configured to lower the temperature of at least one cell piercing device, the thermal controller being a cooling element selected from the group consisting of liquid flow, evaporative cooler, and Peltier device. 【0064】 In another embodiment, the present invention provides a kit for electroperforating a plurality of cells suspended in a liquid, the kit comprising a plurality of cell perforation devices, each of which comprises a plurality of cell perforation devices, each comprising a device of the above-described embodiment; and a plurality of outer structures configured to enclose the plurality of cell perforation devices, each of which comprises a housing configured to enclose a first electrode, a second electrode, and an electroperforation zone of at least one cell perforation device, and a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the plurality of outer structures are integrated into the plurality of cell perforation devices. In some embodiments, the plurality of outer structures are releasably connected to the plurality of cell perforation devices. In some embodiments, the housing further comprises a thermal controller configured to raise the temperature of at least one cell perforation device, the thermal controller being a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin-film heater. In some embodiments, the housing further includes a thermal controller configured to lower the temperature of at least one cell piercing device, the thermal controller being a cooling element selected from the group consisting of liquid flow, evaporative cooler, and Peltier device. 【0065】 In another embodiment, the present invention provides a device for electroperforating a plurality of cells suspended in a fluid, the device comprising: a first electrode having a first inlet and a first outlet, the lumen of which defines an entry zone; a second electrode having a second inlet and a second outlet, the lumen of which defines a recovery zone; and an electroperforation zone, the electroperforation zone being fluid-connected to the first outlet of the first electrode and the second inlet of the second electrode, the electroperforation zone having substantially uniform cross-sectional dimensions, and the application of a potential difference to the first electrode and the second electrode generating an electric field within the electroperforation zone. In the device, a plurality of cells suspended in a fluid are electroperforated upon entering the electroperforation zone. 【0066】 In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir may be fluidly connected to the entry zone, and the second reservoir may be fluidly connected to the recovery zone. 【0067】 In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, elliptical, polygonal, star-shaped, parallelogram, trapezoidal, and irregular shapes. 【0068】 In some cases, the cross-sectional dimensions of the entry zone or the recovery zone are 0.01% to 100,000% of the cross-sectional dimensions of the electroporation zone. For example, the cross-sectional dimensions of the entry zone or the recovery zone could be approximately 0.01% to approximately 1000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 10%, approximately 5% to approximately 25%, approximately 10% to approximately 50%, approximately 10% to approximately 1000%, approximately 25% to approximately 75%, approximately 25% to approximately 750%, or approximately 50% to approximately 1000%. Alternatively, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 100% to 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the cross-sectional dimensions of the electroporation zone. 【0069】 In some embodiments, the cross-sectional dimensions of the electroporation zone are 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 50 mm. In certain embodiments, the length of the electroporation zone is 0.005 mm to 25 mm. In some embodiments, the cross-sectional dimensions of either the first electrode or the second electrode are 0.1 mm to 500 mm. In certain embodiments, the entry zone, recovery zone, or electroporation zone does not reduce the cross-sectional dimensions of any of the multiple cells suspended in the fluid, for example, allowing cells to pass through the device without deformation. 【0070】 In some embodiments, upon exiting the electroporation zone, the phenotype of multiple cells changes by 0% to approximately 25% relative to baseline measurements of cell phenotype. In some embodiments, the phenotype of multiple cells does not change upon exiting the electroporation zone. 【0071】 In further embodiments, the device includes an outer structure having a housing configured to enclose a first electrode, a second electrode, and an electroporation zone of the device. In some embodiments, the outer structure is integrated into the device. In certain embodiments, the outer structure is releasably connected to the device. 【0072】 In another aspect, the present invention provides a device for electroperforating a plurality of cells suspended in a fluid, the device comprising: a first electrode having a first inlet and a first outlet, the lumen of which defines an entry zone; a second electrode having a second inlet and a second outlet, the lumen of which defines a recovery zone; and a third inlet and a third outlet, the third inlet and third outlet intersecting the first electrode between the first inlet and the second outlet. The device includes a third inlet and a third outlet, a fourth inlet and a fourth outlet, the fourth inlet and the fourth outlet intersecting with the second electrode between the second inlet and the second outlet, and an electroporation zone, the electroporation zone being fluidly connected to the first outlet of the first electrode and the second inlet of the second electrode, the electroporation zone having substantially uniform cross-sectional dimensions, and the application of a potential difference to the first electrode and the second electrode generating an electric field within the electroporation zone. In the device, multiple cells suspended in a fluid are electroporated when they enter the electroporation zone. 【0073】 In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to zones of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir may be fluidly connected to an entry zone, and the second reservoir may be fluidly connected to a recovery zone. In certain embodiments, the device includes a third reservoir fluidly connected to a third inlet and a third outlet, and a fourth reservoir fluidly connected to a fourth inlet and a fourth outlet. 【0074】 In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, elliptical, polygonal, star-shaped, parallelogram, trapezoidal, and irregular shapes. 【0075】 In some cases, the cross-sectional dimensions of the entry zone or the recovery zone are 0.01% to 100,000% of the cross-sectional dimensions of the electroporation zone. For example, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 0.01% to approximately 1,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 10%, approximately 5% to approximately 25%, approximately 10% to approximately 50%, approximately 10% to approximately 1,000%, approximately 25% to approximately 75%, approximately 25% to approximately 750%, or approximately 50% to approximately 100%. Alternatively, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 100% to 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the cross-sectional dimensions of the electroporation zone. 【0076】 In some embodiments, the cross-sectional dimensions of the electroporation zone are 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 50 mm. In certain embodiments, the length of the electroporation zone is 0.005 mm to 25 mm. In some embodiments, the cross-sectional dimensions of either the first electrode or the second electrode are 0.1 mm to 500 mm. In certain embodiments, the entry zone, recovery zone, or electroporation zone does not reduce the cross-sectional dimensions of any of the multiple cells suspended in the fluid, for example, allowing cells to pass through the device without deformation. 【0077】 In certain embodiments, the first electrode and / or the second electrode are porous or conductive fluids (e.g., liquids). 【0078】 In some embodiments, upon exiting the electroporation zone, the phenotype of multiple cells changes by 0% to approximately 25% relative to baseline measurements of cell phenotype. In some embodiments, the phenotype of multiple cells does not change upon exiting the electroporation zone. 【0079】 In further embodiments, the device includes an outer structure having a housing configured to enclose a first electrode, a second electrode, and an electroporation zone of the device. In some embodiments, the outer structure is integrated into the device. In certain embodiments, the outer structure is releasably connected to the device. 【0080】 In another embodiment, the present invention provides a system for electroperforating a plurality of cells suspended in a fluid, the system including a cell perforation device comprising: a first electrode having a first inlet and a first outlet, the lumen of which defines an entry zone; a second electrode having a second inlet and a second outlet, the lumen of which defines a recovery zone; and an electroperforation zone, the electroperforation zone being fluidly connected to the first outlet of the first electrode and the second inlet of the second electrode, the electroperforation zone having substantially uniform cross-sectional dimensions, and the application of a potential difference to the first and second electrodes generating an electric field within the electroperforation zone. The system further comprises a potential source, the first and second electrodes of the device being releasably connected to the potential source. In the system, a plurality of cells suspended in a fluid are electroperforated upon entering the electroperforation zone. 【0081】 In some embodiments, the phenotype of cells changes by 0% to approximately 25% relative to baseline measurements after leaving the electroporation zone. In some embodiments, the phenotype of multiple cells does not change after leaving the electroporation zone. 【0082】 In further embodiments, the device includes an outer structure having a first electrode, a second electrode, and a housing configured to enclose the electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the releasable connection between the first or second electrical input and the potential source is selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. 【0083】 In some embodiments, the outer structure is integrated into the device. In certain embodiments, the outer structure is removably connected to the device. 【0084】 In some cases, the system induces reversible or irreversible electroporation. In certain embodiments, the electroporation is substantially non-thermally reversible electroporation, substantially non-thermally irreversible electroporation, or substantially thermally irreversible electroporation. 【0085】 In some embodiments, the releasable connection between the device and the potential source is selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. In certain embodiments, the releasable connection between the device and the potential source is a spring. 【0086】 In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir may be fluidly connected to the entry zone, and the second reservoir may be fluidly connected to the recovery zone. 【0087】 In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, elliptical, polygonal, star-shaped, parallelogram, trapezoidal, and irregular shapes. 【0088】 In some cases, the cross-sectional dimensions of the entry zone or the recovery zone are 0.01% to 100,000% of the cross-sectional dimensions of the electroporation zone. For example, the cross-sectional dimensions of the entry zone or the recovery zone could be approximately 0.01% to approximately 1000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 10%, approximately 5% to approximately 25%, approximately 10% to approximately 50%, approximately 10% to approximately 1,000%, approximately 25% to approximately 75%, approximately 25% to approximately 750%, or approximately 50% to approximately 100%. Alternatively, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 100% to 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the cross-sectional dimensions of the electroporation zone. 【0089】 In some embodiments, the cross-sectional dimensions of the electroporation zone are 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 50 mm. In certain embodiments, the length of the electroporation zone is 0.005 mm to 25 mm. In some embodiments, the cross-sectional dimensions of either the first electrode or the second electrode are 0.1 mm to 500 mm. In certain embodiments, the entry zone, recovery zone, or electroporation zone does not reduce the cross-sectional dimensions of any of the multiple cells suspended in the fluid, for example, allowing cells to pass through the device without deformation. 【0090】 In further embodiments, the system includes a fluid delivery source fluid-connected to the entry zone, which is configured to deliver a plurality of cells suspended in the fluid through the entry zone to the recovery zone. In some embodiments, the delivery rate from the fluid delivery source is 0.001 mL / min to 1,000 mL / min, for example, 25 mL / min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is 0.5 ms to 50 ms. In some embodiments, the conductivity of the fluid is 0.001 mS / cm to 500 mS / cm, for example, 1 to 20 mS / cm. 【0091】 In further embodiments, the system includes a controller operably coupled to a potential source to deliver voltage pulses to a first electrode and a second electrode to generate a potential difference between the first electrode and the second electrode. In some embodiments, the voltage pulse has an amplitude of -3kV to 3kV, e.g., 0.01kV to 3kV, e.g., 0.2 to 0.6kV. In some cases, the duty cycle of electroporation is 0.001% to 100%, e.g., 10 to 95%. In some embodiments, the voltage pulse has a duration of 0.01ms to 1,000ms, e.g., 1 to 10ms. In certain embodiments, the voltage pulse is applied to the first electrode and the second electrode at a frequency of 1Hz to 50,000Hz, e.g., 100 to 500Hz. The waveform of the voltage pulse can be DC, square, pulsed, bipolar, sinusoidal, ramped, asymmetrical bipolar, arbitrary, or a superposition or combination of any of these. In certain embodiments, the electric field generated from the voltage pulse has a magnitude of 1 V / cm to 50,000 V / cm, for example, 100 to 1,000 V / cm. 【0092】 In further embodiments, the system includes a housing (e.g., a housing structure) configured to accommodate the electroporation device described herein. In further embodiments, the housing (e.g., a housing structure) includes a thermal controller configured to raise or lower the temperature of the housing or any component of the system. In some embodiments, the thermal controller is a heating element, e.g., a heating block, a liquid flow, a battery-powered heater, or a thin-film heater. In other embodiments, the thermal controller is a cooling element, e.g., a liquid flow, an evaporative cooler, or a thermoelectric, e.g., a Peltier device. 【0093】 In further embodiments, the system includes multiple cell piercing devices, for example, in series or parallel. In specific embodiments, the system includes multiple external structures for the multiple cell piercing devices. 【0094】 In a related aspect, the present invention provides a system for electroperforating a plurality of cells suspended in a fluid, the system comprising: a first electrode having a first inlet and a first outlet, the lumen of which defines an entry zone; a second electrode having a second inlet and a second outlet, the lumen of which defines a recovery zone; and a third inlet and a third outlet, the third inlet and third outlet intersecting with the first electrode between the first inlet and the second outlet. The cell perforation device includes an inlet and a third outlet, a fourth inlet and a fourth outlet, the fourth inlet and fourth outlet intersecting with the second electrode between the second inlet and the second outlet, and an electroperforation zone, the electroperforation zone being fluidly connected to the first outlet of the first electrode and the second inlet of the second electrode, the electroperforation zone having substantially uniform cross-sectional dimensions, and the application of a potential difference to the first electrode and the second electrode generating an electric field within the electroperforation zone. In the device, multiple cells suspended in a fluid are electroperforated upon entering the electroperforation zone. 【0095】 In some embodiments, upon exiting the electroporation zone, the phenotype of multiple cells changes by 0% to approximately 25% relative to baseline measurements of cell phenotype. In some embodiments, the phenotype of multiple cells does not change upon exiting the electroporation zone. 【0096】 In further embodiments, the device includes an outer structure having a housing (e.g., a housing structure) configured to enclose a first electrode, a second electrode, and an electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the releasable connection between the first or second electrical input and the potential source is selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. 【0097】 In some embodiments, the outer structure is integrated into the device. In certain embodiments, the outer structure is removably connected to the device. 【0098】 In some cases, the system induces reversible or irreversible electroporation. In certain embodiments, the electroporation is substantially non-thermally reversible electroporation, substantially non-thermally irreversible electroporation, or substantially thermally irreversible electroporation. 【0099】 In some embodiments, the releasable connection between the device and the potential source is selected from the group consisting of clamps, clips, springs, sheaths, wire brushes, mechanical connections, inductive connections, or combinations thereof. In certain embodiments, the releasable connection between the device and the potential source is a spring. 【0100】 In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir may be fluidly connected to the entry zone, and the second reservoir may be fluidly connected to the recovery zone. 【0101】 In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, elliptical, polygonal, star-shaped, parallelogram, trapezoidal, and irregular shapes. 【0102】 In some cases, the cross-sectional dimensions of the entry zone or the recovery zone are 0.01% to 100,000% of the cross-sectional dimensions of the electroporation zone. For example, the cross-sectional dimensions of the entry zone or the recovery zone could be approximately 0.01% to approximately 1000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 10%, approximately 5% to approximately 25%, approximately 10% to approximately 50%, approximately 10% to approximately 1,000%, approximately 25% to approximately 75%, approximately 25% to approximately 750%, or approximately 50% to approximately 100%. Alternatively, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 100% to 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the cross-sectional dimensions of the electroporation zone. 【0103】 In some embodiments, the cross-sectional dimensions of the electroporation zone are 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 50 mm. In certain embodiments, the length of the electroporation zone is 0.005 mm to 25 mm. In some embodiments, the cross-sectional dimensions of either the first electrode or the second electrode are 0.01 mm to 500 mm. In certain embodiments, the entry zone, recovery zone, or electroporation zone does not reduce the cross-sectional dimensions of any of the multiple cells suspended in the fluid, for example, allowing cells to pass through the device without deformation. 【0104】 In a further embodiment, the system includes a fluid delivery source fluidly connected to the entry zone, and the fluid delivery source is configured to deliver a plurality of cells suspended in the fluid through the entry zone to the collection zone. In some embodiments, the delivery rate from the fluid delivery source is from 0.001 mL / min to 1,000 mL / min, such as 25 mL / min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is from 0.5 ms to 50 ms. In some embodiments, the conductivity of the fluid is from 0.001 mS / cm to 500 mS / cm, such as from 1 mS / cm to 20 mS / cm. 【0105】 In a further embodiment, the system includes a controller operatively coupled to a potential source to deliver voltage pulses to a first electrode and a second electrode to generate a potential difference between the first electrode and the second electrode. In some embodiments, the voltage pulses have an amplitude of from -3 kV to 3 kV, such as from 0.01 kV to 3 kV, such as from 0.2 to 0.6 kV. In some cases, the duty cycle of the electroporation is from 0.001% to 100%, such as from 10 to 95%. In some embodiments, the voltage pulses have a duration of from 0.01 ms to 1,000 ms, such as from 1 to 10 ms. In certain embodiments, the voltage pulses are applied to the first electrode and the second electrode at a frequency of from 1 Hz to 50,000 Hz, such as from 100 to 500 Hz. The waveform of the voltage pulse can be DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, arbitrary, and any superposition or combination of these. In certain embodiments, the electric field generated from the voltage pulse has a magnitude of from 1 V / cm to 50,000 V / cm, such as from 100 V / cm to 1,000 V / cm. 【0106】 In further embodiments, the system includes a housing (e.g., a housing structure) configured to house the electroporation device described herein. In further instances, the housing structure includes a thermal controller configured to increase or decrease the temperature of the housing or any component of the system. In some embodiments, the thermal controller is a heating element, such as a heating block, a liquid flow, a battery-driven heater, or a thin film heater. In other embodiments, the thermal controller is a cooling element, such as a liquid flow, an evaporative cooler, or a thermoelectric, such as a Peltier device. 【0107】 In further embodiments, the system includes a plurality of cell perforation devices, such as in series or in parallel. In certain embodiments, the system includes a plurality of outer structures for the plurality of cell perforation devices. 【0108】 In another aspect, the present invention provides a method for introducing a composition into at least a portion of a plurality of cells suspended in a fluid, the method comprising: a. providing a device comprising a first electrode having a first inlet and a first outlet, wherein the lumen of the first electrode defines an entry zone, a second electrode having a second inlet and a second outlet, wherein the lumen of the second electrode defines a recovery zone, and an electroporation zone fluidly connected to the first outlet of the first electrode and the second inlet of the second electrode, wherein the application of a potential difference to the first electrode and the second electrode generates an electric field within the electroporation zone; b. generating an electric field within the electroporation zone by applying an electric current to the first electrode and the second electrode to generate a potential difference between the first electrode and the second electrode; and c. passing a plurality of cells suspended in the fluid together with the composition through the electric field within the electroporation zone of the device. In the method, the flow of the plurality of cells suspended in the fluid together with the composition through the electric field within the electroporation zone introduces the composition into at least a portion of the plurality of cells by increasing the temporary permeability of the plurality of cells. 【0109】 In further embodiments, the method includes evaluating the integrity of a portion of several cells suspended in a fluid. In certain embodiments, the evaluation includes measuring the viability of a portion of several cells suspended in a fluid. In some embodiments, the evaluation includes measuring the transfection efficiency of a portion of several cells suspended in a fluid. In some embodiments, the evaluation includes measuring the cell recovery rate of a portion of several cells suspended in a fluid. In certain embodiments, the evaluation includes flow cytometry analysis of cell surface marker expression. 【0110】 In some cases, upon exiting the device's electroporation zone, the phenotype of multiple cells changes by 0% to approximately 25% relative to baseline measurements of the cell phenotype. In other cases, the phenotype of multiple cells does not change upon exiting the device's electroporation zone. 【0111】 In some cases, the method induces reversible or irreversible electroporation. In certain embodiments, the electroporation is substantially non-thermally reversible electroporation, substantially non-thermally irreversible electroporation, or substantially thermally irreversible electroporation. 【0112】 In some embodiments, cells suspended in a fluid along with the composition are passed through an electric field within the electroporation zone of the device by applying positive pressure, for example, a pump, such as a syringe pump or peristaltic pump. 【0113】 In certain embodiments, the cells among the multiple cells in the sample may be mammalian cells, eukaryotes, human cells, animal cells, plant cells, synthetic cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, activated cells, immune cells, stem cells, blood cells, erythrocytes, T cells, B cells, neutrophils, dendritic cells, antigen-presenting cells (APCs), natural killer (NK) cells, monocytes, macrophages, or peripheral blood mononuclear cells (PBMCs), human embryonic kidney cells, e.g., HEK-293 cells, or Chinese hamster ovary (CHO) cells. In certain embodiments, the multiple cells include Jurkat cells. In certain embodiments, the multiple cells include primary human T cells. In certain embodiments, the multiple cells include THP-1 cells. In certain embodiments, the multiple cells include primary human macrophages. In certain embodiments, the multiple cells include primary human monocytes. In certain embodiments, the multiple cells include natural killer (NK) cells. In certain embodiments, the cells include Chinese hamster ovary cells. In certain embodiments, the cells include human embryonic kidney cells. In certain embodiments, the cells include B cells. In certain embodiments, the cells include primary human T cells. In certain embodiments, the cells include primary human monocytes. In certain embodiments, the cells include primary human macrophages. In certain embodiments, the cells include embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). In certain embodiments, the cells include primary human induced pluripotent stem cells (iPSCs). 【0114】 In some cases, the composition comprises at least one compound selected from the group consisting of therapeutic agents, vitamins, nanoparticles, charged therapeutic agents, nanoparticles, charged molecules, e.g., ions in solution, uncharged molecules, nucleic acids, e.g., DNA or RNA, CRISPR-Cas complexes, proteins, polymers, ribonucleoproteins (RNPs), manipulated nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing nucleases, meganucleases (MNs), megaTALs, enzymes, peptides, transposons, or polysaccharides, e.g., dextran, e.g., dextran sulfate. Compositions that can be delivered to cells in suspension include nucleic acids (e.g., oligonucleotides, mRNA, or DNA), antibodies (or antibody fragments, e.g., bispecificity fragments, trispecificity fragments, Fab, F(ab')2, or single-stranded variable fragments (scFv)), amino acids, polypeptides (e.g., peptides or proteins), cells, bacteria, gene therapies, genome-engineered therapeutics, epigenomically engineered therapeutics, carbohydrates, chemical drugs, contrast agents, magnetic particles, polymer beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotics, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, anti-inflammatory agents, antimicrobial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacteriophages, adjuvants, vitamins, minerals, organelles, and combinations thereof. In certain embodiments, the composition is nucleic acid (e.g., oligonucleotides, mRNA, or DNA). In certain embodiments, the composition is an antibody. In certain embodiments, the composition is a polypeptide (e.g., peptides or proteins). 【0115】 In a particular embodiment, the composition ranges from 0.0001 μg / mL to 1,000 μg / mL (for example, approximately 0.0001 μg / mL to approximately 0.001 μg / mL, approximately 0.001 μg / mL to approximately 0.01 μg / mL, approximately 0.001 μg / mL to approximately 5 μg / mL, approximately 0.005 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 5 μg / mL, approximately 1 μg / mL to approximately 10 μg / mL, approximately 1 μg / mL to approximately 50 μg / mL, approximately 1 μg / mL~about 100μg / mL, about 2.5μg / mL~about 15μg / mL, about 5μg / mL~about 25μg / mL, about 5μg / mL~about 50μg / mL, about 5μg / mL~about 500μg / mL, about 7.5μg / mL~about 75μg / mL, about 10μg / mL~about 100μg / mL, Approximately 10μg / mL to approximately 1,000μg / mL, approximately 25μg / mL to approximately 50μg / mL, approximately 25μg / mL to approximately 250μg / mL, approximately 25μg / mL to approximately 500μg / mL, approximately 50μg / mL to approximately 100μg / mL, approximately 50μg / mL to approximately 250μg / mL, approximately 50μg / mL ~750 μg / mL, approximately 100 μg / mL to approximately 300 μg / mL, approximately 100 μg / mL to approximately 1,000 μg / mL, approximately 200 μg / mL to approximately 400 μg / mL, approximately 250 μg / mL to approximately 500 μg / mL, approximately 350 μg / mL to approximately 500 μg / mL, approximately 400 μg / mL to approximately 1,000 μg / mL, approximately 500 μg / mL to approximately 750 μg / mL, approximately 650 μg / mL to approximately 1,000 μg / mL, or approximately 800 μg / mL to approximately 1,000 μg / mL, for example, approximately 0.0001 μg / mL, approximately 0.0005 μg / mL, approximately 0.001 μg / mL, approximately 0 .005μg / mL, approximately 0.01μg / mL, approximately 0.02μg / mL, approximately 0.03μg / mL, approximately 0.04μg / mL, approximately 0.05μg / mL, approximately 0.06μg / mL, approximately 0.07μg / mL, approximately 0.08μg / mL, approximately 0.09μg / mL, approximately 0.1μg / mL, approximately 0.2 μg / mL, approximately 0.3 μg / mL, approximately 0.4 μg / mL, approximately 0.5 μg / mL, approximately 0.6 μg / mL, approximately 0.7 μg / mL, approximately 0.8 μg / mL, approximately 0.9 μg / mL, approximately 1 μg / mL, approximately 1.5 μg / mL, approximately 2 μg / mL, approximately 2.5 μg / mL, approximately 3 μg / mL, approximately 3.5μg / mL, approximately 4μg / mL, approximately 4.5μg / mL, approximately 5μg / mL, approximately 5.5μg / mL, approximately 6μg / mL, approximately 6.5μg / mL, approximately 7μg / mL, approximately 7.5μg / mL, approximately 8μg / mL, approximately 8.5μg / mL, approximately 9μg / mL, approximately 9.5μg / mL, approximately 10 μg / mL, approximately 15 μg / mL, approximately 20 μg / mL, approximately 25 μg / mL, approximately 30 μg / mL, approximately 35 μg / mL, approximately 40 μg / mL, approximately 45 μg / mL, approximately 50 μg / mL, approximately 55 μg / mL, approximately 60 μg / mL, approximately 65 μg / mL, approximately 70 μg / mL, approximately 75 μg It has concentrations in the fluid of approximately 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, or 1,000 μg / mL. 【0116】 In some embodiments, the device further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir may be fluidly connected to the entry zone, and the second reservoir may be fluidly connected to the recovery zone. 【0117】 In some embodiments, the electroporation zones of the device have uniform cross-sectional dimensions. In other embodiments, the electroporation zones of the device have non-uniform cross-sectional dimensions. In further embodiments, the device further includes a plurality of electroporation zones, each of which may have a uniform or non-uniform cross-section. In certain embodiments, the cross-section of the electroporation zone is selected from the group consisting of cylindrical, elliptical, polygonal, star-shaped, parallelogram, trapezoidal, and irregular shapes. 【0118】 In some cases, the cross-sectional dimensions of the entry zone or the recovery zone are 0.01% to 100,000% of the cross-sectional dimensions of the electroporation zone. For example, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 0.01% to approximately 100% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 10%, approximately 5% to approximately 25%, approximately 10% to approximately 50%, approximately 25% to approximately 75%, or approximately 50% to approximately 100% of the cross-sectional dimensions of the electroporation zone. Alternatively, the cross-sectional dimensions of the entry zone or the recovery zone may be approximately 100% to 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the cross-sectional dimensions of the electroporation zone. 【0119】 In some embodiments, the cross-sectional dimensions of the electroporation zone are 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 50 mm. In some embodiments, the length of the electroporation zone is 0.005 mm to 25 mm. In some embodiments, the cross-sectional dimensions of either the first electrode or the second electrode are 0.1 mm to 500 mm. In certain embodiments, the entry zone, recovery zone, or electroporation zone does not reduce the cross-sectional dimensions of any of the multiple cells suspended in the fluid, for example, allowing cells to pass through the device without deformation. 【0120】 In further embodiments, the device includes an outer structure having a first electrode, a second electrode, and a housing configured to enclose the electroporation zone of the device. In some embodiments, the outer structure includes a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the outer structure is integrated into the device. In certain embodiments, the outer structure is releasably connected to the device. 【0121】 In some embodiments, the delivery rate from the fluid delivery source is 0.001 mL / min to 1,000 mL / min, e.g., 20 to 30 mL / min, e.g., 25 mL / min. In certain embodiments, the residence time of any of the multiple cells suspended in the fluid is 0.5 ms to 50 ms. In some embodiments, the conductivity of the fluid is 0.001 mS / cm to 500 mS / cm, e.g., 1 to 20 mS / cm. 【0122】 In further embodiments, the method includes a controller operably coupled to a potential source to deliver voltage pulses to a first electrode and a second electrode to generate a potential difference between the first electrode and the second electrode. In some embodiments, the voltage pulse has an amplitude of -3kV to 3kV, e.g., 0.2 to 0.6kV. In some cases, the duty cycle of electroporation is 0.001% to 100%, e.g., 10% to 95%. In some embodiments, the voltage pulse has a duration of 0.01ms to 1,000ms, e.g., 1ms to 10ms. In certain embodiments, the voltage pulse is applied to the first electrode and the second electrode at a frequency of 1Hz to 50,000Hz, e.g., 100 to 500Hz. The waveform of the voltage pulse can be DC, square, pulsed, bipolar, sinusoidal, ramped, asymmetrical bipolar, arbitrary, or a superposition or combination of any of these. In certain embodiments, the electric field generated from the voltage pulse has a magnitude of 1 V / cm to 50,000 V / cm, for example, 100 V / cm to 1,000 V / cm. 【0123】 In further embodiments, the method includes a housing structure configured to accommodate the electroporation device described herein. In further embodiments, the housing structure includes a thermal controller configured to raise or lower the temperature of any component of the housing or system. In some embodiments, the thermal controller is a heating element, e.g., a heating block, a liquid flow, a battery-powered heater, or a thin-film heater. In other embodiments, the thermal controller is a cooling element, e.g., a liquid flow, an evaporative cooler, or a thermoelectric, e.g., a Peltier device. In certain embodiments, the temperature of a plurality of cells suspended in a fluid is between 0°C and 50°C. 【0124】 In further embodiments, the device includes multiple cell-piercing devices, for example, in series or parallel. In specific embodiments, the device includes multiple external structures for multiple devices. 【0125】 In some cases, the method further includes storing a plurality of cells suspended in a fluid in a recovery buffer after perforation. In certain embodiments, the electroperforated cells have a viability of 0.1% to 99.9%, e.g., 25% to 85%, after introduction of the composition. In other embodiments, the efficiency of introduction of the composition into the cells is 0.1% to 99.9%, e.g., 25% to 85%. In certain embodiments, the cell recovery rate is 0.1% to 100%. In certain embodiments, the cell recovery yield is 0.1% to 500%. In some embodiments, the number of recovered cells (e.g., viable cells) is 10 4 ~10 12 That is the case. 【0126】 In another embodiment, the present invention provides a kit for electroperforating a plurality of cells suspended in a fluid, the kit comprising a plurality of cell perforation devices described herein, a plurality of external structures described herein, and a transfection buffer. 【0127】 In some embodiments, the outer structure is integrated with a plurality of cell-poration devices. In certain embodiments, the outer structure is releasably connected to a plurality of cell-poration devices. 【0128】 This application document includes at least one color printed drawing. Copies of this patent application containing color drawings will be provided by the Patent Office upon request for payment of the necessary fees. 【Brief Description of the Drawings】 【0129】 [Figure 1A-1C] Figures 1A - 1C are schematic views of an embodiment of a single electroporation device of the present invention. Figure 1A shows a schematic view of the operation of the device of the present invention. Figure 1B shows a schematic view of the components of the present invention. Figure 1C shows a photograph of the embodiment of the device of the present invention shown in Figure 1B. [Figure 2A-2B] Figures 2A - 2B are exemplary schematic views of a housing for parallel delivery of electrical energy to an embodiment of the electroporation device of the present invention. Figure 2A shows an isometric view of a housing having an electrical grid concept for energizing 96 electroporation devices of the present invention in parallel. Figure 2B shows an enlarged view of the interface between a single electroporation device of the present invention and a housing having an electrical grid, the housing using spring-biased electrodes to securely hold the first and second electrodes of each electroporation device against the electrical grid of the housing. [Figure 3A-3B] Figures 3A - 3B are bar graphs of the optimization of fluid flow rate (mL / min) for electroporation of Jurkat cells (1×107 cells / mL) using the device of the present invention. The recovered cells were cultured for 24 hours at 37°C in RPMI with 10% FBS prior to flow cytometer analysis using LSR II HTS (BD Bioscience). Figure 3A shows the viability of Jurkat cells evaluated using the 7-AAD exclusion dye. Figure 3B shows the transfection efficiency of Jurkat cells evaluated using GFP expression. [Figure 4A-4D]Figures 4A to 4D are explanatory diagrams of flow rate simulations along the active zone of the device. Figure 4A is a 3D model representing a liquid volume flow rate of 10 mL per minute. Figure 4C is a 3D model representing a liquid volume flow rate of 100 mL per minute. Figures 4B and 4D are 2D models corresponding to Figures 4A and 4C, respectively. [Figure 5A-5B] Figures 5A and 5B are bar graphs for optimizing the electric field in the electroporation zone of the device of the present invention for electroporation of Jurkat cells. The recovered cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 5A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. Figure 5B shows the transfection efficiency of Jurkat cells as assessed using GFP expression. [Figure 6A-6B] Figures 6A and 6B are bar graphs showing the effect of temperature on the transfection of Jurkat cells using the device of the present invention. "RT" in these figures represents room temperature. The harvested cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 6A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. Figure 6B shows the transfection efficiency of Jurkat cells as assessed using GFP expression. [Figures 7A-7D] Figures 7A to 7D are explanatory diagrams of simulations showing the electric field distribution along the active zone of the device. Figure 7A shows the electric field distribution map of the device with an applied voltage of 225V. Figure 7B is a longitudinal cross-section of the 2D model of Figure 7A. Figure 7C shows the electric field distribution map of the device with an applied voltage of 275V. Figure 7D is a longitudinal cross-section of the 2D model of Figure 7C. [Figures 8A-8D]Figures 8A to 8D are explanatory diagrams of the simulation showing the effect of temperature distribution along the active zone of the device. Figure 8A shows the temperature distribution map of the liquid in the active zone of the device at time = 0 ms. Figure 8B shows the temperature distribution map of the liquid in the active zone of the device at time = 100 ms. Figure 8C shows the temperature distribution map of the liquid in the active zone of the device at time = 200 ms. Figure 8D shows the temperature distribution map of the liquid in the active zone of the device at time = 300 ms. [Figure 9A-9B] Figures 9A and 9B are bar graphs showing the optimization of voltage pulse duration and pulse number for electroporation of Jurkat cells using the device of the present invention. The harvested cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 8A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. Figure 9B shows the transfection efficiency of Jurkat cells as assessed using 7GFP expression. [Figure 10A-10B] Figures 10A and 10B are bar graphs showing the optimization of sample volume for electroporation of Jurkat cells using the device of the present invention. The recovered cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 10A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. Figure 10B shows the transfection efficiency of Jurkat cells as assessed using GFP expression. [Figures 11A-11B]Figures 11A and 11B are bar graphs showing the optimization of the electroporation zone diameter for electroporation of Jurkat cells using the device of the present invention. Electroporation was performed at a fixed voltage with variable flow rate to substantially match the total cell residence time across different channel dimensions. The harvested cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 11A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. Figure 11B shows the transfection efficiency of Jurkat cells as assessed using GFP expression. [Figure 12A] Figures 12A–12L show bar graphs illustrating the effect of selective voltage pulse waveforms for electroporation of Jurkat cells using the device and exemplary waveform shapes of the present invention. The harvested cells were cultured for 24 hours in RPMI with 10% FBS at 37°C prior to flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 12A shows the viability of Jurkat cells as assessed using 7-AAD exclusion dye. [Figure 12B] Figure 12B shows the transfection efficiency of Jurkat cells as evaluated using GFP expression. [Figure 12C] Figure 12C shows the always-on waveform of direct current (DC). [Figure 12D] Figure 12D shows a square wave waveform with a 50% duty cycle including an offset. [Figure 12E] Figure 12E shows a 75% asymmetric ramp waveform. [Figure 12F] Figure 12F shows a pulse waveform with a duty cycle of 95%. [Figure 12G] Figure 12G shows a square wave waveform with a 75% duty cycle including the offset. [Figure 12H] Figure 12H shows a sinusoidal waveform. [Figure 12I] Figure 12I shows a 25% asymmetric ramp waveform. [Figure 12J]Figure 12J shows a square wave waveform with a 25% duty cycle including an offset. [Figure 12K] Figure 12K shows a bipolar square wave waveform without offset. [Figure 12L] Figure 12L shows a symmetrical ramp waveform. [Figure 13A] Figures 13A and 13B are bar graphs comparing the transfection efficiency and resulting cell viability of Jurkat cells using the device of the present invention and commercially available cell transfection equipment. Jurkat cell viability was assessed using 7-AAD exclusion dye, and Jurkat cell transfection efficiency was assessed using GFP expression. Figure 13A shows the results of transfection experiments performed using published parameters for Jurkat cell transfection (sample in 100 μL tip; 3 pulses / 10 ms / 450 V / cm). [Figure 13B] Figure 13B is a duplicate of Figure 13A, showing the reproducibility of experiments performed using optimized parameters for the device of the present invention compared to published parameters for Jurkat cell transfection. [Figure 13C] Figure 13C shows a schematic workflow for Cas9 ribonucleoprotein array library screening, using a commercially available single-stranded sgRNA array library to anneal purified Cas9 protein to form an arrayed Cas9 ribonucleoprotein library. Using the device of the present invention, Cas9 ribonucleoprotein array library screening leads to the identification of gene targets for future immunotherapy research using plate-based analysis. In addition, Cas9 ribonucleoprotein pool library screening can be used to perform assays necessary to identify gene targets for future therapies. [Figures 14A-14B]Figures 14A and 14B are bar graphs showing the viability and efficiency of FITC dextran delivery to primary human T cells using the device of the present invention, which evaluates arbitrary size constraints for dextran delivery using variable molecular weight dextran polymers. Recovered cells were cultured for 24 hours in RPMI with 10% FBS at 37°C prior to flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 14A shows the viability of primary human T cells as assessed using 7-AAD exclusion dye. Figure 14B shows the transfection efficiency of primary human T cells as assessed using GFP expression. [Figures 15A-15B] Figures 15A and 15B are bar graphs comparing the transfection efficiency and viability of THP-1 monocytes using the device of the present invention with that of a commercially available cell transfection device (NEON®) using a published transfection protocol for THP-1 monocytes. Recovered cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 15A shows the viability of THP-1 monocytes as assessed using 7-AAD exclusion dye. Figure 15B shows the transfection efficiency of THP-1 monocytes as assessed using GFP expression. [Figures 16A-16B] Figures 16A and 16B are bar graphs comparing the transfection efficiency and viability of primary human monocytes using the device of the present invention with that of commercially available cell transfection devices using published transfection protocols for primary human monocytes. Primary human monocytes were isolated from peripheral blood using negative selection. The recovered cells were cultured for 24 hours in RPMI with 10% FBS at 37°C before flow cytometry analysis using LSR II HTS (BD Bioscience). Figure 16A shows the viability of primary human monocytes as assessed using 7-AAD exclusion dye. Figure 16B shows the transfection efficiency of primary human monocytes as assessed using GFP expression. [Figures 17A-17B]Figures 17A and 17B are bar graphs comparing the transfection efficiency and viability of NK-92 cell lines using the device of the present invention with commercially available cell transfection equipment using a published transfection protocol for NK-92 cell lines. After electroporation, cells were cultured for 24 hours at 37°C in complete aMEM (aMEM containing 25% serum, 0.2 mM inositol, 0.02 mM folic acid, and 0.1 mM mercaptoethanol) before flow cytometry analysis using iQue (Intellicyt). Figure 17A shows viability as assessed using 7-AAD exclusion dye. Figure 17B shows transfection efficiency as assessed by GFP expression. [Figures 18A-18B] Figures 18A and 18B are bar graphs comparing the transfection efficiency and viability of NK-92MI cell lines using the device of the present invention with commercially available cell transfection equipment using a published transfection protocol for NK-92MI cell lines. After electroporation, cells were cultured for 24 hours at 37°C in complete aMEM (aMEM containing 25% serum, 0.2 mM inositol, 0.02 mM folic acid, and 0.1 mM mercaptoethanol) before flow cytometry analysis using iQue (Intellicyt). Figure 18A shows viability as assessed using 7-AAD exclusion dye. Figure 18B shows transfection efficiency as assessed by GFP expression. [Figure 19A] Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells that proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression was evaluated over 24 hours. Figure 19A is a representative graph of viability measured as 7AAD-negative cells. Monocytes isolated from PBMCs were transfected with 20 μg of SIRPα mRNA, and overexpression was evaluated over 24 hours. The graphs represent mean ± SEM values. [Figure 19B]Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression over 24 hours was evaluated. Figure 19B is a representative graph of transfection efficiency measured as SIRPα-positive cells. Monocytes isolated from PBMCs were transfected with 20 μg of SIRPα mRNA, and overexpression over 24 hours was evaluated. The graphs represent mean ± SEM values. [Figure 19C] Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression was evaluated over 24 hours. Figure 19C is a representative graph of SIRPα expression measured as mean fluorescence intensity (MFI). Monocytes isolated from PBMCs were transfected with 20 μg of SIRPα mRNA, and overexpression was evaluated over 24 hours. The graphs represent mean ± SEM values. [Figure 19D] Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells that proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression over 24 hours was evaluated. Figure 19D is a representative graph of viability measured as 7AAD-negative cells. The graphs represent the mean ± SEM value. [Figure 19E]Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression over 24 hours was evaluated. Figure 19E is a representative graph of transfection efficiency measured as SIRPα-positive cells. The graphs represent the mean ± SEM value. [Figure 19F] Figures 19A to 19F are bar graphs comparing T cells (Figures 19A to 19C) with primary human monocytes (Figures 19D to 19F) that were electropermeable using the device of the present invention and transfected with SIRPα custom mRNA, compared with cells that were not electropermeable. T cells that proliferated on day 11 were transfected with 20 μg of SIRPα mRNA, and overexpression over 24 hours was evaluated. Figure 19F is a representative graph of SIRPα expression measured as mean fluorescence intensity (MFI). The graphs represent mean ± SEM values. [Figures 20A-20D] Figures 20A–20D are bar graphs showing the delivery of GFP nRMA to human primary native T cells. Figure 20A shows the recovered cells. Figure 20B shows the naive T cell efficiency. Figure 20C shows the naive T cell viability. Figure 20D shows the total yield. Naive T cells were transfected with 10 μg of commercial GFP mRNA and expression was evaluated over 24 hours. Representative graphs of count, viability, efficiency, and yield are shown. Graphs are mean ± SEM values. [Figures 21A-21B]Figures 21A and 21B are FACS plots showing that electroporation does not alter the phenotype of human primary naive T cells. Figure 21A shows untreated cells. Figure 21B shows electroporated cells. Naive T cells were transfected with 10 μg of commercial GFP mRNA and then stained for CD45RA and CD45RO over 24 hours, as shown in the dot plot. The CD45RA / CD45RO phenotype is equivalent between untreated naive T cells and naive T cells electroporated with Flowfect®. [Figure 22] Figure 22 is a dynamic plot showing the proliferation of naive T cells using the device of the present invention compared to untreated cells. Electroporation does not alter the proliferation of human primary naive T cells. Naive T cells were transfected with 10 μg of commercial GFP mRNA and then grown with a soluble CD3 / CD28 activator. Cell counts were obtained on days 1, 4, and 6 after activation. The proliferation rates were equivalent between untreated and electroporated naive T cells. [Figures 23A-23C] This shows an exemplary embodiment of the electroporation device of the present invention, integrated into an electron discharge device configured to simultaneously energize and electroporate multiple cell samples. Figure 23A shows a top isometric view of the electron discharge device. Figure 23B shows a side view of the device of the present invention installed within the electron discharge device, showing how electrical contacts are made in the system using pogo pin-style electrical contacts. Figure 23C shows a side view of the complete electron discharge device. [Figures 23D-23F]Figure 23D shows an exemplary embodiment of the electroporation device of the present invention, integrated into an electron discharge device configured to simultaneously energize and electroporate multiple cell samples. Figure 23E shows a top isometric view of an alternative embodiment of the electron discharge device. Figure 23E shows a side view of the device of the present invention installed within the electron discharge device, showing how electrical contacts are made in the system using flexible spring-type electrical contacts. Figure 23F shows a top view of the electron discharge device configured to simultaneously energize and electroporate multiple cell samples. [Figures 24A-24B] Figures 24A and 24B illustrate embodiments of a temperature-controlled electroporation device that uses a thermal liquid for temperature control. Figure 24A shows a schematic diagram of the components of the temperature-controlled electroporation device. Figure 24B shows a side view of the temperature-controlled electroporation device, showing the device in its external frame. [Figures 25A-25B] Figures 25A and 25B show embodiments of a fluid tip-based electroporation device configured to accept industry-standard pipette tips for sample introduction. Figure 25A shows an embodiment of a fluid tip incorporating an embedded electrode and fluid channel. Figure 25B shows a schematic diagram of the components of the fluid tip-based electroporation device. [Figures 26A-26B] Figures 26A and 26B show embodiments of a continuous flow electroporation device. Figure 26A shows a schematic cross-section of the components of the continuous flow electroporation device. Figure 26B shows a transparent exterior for illustrating the components of the continuous flow electroporation device. [Figure 27A] Figures 27A to 27F show the simulated electric field generated using computational modeling of an embodiment of a helical electrode. Figure 27A shows the simulated electric field of the helical electrode shown along all three Cartesian axes. [Figure 27B] Figure 27B shows the simulated electric field of a helical electrode, as seen from a cross-section along the Z-axis. [Figure 27C] Figure 27C shows the simulated electric field of a helical electrode along the XY axes, shown from four different positions along the Z axis. [Figure 27D] Figure 27D shows the simulated electric field of a helical electrode along the XY axes, shown from four different positions along the Z axis. [Figure 27E] Figure 27E shows the simulated electric field of a helical electrode along the XY axes, shown from four different positions along the Z axis. [Figure 27F] Figure 27F shows the simulated electric field of a helical electrode along the XY axes, shown from four different positions along the Z axis. [Figures 28A-28C] Figures 28A to 28C illustrate embodiments of the two-part electroporation device of the present invention configured for manufacturing scalability. Figure 28A shows a top isometric 3D rendering of an embodiment of the two-part electroporation device of the present invention. Figure 28B shows a vertical cross-section of the embodiment depicted in Figure 28A, showing how the two components fit together. Figure 28C shows the same diagram of the embodiment depicted in Figure 28B with the device dimensions (in mm) superimposed. [Figures 29A-29B] Figures 29A and 29B illustrate embodiments of the two-part electroporation device of the present invention, including an implanted electrode having an interface for a liquid handling cannula. Figure 29A shows a top isometric 3D rendering of an embodiment of the two-part electroporation device of the present invention having an implanted electrode. Figure 29B shows a vertical cross-section of the embodiment depicted in Figure 29A, showing the location of the implanted electrode relative to the electroporation zone of the device of the present invention. [Figures 30A-30B] Figures 30A and 30B show embodiments of the outer housing of the present invention configured to house multiple devices, liquid handling components, controllers, and optional electrical components of the present invention. Figure 30A shows an embodiment of the outer housing of the present invention having a user interface. Figure 30B shows an embodiment of the device of the present invention connected to a liquid dispensing manifold and sample plate. [Figure 31]Figure 31 shows a comparison between a conventional flow cytometry gating strategy (bottom, using a commercially available Lonza NUCLEOFECTOR 4D® electroporation system) and a flow cytometry gating strategy (top, using the devices and systems of the present invention) employed for post-transfection analysis for cell counting, viability, transfection efficiency, and detection of surface / intracellular markers. [Figures 32A-32B] Figures 32A and 32B are bar graphs showing the viability and efficiency of GFP-coding plasmid DNA delivery into CHO-K1 cells using the device of the present invention, 24 hours after electroporation. Figure 32A shows the viability of CHO-K1 cells. Figure 32B shows the transfection efficiency of CHO-K1 cells as evaluated using GFP expression. [Figures 33A-33D] Figures 33A to 33D are bar graphs showing the viability and efficiency of GFP-coding plasmid DNA delivery into HEK-293T cells using the device of the present invention, 24 and 48 hours after electroporation. Figure 33A shows the viability of HEK-293T cells 24 hours after electroporation. Figure 33B shows the transfection efficiency of HEK-293T cells, as evaluated using GFP expression, 24 hours after electroporation. Figure 33C shows the viability of HEK-293T cells 48 hours after electroporation. Figure 33D shows the transfection efficiency of HEK-293T cells, as evaluated using GFP expression, 48 hours after electroporation. [Figures 34A-34B] Figures 34A and 34B show the collected GFP fluorescence signals of Chinese hamster ovary (CHO-K1) cells before (Figure 34A) and after (Figure 34B) electroporation using the device and system of the present invention. GFP fluorescence images were captured using an ECHO Revolve microscope equipped with a 10x objective lens. [Figures 35A-35B] Figures 35A and 35B show the collected GFP fluorescence signals of HEK-293T cells before (Figure 35A) and after (Figure 35B) electroporation using the device and system of the present invention. GFP fluorescence images were captured using an ECHO Revolve microscope equipped with a 10x objective lens. [Figures 36A-36D] Figures 36A to 36D are bar graphs showing the total cell count, viability, efficiency, and relative biopositive transfected cells after electroporation for the delivery of 40kD FITC dextran to primary human T cells using a commercially available NEON® transfection system and the device of the present invention. Figure 36A shows the total cell count after electroporation. Figure 36B shows the viability of primary human T cells. Figure 36C shows the efficiency of delivery into primary human T cells. Figure 36D shows the relative biopositive transfected cell population. [Figure 37] Figure 37 is a bar graph showing a comparison between the NEON® transfection system and the device of the present invention for a relative biopositive transfected cell population after delivery of the GFP plasmid to primary human T cells. [Figures 38A-38D] Figures 38A–38D are bar graphs showing the recovery, viability, efficiency, and yield of mRNA delivery to 9-day-old primary human T cells. Electroporation was performed using two commercially available transfection systems (Lonza NUCLEOFECTOR 4D® and Thermo Fisher NEON®) and the device of the present invention. Either 1 million (10⁶ cells / mL) or 5 million (5 × 10⁶ cells / mL) cells were electroporated in 100 μL containing 10 μg of mRNA encoding EGFP. Flow cytometry analysis was performed 24 hours after electroporation. Cell counts were normalized to 1 million cell inputs, and yields were normalized to results collected using the device of the present invention. Figure 38A shows the recovery at both cell densities. Figure 38B shows the viability at both cell densities. Figure 38C shows the efficiency at both cell densities. Figure 38D shows the yield at both cell densities. [Figures 39A-39D]Figures 39A–39D are line graphs showing the recovery, viability, efficiency, and MFI of Cas9 ribonucleoprotein complex (RNP) delivery targeting CXCR3 in primary human T cells. Cas9 RNP was formulated using two commercially available sources: commercially available Cas9 protein and sgRNA. Flow cytometry analysis was performed 24–72 hours after electroporation. Figure 39A shows cell recovery. Figure 39B shows viability. Figure 39C shows efficiency. Figure 39D shows the total yield of targeted KO cells grown up to 72 hours after electroporation. [Figures 40A-40B] Figures 40A and 40B are bar graphs showing live cell counts for GFP expression from THP-1 cells and for FITC-labeled dextran delivery to NK-92MI cells for electroporation using the commercial NEON® transfection system and the device of the present invention. Figure 40A shows the live cell count for GFP expression in THP-1 cells. Figure 40B shows the live cell count for FITC-labeled dextran delivery to NK-92MI cells. [Figure 41A-41B] Figures 41A and 41B are bar graphs showing a comparison of the resulting viability and efficiency of GFP mRNA delivery to THP-1 monocytes using the commercial NEON® transfection system and the device of the present invention. Figure 41A shows the viability of THP-1 monocytes evaluated 24 hours after transfection. Figure 41B shows the transfection efficiency of THP-1 monocytes evaluated using GFP expression 24 hours after electroporation. [Figures 42A-42C] Figures 42A to 42C are bar graphs showing the viability, efficiency, and yield of GFP mRNA delivery into THP-1 monocytes using the device of the present invention with control samples of cells that were not electroporated. Figure 42A shows the viability of transfected cells evaluated 24 to 72 hours after electroporation. Figure 42B shows the efficiency of GFP mRNA uptake evaluated 24 to 72 hours after electroporation. Figure 42C shows the yield of transfected cells evaluated 24 to 72 hours after electroporation. [Figures 43A-43B]Figures 43A and 43B are bar graphs showing the viability and efficiency of GFP mRNA delivery into LPS-activated THP-1 cells using the device of the present invention. Figure 43A shows the viability of LPS-activated THP-1 cells evaluated 24 hours after transfection. Figure 43B shows the transfection efficiency of LPS-activated THP-1 cells evaluated using GFP expression 24 hours after electroporation. [Figures 44A-44D] Figures 44A to 44D are bar graphs showing the survival rate and efficiency of delivery of 40kD FITC dextran and GFP mRNA into primary peripheral blood monocytes using the device of the present invention. Figure 44A shows the survival rate of primary peripheral blood monocytes transfected with FITC dextran. Figure 44B shows the transfection efficiency of primary peripheral blood monocytes transfected with FITC dextran. Figure 44C shows the survival rate of primary peripheral blood monocytes transfected with GFP mRNA. Figure 44B shows the transfection efficiency of primary peripheral blood monocytes transfected with GFP mRNA. [Figures 45A-45B] Figures 45A and 45B are bar graphs showing the expression of CD80 and CD86 in primary peripheral blood monocytes transfected with GFP via LPS stimulation using the device of the present invention. The expression of CD80 and CD86 was measured 24 hours and 96 hours after electroporation. Figure 45A shows the expression of the activation marker CD80. Figure 45B shows the expression of the lineage marker CD86. [Figures 46A-46C] Figures 46A to 46C are bar graphs showing the macrophage phenotype, viability, and GFP expression of primary peripheral blood monocytes transfected with GFP mRNA using the device of the present invention, which differentiated into macrophages over days 4 to 8. Figure 46A shows the macrophage phenotype as evaluated by flow cytometry analysis of FSCs and SSCs. Figure 46B shows the viability of transfected macrophages. Figure 46C shows the GFP expression percentage of transfected macrophages. [Figures 47A-47D]Figures 47A to 47D are bar graphs showing the survival rate and efficiency of delivery of 40kD FITC dextran and GFP mRNA to peripheral blood differentiated macrophages using the device of the present invention. Figure 47A shows the survival rate of peripheral blood differentiated macrophages transfected with FITC dextran. Figure 47B shows the transfection efficiency of peripheral blood differentiated macrophages transfected with FITC dextran. Figure 47C shows the survival rate of peripheral blood differentiated macrophages transfected with GFP mRNA. Figure 47D shows the transfection efficiency of peripheral blood differentiated macrophages transfected with GFP mRNA. [Figures 48A-48B] Figures 48A and 48B are bar graphs showing the ability of peripheral blood differentiated macrophages to polarize into M1 and M2 macrophages after transfection with GFP mRNA using the device of the present invention. Figure 48A shows M1-polarized macrophages, where M1 polarization induced by IFNg + LPS stimulation is indicated by enhanced CD86 expression. Figure 48B shows M2-polarized macrophages, where M2 polarization induced by IL-4 stimulation is indicated by CD206 expression. [Figures 49A-49C] Figures 49A to 49C are bar graphs showing the viability, efficiency, and viable cell count of primary human monocytes transfected with FITC dextran using the commercial NEON® transfection system and the device of the present invention. Figure 49A shows the viability of primary human monocytes. Figure 49B shows the efficiency of FITC dextran delivery into primary human monocytes. Figure 49C shows the viable cell count of transfected primary human monocytes. [Figures 50A-50D]Figures 50A to 50D are bar graphs comparing the recovery, viability, efficiency, and yield of DNA transfection into Jurkat cells at various cell densities using the single-channel and continuous flow devices of the present invention. Figure 50A shows the recovery of transfected Jurkat cells. Figure 50B shows the viability of transfected Jurkat cells. Figure 50C shows the efficiency of DNA transfection into Jurkat cells. Figure 50D shows the yield of transfected Jurkat cells. [Figures 51A-51B] Figures 51A and 51B are bar graphs comparing the GFP and FITC yields of Jurkat cells transfected using the single-channel and continuous-flow devices of the present invention. Figure 51A shows the GFP yield of transfected Jurkat cells. Figure 51B shows the FITC yield of transfected Jurkat cells. [Figures 52A-52D] Figures 52A to 52D are bar graphs showing the delivery of FITC dextran into a high-cell-density suspension using the continuous flow device of the present invention. Flow cytometry analysis was performed 24 hours after electroporation. Figure 52A shows the total number of cells recovered for a 1 million cell input. Figure 52B shows the viability of transfected Jurkat cells. Figure 52C shows the efficiency of FITC dextran transfection into Jurkat cells. Figure 52D shows the FITC yield of transfected Jurkat cells. [Figures 53A-53D]Figures 53A to 53D are bar graphs showing the recovery, viability, efficiency, and yield of mRNA transfection into Jurkat cells at a cell count of 100 million cells using various amounts of mRNA and various cell concentrations in the continuous flow device of the present invention. Flow cytometry analysis was performed 24 hours after electroporation. Figure 53A shows the number of Jurkat cells recovered at different mRNA and cell concentrations. Figure 53B shows the viability of transfected Jurkat cells at different mRNA and cell concentrations. Figure 53C shows the efficiency of mRNA transfection into Jurkat cells at different mRNA and cell concentrations. Figure 53D shows the yield of transfected Jurkat cells at different mRNA and cell concentrations. [Figure 54] Figure 54 shows flow cytometry analysis of untreated and electroporated T cells comparing the commercial Lonza NUCLEOFECTOR 4D® transfection system with the device of the present invention. The upper panel shows the FSC / SSC total cell plot, and the lower panel shows viability staining. Dead cell populations are indicated by red arrows and red squares. Morphological shifts of cells transfected with Lonza NUCLEOFECTOR 4D® over 24 hours compared to untreated cells are also observed, indicating phenotypic changes occur during electroporation by the Lonza platform. [Figure 55] Figure 55 shows a bar graph of total cell yield from electroporation of 50 million primary T cells using either FITC-dextran or EGFP mRNA, using a commercial Lonza LV transfection system and the continuous flow device of the present invention. [Figures 56A-56B] Figures 56A and 56B are bar graphs showing the viability and efficiency of FITC dextran delivery to a suspension of 1 billion THP-1 cells using the continuous flow device of the present invention over a maximum electroporation period of 72 hours. Figure 56A shows the viability of THP-1 cells. Figure 56B shows the efficiency of FITC dextran delivery into THP-1 cells. [Figure 57]Figure 57 is a bar graph showing the yield of recoverable live FITC dextran-transfected cells starting from a suspension of 1 billion THP-1 cells using the continuous flow device of the present invention. The yield was tracked over a period of up to 72 hours after electroporation culture, and the yield represents approximately 50% of the number of cells introduced. Flow cytometry analysis was performed 4, 24, 48, and 72 hours after electroporation. [Figures 58A-58D] Figures 58A to 58D are bar graphs comparing waveform shapes and waveform voltages with respect to total cell count, viability, efficiency, and yield of FITC dextran transfection into Jurkat cells using the device of the present invention. Figure 58A shows the number of Jurkat cells recovered at different waveform shapes and voltages. Figure 58B shows the viability of transfected Jurkat cells at different waveform shapes and voltages. Figure 58C shows the efficiency of FITC dextran transfection into Jurkat cells at different waveform shapes and voltages. Figure 58D shows the yield of transfected Jurkat cells at different waveform shapes and voltages. [Figures 59A-59D] Figures 59A to 59D are bar graphs comparing waveform maximum voltage and duty cycle with respect to total cell count, viability, efficiency, and yield of FITC dextran transfection into primary T cells using the device of the present invention. Figure 59A shows the number of primary T cells recovered at different waveform maximum voltages and duty cycles. Figure 59B shows the viability of transfected primary T cells at different waveform maximum voltages and duty cycles. Figure 59C shows the efficiency of FITC dextran transfection into primary T cells at different waveform maximum voltages and duty cycles. Figure 59D shows the yield of transfected primary T cells at different waveform maximum voltages and duty cycles. [Figures 60A-60D]Figures 60A to 60D are bar graphs comparing waveform maximum voltage and duty cycle with respect to total cell count, viability, efficiency, and yield of mRNA transfection into primary T cells using the device of the present invention. Figure 60A shows the number of primary T cells recovered at different waveform maximum voltages and duty cycles. Figure 60B shows the viability of transfected primary T cells at different waveform maximum voltages and duty cycles. Figure 60C shows the efficiency of mRNA transfection into primary T cells at different waveform maximum voltages and duty cycles. Figure 60D shows the yield of transfected primary T cells at different waveform maximum voltages and duty cycles. [Figure 61] Figure 61 is a bar graph showing the efficiency of CD3 / CD28 Dynabead delivery to a suspension of 1 million primary human T cells using the device of the present invention. Electroporation was performed with or without Dynabead, and Dynabead integration was performed for 5 minutes or overnight. Flow cytometry analysis was performed 24 hours after electroporation. [Figures 62A-62B] Figures 62A and 62B show embodiments of an outer structure configured to enclose the electrodes of the device of the present invention. Figure 62A shows an outer structure comprising a latch and a clamshell hinge for enclosing the device of the present invention. Figure 62B shows the outer structure of Figure 62A with the device of the present invention placed in the corresponding internal recess of the outer structure. [Figures 63A-63B] Figures 63A and 63B are bar graphs showing the viability and efficiency of FITC dextran delivery into THP-1 monocytes using the device of the present invention, both with and without an outer structure covering the device electrodes. Flow cytometry analysis was performed 24 hours after electroporation. Figure 63A shows the viability of THP-1 monocytes. Figure 63B shows the efficiency of THP-1 monocyte transfection. [Figures 64A-64B]Figures 64A and 64B are bar graphs showing the viability and efficiency of FITC dextran delivery into THP-1 monocytes using the devices of the present invention manufactured from different polymer resins. Figure 64A shows the viability of transfected THP-1 monocytes. Figure 64B shows the efficiency of FITC dextran transfection into THP-1 monocytes. [Figures 65A-65B] Figures 65A and 65B are bar graphs comparing the viability and efficiency of delivery of both GFP-encoding DNA and mRNA into Jurkat cells using the manually operated device of the present invention or an automated fluid handling platform. Figure 65A shows the viability of transfected Jurkat cells. Figure 65B shows the efficiency of transfection of GFP-encoding DNA and mRNA into Jurkat cells. [Figures 66A-66E] Figures 66A–66E are bar graphs and dot plots comparing the viability and efficiency of delivery into T cells of multiple mRNAs encoding both GFP and mCherry, either in parallel (on the same day) or in series (with a 2-day difference), using a manually operated device of the present invention or an automated fluid handling platform. Figure 66A shows T cell viability 24 hours after electroporation for delivery of multiple mRNAs encoding mCherry. Figure 66B shows GFP efficiency 24 hours after electroporation. Figure 66C shows mCherry efficiency 24 hours after electroporation. Figure 66D shows dual GFP and mCherry efficiency 24 hours after electroporation. Figure 66E shows dot plots of both GFP (x axis) and mCherry (y axis) expression over 24 hours. [Figures 67A-67B]Figures 67A and 67B are bar graphs demonstrating the efficiency of mRNA delivery into peripheral blood mononuclear cells (PBMCs) using the device of the present invention. These experiments were performed using commercially available mRNA encoding GFP, followed by identification of specific cell populations by phenotypic staining of surface receptors. Figure 67A shows the efficiency in the T cell subpopulation. Figure 67B shows the efficiency in the non-T cell population derived from PBMCs. Flow cytometry analysis was performed 24 hours after electroporation. [Figure 68] Figure 68 is a photograph of an embodiment of the system of the present invention having a reservoir (bag) that is in fluid communication with a first inlet and a reservoir (bag) that is in fluid communication with a second outlet. [Figures 69A-69C] Figure 69A is a set of micrographs showing a comparison between eGFP-mRNA expression using the device of the present invention and eGFP-mRNA expression using an untreated control. Figure 69B is a bar graph showing the percentage of viable cells. Figure 69C is a bar graph showing the percentage of GFP+ cells. [Figures 70A-70D] Figure 70A is a bar graph showing total NK cell recovery. Figure 70B is a bar graph showing viability. Figure 70C is a bar graph showing transfection efficiency. Figure 70D is a bar graph showing GFP+ cell yield. [Modes for carrying out the invention] 【0130】 If a value is stated as a range, it will be understood that such disclosure includes all possible subranges within that range, as well as any specific numerical values ​​that fall within that range, regardless of whether a particular numerical value or subrange is explicitly stated. 【0131】 As used herein, the term “approximately” refers to a range of + / - 10% of the listed values. 【0132】 As used herein, the term “plural” refers to two or more. 【0133】 As used herein, the term “substantially uniform” refers to a variation of + / - 5%. 【0134】 As used herein, the term “minimum cross-sectional dimension” refers to the minimum length of a straight line that passes through the geometric center of the cross-section of the lumen and intersects the inner wall of the lumen twice on the same plane as the cross-section. 【0135】 The term "cross-sectional area" refers to the cross-sectional area (for example, along a plane perpendicular to the longitudinal axis or the direction of flow) unless otherwise specified. 【0136】 As used herein, the term “fluidically connected” refers to a direct connection between at least two device elements, such as an electroporating device, a reservoir, etc., that allows a fluid to move between such device elements without passing through an intervening element. 【0137】 As used herein, the term “fluid communication” refers to an indirect connection between at least two device elements, such as an electroporated zone, a reservoir, etc., that allows a fluid to move between such device elements, for example, through an intervening element (e.g., through an intervening tube, an intervening channel, etc.). For example, in an embodiment in which a fluid flows from the lumen of a first electrode through an electroporated zone into the lumen of a second electrode, the first electrode is in fluid communication with the second electrode. 【0138】 As used herein, the term “lumen” refers to the internal cavity of an electrode in the device of the present invention that allows fluid to pass through. Part or all of the electrode lumen may be conductive or nonconductive. For example, the electrode lumen may contain a C-shaped conductive element that does not completely enclose the lumen. In other embodiments, the electrode is substantially composed of a conductive material that conducts electric current. When a potential difference is applied to the first and second electrodes of the device of the present invention, the electric field that may be generated in the lumen of either the first or second electrode is not high enough to cause cellular electroperforation within the lumen. 【0139】 As used herein, the term “entry zone” includes the lumen of the first electrode of the device of the present invention, through which a fluid and a plurality of cells suspended in the fluid can pass before electroporation. The entry zone may further include an additional reservoir that is in fluid communication with the lumen of the first electrode of the device of the present invention. When a potential difference is applied to the first and second electrodes of the device of the present invention, the electric field that may be generated within the entry zone of the device of the present invention is not sufficiently high to cause cellular electroporation. 【0140】 As used herein, the term “recovery zone” includes the lumen of the second electrode of the device of the present invention through which a fluid and a plurality of cells suspended in the fluid can pass after electroporation. The recovery zone may further include an additional reservoir in fluid communication with the lumen of the second electrode of the device of the present invention. When a potential difference is applied to the first and second electrodes of the device of the present invention, the electric field that may be generated within the recovery zone of the device of the present invention is not sufficiently high to cause cell electroporation. 【0141】 The present invention provides devices, systems, and methods for transfection of cells, such as primary T cells, with greater volume electroporation, higher transfection efficiency, higher throughput, higher recovery, higher yield, and higher cell viability compared to traditional cuvette-based electroporation approaches or commercially available electroporation equipment. In particular, systems and methods are provided that allow electroporation to be performed in a flow-through manner, a continuous manner, or using multiple electroporation devices of the present invention to increase throughput and cell number. 【0142】 device Generally, the devices of the present invention are configured to be flow-through devices capable of interacting with existing liquid handling, pumping, or fluid transport devices, such as conventional pipette tip robots or large-scale liquid handling systems, in order to provide continuous electroporation of cells suspended in a fluid. The devices of the present invention typically feature three distinct regions: a first electrode having a first inlet and a first outlet, the lumen of which defines an entry zone; a second electrode having a second inlet and a second outlet, the lumen of which defines a recovery zone; and an electroporation zone fluid-connected to the first outlet of the first electrode and the second inlet of the second electrode. An example of an embodiment of the devices of the present invention is shown in Figure 1A, in which the first electrode and the second electrode are fluid-connected by the electroporation zone between them. When a potential difference is applied to the first and second electrodes, a local electric field is generated in the space between the two electrodes, for example, an electroporation zone, and cells exposed to the electric field are electroporated. Individual devices of the present invention may include two electrodes, as shown in Figures 1A to 1C, and alternatively, individual devices of the present invention may include three or more electrodes defining multiple electroporation zones, thus enabling multiple electroporations of cells suspended in a fluid. Devices of the present invention may include multiple electroporation zones between the first and second electrodes, allowing cells to receive different electric fields, for example, generated by the different geometries of each of the multiple electroporation zones, while flowing in a single device or multiple devices. 【0143】 In some cases, the first and second electrodes may be conductive wires, hollow cylinders, conductive thin films, metal foams, mesh electrodes, liquid diffusion films, conductive liquids, or any combination thereof, and may be included in the device. The electrodes may be aligned parallel to the fluid flow axis of the device or perpendicular to the fluid flow axis of the device. For example, the first and second electrodes may be hollow cylindrical electrodes positioned parallel to the axis of fluid flow in the device, as in the devices of Figures 1A-1C, so that fluid flows through the electrodes. In alternative examples, the first and / or second electrodes may be made of a porous conductor, such as a metal mesh, with holes aligned with the fluid flow axis of the device. In alternative examples, the first and / or second electrodes may be a conductive fluid, such as a liquid. In some cases, the first and second electrodes may be configured as a helical structure, such as a double helix, of a solid conductor, such as a wire, around an electroporated zone. In this configuration, the cross-sectional dimensions of the electroporation zone remain substantially uniform, but the positions of the first and second electrodes change along the length of the electroporation zone. The first and second electrodes are in fluid communication with the electroporation zone, but the electric field generated when a potential difference is applied to the electrodes rotates as cells suspended in the fluid move through the device of the present invention. In certain embodiments, the first and second electrodes are embedded in the device of the present invention and have an active area located at or near the fluid connection to the electroporation zone, so that the fluid transporting the cells in suspension comes into contact with a portion of the electrodes where the electric field is generated within the electroporation zone. 【0144】 When configured as a hollow cylindrical electrode, the electrode diameter is approximately 0.1 mm to 5 mm, for example, approximately 0.1 mm to 1 mm, approximately 0.5 mm to 1.5 mm, approximately 1 mm to 2 mm, approximately 1.5 mm to 2.5 mm, approximately 2 mm to 3 mm, approximately 2.5 mm to 3.5 mm, approximately 3 mm to 4 mm, approximately 3.5 mm to 4.5 mm, or approximately 4 mm to 5 mm, for example, approximately 0.1 mm, approximately 0.2 mm, approximately 0.3 mm, approximately 0.4 mm, approximately 0.5 mm, approximately 0.6 mm, approximately 0.7 mm, approximately 0.8 mm, approximately 0.9 mm, approximately 1 mm, approximately 1.1 mm, approximately 1.2 mm, approximately 1.3 mm, approximately 1.4 mm. The outer diameter of the electrode can be approximately 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4.5mm, 4.6mm, 4.7mm, 4.8mm, 4.9mm, or 5mm. An exemplary electrode outer diameter is 1.3mm, corresponding to a 16 gauge electrode. 【0145】 In some embodiments, when the device of the present invention is configured to include a hollow cylindrical electrode, the lumen of the electrode, for example, the first electrode or the second electrode, may include a zone not exposed to the electric field of the electroporation zone, for example, an entry zone or a retrieval zone. As shown in Figure 1A, the entry zone may be the lumen of the first electrode immediately before the entrance to the electroporation zone where cells in a suspension to be electroporated with a composition to be delivered into the cells are located. The retrieval zone may be the lumen of the second electrode immediately after the exit to the electroporation zone where cells having the delivered composition are moved so that pores in the cell membrane can be closed, thus ensuring that the delivered composition remains inside the cell. In this configuration, as the cell passes toward the lumen of the first electrode and the lumen of the second electrode, the first electrode is energized and the second electrode is kept ground, creating a local electric field within the electroporation zone, and thus the cell passing through the device is electroporated. 【0146】 The electroporation zone fluidly connects the first and second electrodes of the device of the present invention, and when the electrodes are energized, a local electric field is applied between the first and second electrodes. The cross-sectional shape of the electroporation zone can be any preferred shape that allows cells to pass through the electroporation zone and the electric field within the electroporation zone. The cross-sectional shape can be, for example, circular, elliptical, or polygonal, e.g., square, rectangular, triangular, n-sided (e.g., regular or non-regular polygons having 4, 5, 6, 7, 8, 9, 10 or more sides), star-shaped, parallelogram, trapezoidal, or irregular, e.g., oval, or curved. In some cases, the electroporation zone is a channel having substantially uniform cross-sectional dimensions along the length of the channel, for example, the electroporation zone may have a circular cross-section, with a diameter constant from the fluid connection to the entry zone to the fluid connection to the recovery zone. In this configuration, the resulting electric field is more uniform, thus allowing for more predictable electric field exposure of cells suspended in the fluid. Alternatively, the cross-sectional dimensions of the electroporation zone may vary along its length. For example, the cross-sectional dimensions of the electroporation zone may either increase or decrease along the length of the electroporation zone, or may have two or more dimensional changes along the length of the electroporation zone, for example, the cross-sectional dimensions, e.g., diameter, may increase or decrease by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or by up to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In this configuration, the electroporation zone may have a truncated conical cross-section in which the diameter increases from the upper opening to the lower opening, or decreases from the upper opening to the lower opening. In some cases, the device of the present invention may include a plurality of electroporation zones fluidly connected in series, each having either a uniform or non-uniform cross-section, and each having a different cross-sectional shape. In a non-limiting example, the device of the present invention may include a plurality of electroporation zones connected in series, each of which has a cylindrical cross-section of different cross-sectional dimensions, for example, each having a different diameter. 【0147】 In some embodiments, the cross-sectional dimensions of the electroporation zone are approximately 0.005 mm to approximately 50 mm, for example, approximately 0.005 mm to approximately 0.05 mm, approximately 0.01 mm to approximately 0.1 mm, approximately 0.05 mm to approximately 0.5 mm, approximately 0.1 mm to approximately 1 mm, approximately 0.5 mm to approximately 2 mm, approximately 1 mm to approximately 5 mm, approximately 3 mm to approximately 7 mm, approximately 5 mm to approximately 10 mm, approximately 7 mm to approximately 12 mm, approximately 10 mm to approximately 15 mm, approximately 13 mm to approximately 18 mm, approximately 1 5mm to approximately 20mm, approximately 22mm to approximately 30mm, approximately 25mm to approximately 35mm, approximately 30mm to approximately 40mm, approximately 35mm to approximately 45mm, or approximately 40mm to approximately 50mm, for example, approximately 0.005mm, approximately 0.006mm, approximately 0.007mm, approximately 0.008mm, approximately 0.009mm, approximately 0.01mm, approximately 0.02mm, approximately 0.03mm, approximately 0.04mm, approximately 0.05mm, approximately 0.06mm, approximately 0.07mm, approximately 0.08mm, approximately 0.09mm, approximately 0.1mm, approximately 0.2mm, approximately 0.3mm, approximately 0.4mm, approximately 0.5mm, approximately 0.6mm, approximately 0.7mm, approximately 0.8mm, approximately 0.9mm, approximately 1mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5 mm, approximately 6mm, approximately 7mm, approximately 8mm, approximately 9mm, approximately 10mm, approximately 11mm, approximately 12mm, approximately 13mm, approximately 14mm, approximately 15mm, approximately 16mm, approximately 17mm, approximately 18mm, approximately 19mm, approximately 20mm, approximately 21m m, approximately 22mm, 23mm, 24mm, 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 31mm, 32mm, 33mm, 34mm, 35mm, 36mm, 37mm, 38mm, 39mm, 40mm, 41mm, 42mm, 43mm, 44mm, 45mm, 46mm, 47mm, 48mm, 49mm, or approximately 50mm. Generally, the diameter of the electroporation zone is sized such that the electroporation zone does not have a contraction area that contacts the cell to deform the cell membrane together with the channel wall, for example, cell perforation is not induced by mechanical deformation resulting from cell constriction, for example, the cell can freely pass through the electroporation zone. 【0148】 In some cases, the electroporation zone length is approximately 0.005 mm to 50 mm, for example, approximately 0.005 mm to 0.05 mm, approximately 0.01 mm to 0.1 mm, approximately 0.05 mm to 0.5 mm, approximately 0.1 mm to 1 mm, approximately 0.5 mm to 2 mm, approximately 1 mm to 5 mm, approximately 3 mm to 7 mm, approximately 5 mm to 10 mm, approximately 7 mm to 12 mm, approximately 10 mm to 15 mm, approximately 13 mm to 18 mm, and approximately 15 mm. ~20mm, 22mm~30mm, 25mm~35mm, 30mm~40mm, 35mm~45mm, or 40mm~50mm, for example, 0.005mm, 0.006mm, 0.007mm, 0.008mm, 0.009mm, 0.01mm, 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0. 09mm, approximately 0.1mm, approximately 0.2mm, approximately 0.3mm, approximately 0.4mm, approximately 0.5mm, approximately 0.6mm, approximately 0.7mm, approximately 0.8mm, approximately 0.9mm, approximately 1mm, approximately 2mm, approximately 3mm, approximately 4mm, approximately 5mm , about 6mm, about 7mm, about 8mm, about 9mm, about 10mm, about 11mm, about 12mm, about 13mm, about 14mm, about 15mm, about 16mm, about 17mm, about 18mm, about 19mm, about 20mm, about 21mm , approximately 22mm, 23mm, 24mm, 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 31mm, 32mm, 33mm, 34mm, 35mm, 36mm, 37mm, 38mm, 39mm, 40mm, 41mm, 42mm, 43mm, 44mm, 45mm, 46mm, 47mm, 48mm, 49mm, or 50mm, not necessarily. 【0149】 The cross-sectional dimensions of the entry zone and / or recovery zone may independently be substantially the same as those of the electroporation zone. Alternatively, the entry zone and / or recovery zone may independently be smaller or larger than the cross-sectional dimensions of the electroporation zone. For example, when the cross-sectional dimensions of the entry zone and / or recovery zone are independently configured to be smaller than the cross-sectional dimensions of the electroporation zone, the cross-sectional dimensions of the entry zone and / or recovery zone are approximately 0.01% to 100%, 0.01% to 1%, 0.1% to 10%, 5% to 25%, 10% to 50%, 25% to 75%, or 50% to 100% of the cross-sectional dimensions of the electroporation zone, for example, approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, Approximately 0.15%, approximately 0.2%, approximately 0.25%, approximately 0.3%, approximately 0.35%, approximately 0.4%, approximately 0.45%, approximately 0.5%, approximately 0.55%, approximately 0.6%, approximately 0.65%, approximately 0.7%, approximately 0.75%, approximately 0.8%, approximately 0.85%, approximately 0.9%, approximately 0.95%, approximately 1%, approximately 2%, approximately 3%, approximately 4% Whether it is approximately 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 【0150】 Alternatively, when the cross-sectional dimensions of the entry zone and / or recovery zone are configured to be independently larger than the cross-sectional dimensions of the electroporation zone, the cross-sectional dimensions of the entry zone and / or recovery zone are approximately 100% to approximately 100,000% of the cross-sectional dimensions of the electroporation zone, for example, approximately 100% to approximately 1000%, approximately 500% to approximately 5,000%, approximately 1,000% to approximately 10,000%, approximately 5,000% to approximately 25,000%, approximately 10,000% to approximately 50,000%, approximately 25,000% to approximately 75,000%, or approximately 50,000% to approximately 100,000%, for example, approximately 100%, approximately 200%, approximately 300%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, approximately 1,000%, approximately 2,000%, approximately 3,000%, approximately 4,000%, approximately 5,000%, approximately 6,000%, approximately 7,000%, approximately 8,000%, approximately 9,000%, approximately 10,000%, approximately 15,000%, approximately 20,000%, approximately 25,000%, approximately 30,000 Possible percentages include approximately 35,000%, 40,000%, 45,000%, 50,000%, 55,000%, 60,000%, 65,000%, 70,000%, 75,000%, 80,000%, 85,000%, 90,000%, 95,000%, or 100,000%. 【0151】 The device of the present invention may also include one or more reservoirs for fluid reagents, such as buffer solutions, or samples, such as cell suspensions and compositions introduced into cells. For example, the device of the present invention may include a reservoir for cells suspended in fluid to flow into the electroporation zone within a first electrode, and / or a reservoir for holding the electroporated cells. Similarly, there may be reservoirs for the flow of liquid in additional components of the device, such as additional inlets that intersect with the first or second electrode. A single reservoir may also be connected to multiple devices of the present invention, for example, when the same liquid is introduced into two or more individual devices of the present invention configured to electroporate cells in parallel or in series. Alternatively, the device of the present invention may be configured to mate with a liquid source, which may be an external reservoir such as a vial, tube, or pouch. Similarly, the device may be configured to mate with a separate component that houses a reservoir. The reservoir can be any suitable size to hold, for example, 10 mL to 5000 mL, 10 mL to 3000 mL, 25 mL to 100 mL, 100 mL to 1000 mL, 40 mL to 300 mL, 1 mL to 100 mL, or 10 mL to 500 mL. If multiple reservoirs are present, each reservoir may be the same size or of a different size. 【0152】 In addition to the components discussed above, the device of the present invention may include additional components. For example, the first and second electrodes of the device of the present invention may include one or more additional fluid inlets to allow the introduction of a non-sample fluid, such as a buffer solution, into a suitable area of ​​the device. For example, the recovery zone of the device of the present invention may include additional inlets and outlets to circulate the recovery buffer to assist in closing the pores opened in the cell membrane from the electroporation process. 【0153】 Systems and kits One or more electroporation devices of the present invention can be combined with various external components, such as a power supply, pump, reservoir (e.g., a bag), controller, reagents, liquids, and / or samples, forming a system. In some embodiments, the system of the present invention includes a plurality of the devices of the present invention and a potential source releasably connected to a first electrode and a second electrode of the device(s) of the present invention. In this configuration, the device(s) of the present invention are connected to the potential source, the first electrode is energized, and the second electrode is held to ground. This creates a localized electric field within the electroporation zone, thus electroporating cells passing through the device(s). An electroporation system incorporating the devices of the present invention can induce either reversible or irreversible electroporation in cells passing through the devices and system of the present invention. For example, the devices and system of the present invention can induce substantially nonthermally reversible electroporation, substantially nonthermally irreversible electroporation, or substantially thermally irreversible electroporation in cells suspended in a fluid. 【0154】 In some cases, the releasable connections to the first and second electrodes may include any practical electromechanical connections that can maintain consistent electrical contact between the potential source and the first and second electrodes. Exemplary electrical connections include, but are not limited to, clamps, clips (e.g., alligator clips), springs (e.g., leaf springs), external sheaths or sleeves, wire brushes, flexible conductors, pogo pins, mechanical connections, inductive connections, or combinations thereof. Other types of electrical connections are known in the art. For example, spring electrodes may be integrated into a conductive platform as shown in Figures 2A-2B. In the embodiments shown in Figures 2A-2B, the device of the present invention is inserted into a housing that incorporates two electrically isolated conductive grids on a base encompassing individual openings for receiving the device of the present invention. The device of the present invention may be positioned within the openings of the conductive grid so that the first and second electrodes of the device can contact the conductive grid. In particular, the conductive grid includes spring-biased electrodes, such as electrodes connected to a spring, such that when the device of the present invention is placed within the opening of the conductive grid, the spring-biased electrodes displace and compress the spring (therefore providing a restoring force to the first and second electrodes of the device of the present invention), thus ensuring electrical contact between the device of the present invention and the potential source. 【0155】 The potential source is configured to deliver an applied voltage to one or more electrodes in order to provide a potential difference between the electrodes and thus establish a uniform electric field within the electroporation zone. In some cases, such as in a two-electrode electroporation circuit, the applied voltage is delivered to the first electrode and the second electrode is held to ground. While we do not wish to be bound by any particular theory, the applied voltage delivered to the electrodes is delivered with a specific amplitude, a specific frequency, a specific pulse shape, a specific duration, a specific number of pulses applied, and a specific duty cycle. Coupled with the geometry of the electroporation zone, these parameters deliver a specific electric field within the electroporation zone that cells suspended in a fluid will experience. The electrical parameters described herein may be optimized for a specific cell line and / or composition to be delivered to a specific cell line. The application of potential to the electrodes of the device(s) of the present invention may be initiated and / or controlled by a controller operably coupled to the potential source, such as a computer with programming. 【0156】 Along with the potential parameters described herein, the geometry of the device of the present invention, for example, the shape and dimensions of the cross-section of the electroporation zone, controls the shape and intensity of the resulting electric field within the electroporation zone. Typically, a device having an electroporation zone with a uniform cross-section will exhibit a uniform electric field along the length of the electroporation zone. To modulate the resulting electric field within the electroporation zone, the electroporation zone may include multiple different cross-sectional dimensions and / or different cross-sectional shapes along the length of the electroporation zone. As a non-limiting example, the device of the present invention may include multiple electroporation zones connected in series, each of which has a circular cross-section of a different cross-sectional dimension, for example, each having a different diameter. In this configuration, each of the circular cross-sections of the electroporation zone with different diameters acts as an independent electroporation zone, each inducing a different electric field for each change in dimension at the same applied voltage, for example, a constant DC voltage. 【0157】 In some cases, the device of the present invention may include a plurality of electroporation zones fluidly connected in series, each having either a uniform or non-uniform cross-section, and each having a different cross-sectional shape. Alternatively, the system of the present invention may include a plurality of the devices of the present invention in a parallel configuration, with each device operating independently of the others to increase the overall electroporation throughput. 【0158】 In some cases, the amplitude of the applied voltage is approximately -3kV to 3kV, for example, 0.01kV to approximately 3kV, for example, approximately 0.01kV to approximately 0.1kV, approximately 0.02kV to approximately 0.2kV, approximately 0.03kV to approximately 0.3kV, approximately 0.04kV to approximately 0.4kV, approximately 0.05kV to approximately 0.5kV, approximately 0.06kV to approximately 0.6kV, and approximately 0.07kV. ~approximately 0.7kV, approximately 0.08kV~approximately 0.8kV, approximately 0.09kV~approximately 0.9kV, approximately 0.1kV~approximately 1kV, approximately 0.15kV~approximately 1.5kV, approximately 0.2kV~approximately 2kV, approximately 0.25kV~approximately 2.5kV, or approximately 0.3kV~approximately 3kV, for example, approximately 0.01~approximately 1kV, approximately 0.1kV~approximately 0.7kV, or approximately 0.2~approximately 0 0.6kV, for example, approximately 0.01kV, approximately 0.02kV, approximately 0.03kV, approximately 0.04kV, approximately 0.05kV, approximately 0.06kV, approximately 0.07kV, approximately 0.08kV, approximately 0.09kV, approximately 0.1kV, approximately 0.2kV, approximately 0.3kV, approximately 0.4kV, approximately 0.5kV, approximately 0.6kV, approximately 0.7kV, approximately 0.8kV, approximately 0.9kV, approximately 1 kV, approximately 1.1kV, 1.2kV, 1.3kV, 1.4kV, 1.5kV, 1.6kV, 1.7kV, 1.8kV, 1.9kV, 2kV, 2.1kV, 2.2kV, 2.3kV, 2.4kV, 2.5kV, 2.6kV, 2.7kV, 2.8kV, 2.9kV, or approximately 3kV. 【0159】 In the case of a flow, the frequency of the applied voltage is approximately 1 Hz to 50,000 Hz, for example, approximately 1 Hz to 1,000 Hz, approximately 100 Hz to 5,000 Hz, approximately 500 Hz to 10,000 Hz, approximately 1,000 Hz to 25,000 Hz, or approximately 5,000 Hz to 50,000 Hz, for example, approximately 10 Hz to 1,000 Hz, approximately 500 Hz to 750 Hz, or approximately 100 Hz to 500 Hz, for example, approximately 1 Hz, approximately 2 Hz, approximately 3 Hz, approximately 4 Hz, approximately 5 Hz, approximately 6 Hz, approximately 7 Hz, approximately 8 Hz, approximately 9 Hz, approximately 10 Hz, approximately 20 Hz, approximately 30 Hz, approximately 40 Hz, approximately 50 Hz, approximately 60 Hz These are approximately 70Hz, 80Hz, 90Hz, 100Hz, 200Hz, 300Hz, 400Hz, 500Hz, 600Hz, 700Hz, 800Hz, 900Hz, 1,000Hz, 2,000Hz, 3,000Hz, 4,000Hz, 5,000Hz, 6,000Hz, 7,000Hz, 8,000Hz, 9,000Hz, 10,000Hz, 15,000Hz, 20,000Hz, 25,000Hz, 30,000Hz, 35,000Hz, 40,000Hz, 45,000Hz, or 50,000Hz. 【0160】 In some embodiments, the shape of the applied pulse, e.g., the waveform, may be a square wave, pulse, bipolar wave, sine wave, ramp, asymmetric bipolar wave, or any other. Other voltage waveforms are known in the art. The selected waveform may be applied in any practical voltage pattern, including but not limited to high-voltage-low-voltage, low-voltage-high-voltage, direct current (DC), alternating current (AC), unipolar, positive (+) polarity only, negative (-) polarity only, (+) / (-) polarity, (-) / (+) polarity, or any superposition or combination thereof. Those skilled in the art will understand that these pulse parameters depend on any electrical properties of the cell line and the composition being delivered to the cells. 【0161】 The applied voltage pulse is approximately 0.01ms to approximately 1,000ms, for example, approximately 0.01ms to approximately 1ms, approximately 0.1ms to approximately 10ms, approximately 1ms to approximately 50ms, approximately 10ms to approximately 100ms, approximately 25ms to approximately 200ms, approximately 50ms to approximately 400ms, approximately 100ms to approximately 600ms, approximately 300ms to approximately 800ms, or approximately 500ms to approximately 1,000ms. 00ms, for example, approximately 0.01ms to 100ms, approximately 0.1ms to approximately 50ms, or approximately 1ms to approximately 10ms, for example, approximately 0.01ms, approximately 0.02ms, approximately 0.03ms, approximately 0.04ms, approximately 0.05ms, approximately 0.06ms, approximately 0.07ms, approximately 0.08ms, approximately 0.09ms, approximately 0.1ms, approximately 0.2ms, approximately 0.3ms, approximately 0 It can be delivered to the electroporation zone with a duration of approximately 0.4ms, 0.5ms, 0.6ms, 0.7ms, 0.8ms, 0.9ms, 1ms, 2ms, 3ms, 4ms, 5ms, 6ms, 7ms, 8ms, 9ms, 10ms, 20ms, 30ms, 40ms, 50ms, 60ms, 70ms, 80ms, 90ms, 10ms, 20ms, 30ms, 40ms, 50ms, 60ms, 70ms, 80ms, 90ms, 100ms, 150ms, 200ms, 250ms, 300ms, 350ms, 400ms, 450ms, 500ms, 550ms, 600ms, 650ms, 700ms, 750ms, 800ms, 850ms, 900ms, 950ms, or 1,000ms. 【0162】 In some cases, the number of applied voltage pulses delivered can range from 0 to approximately 1000 or more, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, or 100 or more, for example, 1 to 4, 2 to 5, 3 to 6, 4 to 7, 5 to 8, 6 to 9, or 7 to 10, for example, approximately 0.01 to approximately 1,000, for example, approximately 0.01 Approximately 1, approximately 0.1 to approximately 10, approximately 1 to approximately 50, approximately 10 to approximately 100, approximately 25 to approximately 200, approximately 50 to approximately 400, approximately 100 to approximately 600, approximately 300 to approximately 800, or approximately 500 to approximately 1,000, for example, approximately 0.01 to 100, approximately 0.1 to approximately 50, or approximately 1 to approximately 10, for example, approximately 0.01, approximately 0.02, approximately 0.03, approximately 0 .04 pieces, about 0.05 pieces, about 0.06 pieces, about 0.07 pieces, about 0.07 pieces, about 0.08 pieces, about 0.09 pieces, about 0.1 pieces, about 0.2 pieces, about 0.3 pieces, about 0.4 pieces, about 0.5 pieces , about 0.6 pieces, about 0.7 pieces, about 0.8 pieces, about 0.9 pieces, about 1 piece, about 2 pieces, about 3 pieces, about 4 pieces, about 5 pieces, about 6 pieces, about 7 pieces, about 8 pieces, about 9 pieces, about 10 pieces, about 20 pieces, about 30 pieces, about 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500, approximately 550, approximately 600, approximately 650, approximately 700, approximately 750, approximately 800, approximately 850, approximately 900, approximately 950, or approximately 1,000. 【0163】 The pulse of the applied voltage is, in some cases, approximately 0.001% to approximately 100%, for example, approximately 0.001% to approximately 0.1%, approximately 0.01% to approximately 1%, approximately 0.1% to approximately 5%, approximately 1% to approximately 10%, approximately 2.5% to approximately 20%, approximately 5% to approximately 40%, approximately 10% to approximately 60%, approximately 30% to approximately 80%, or approximately 50% to approximately 1%. 00%, for example, approximately 0.01% to 100%, approximately 0.1% to 99%, approximately 1% to 97%, or approximately 10% to 95%, for example, approximately 0.001%, approximately 0.002%, approximately 0.003%, approximately 0.004%, approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.0 1%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, Delivery may be made with duty cycles of approximately 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. 【0164】 The device(s) of the present invention generate a local electric field within an electroporation zone that electroporates cells passing through it when electrodes are connected to a potential source and current is applied. In some cases, the electric field generated in the electroporation zone is approximately 2 V / cm to approximately 50,000 V / cm, for example, approximately 2 V / cm to approximately 1,000 V / cm, approximately 100 V / cm to approximately 5,000 V / cm, approximately 500 V / cm to approximately 10,000 V / cm, approximately 1,000 V / cm to approximately 25,000 V / cm, or approximately 5,000 V / cm to approximately 50,000 V / cm, for example, approximately 2 V / cm ~ approximately 20,000 V / cm, approximately 5 V / cm ~ approximately 10,000 V / cm, or approximately 100 V / cm ~ approximately 1,000 V / cm, for example, approximately 2 V / cm, approximately 3 V / cm, approximately 4 V / cm, approximately 5 V / cm, approximately 6 V / cm, approximately 7 V / cm, approximately 8 V / cm, approximately 9 V / cm, approximately 10 V / cm, approximately 20 V / cm, approximately 30 V / cm, approximately 40 V / cm, approximately 50 V / cm, approximately 60 V / cm cm, approx. 70V / cm, approx. 80V / cm, approx. 90V / cm, approx. 100V / cm, approx. 200V / cm, approx. 300V / cm, approx. 400V / cm, approx. 500V / cm, approx. 600V / cm, approx. 700V / cm, approx. 800V / cm, approx. 900V / cm, approx. 1,000V / cm, approx. 2,000V / cm, approx. 3,000V / cm, approx. 4,000V / cm, approx. 5,000V / cm, It has a magnitude of approximately 6,000 V / cm, approximately 7,000 V / cm, approximately 8,000 V / cm, approximately 9,000 V / cm, approximately 10,000 V / cm, approximately 15,000 V / cm, approximately 20,000 V / cm, approximately 25,000 V / cm, approximately 30,000 V / cm, approximately 35,000 V / cm, approximately 40,000 V / cm, approximately 45,000 V / cm, or approximately 50,000 V / cm. 【0165】 The system of the present invention typically includes a fluid delivery source configured to deliver a plurality of cells suspended in a fluid to a second electrode, e.g., a recovery zone, through a first electrode, e.g., an entry zone. The fluid delivery source typically includes, but is not limited to, a syringe pump, a micropump, or a peristaltic pump. Alternatively, the fluid may be delivered by the displacement of a working fluid relative to a reservoir of the fluid to be delivered, or by air displacement. Other fluid delivery sources are known in the art. In some embodiments, the fluid delivery source is configured to flow cells suspended in a fluid by the application of positive pressure. While we do not wish to be bound by any particular theory, the flow rate through which the cells in suspension flow through the device of the present invention, and the specific geometric shape of the electroporation zone of the device of the present invention, will determine the residence time of the cells in the electric field within the electroporation zone. 【0166】 In some cases, the volumetric flow rate of the fluid delivered from the fluid delivery source is approximately 0.001 mL / min to approximately 1,000 mL / min, for example, approximately 0.001 mL / min to approximately 0.1 mL / min, approximately 0.01 mL / min to approximately 1 mL / min, approximately 0.1 mL / min to approximately 10 mL / min, approximately 1 mL / min to approximately 50 mL / min, approximately 10 mL / min to approximately 100 mL / min, approximately 25 mL / min to approximately 200 mL / min, approximately 50 mL / min to approximately 400 mL / min, approximately 100 mL / min to approximately 600 mL / min, approximately 300 mL / min to approximately 800 mL / min, or approximately 500 mL / min to Approximately 1,000 mL / min, for example, approximately 0.001 mL / min, approximately 0.002 mL / min, approximately 0.003 mL / min, approximately 0.004 mL / min, approximately 0.005 mL / min, approximately 0.006 mL / min, approximately 0.007 mL / min, approximately 0.008 mL / min, approximately 0.009 mL / min, approximately 0.01 mL / min, approximately 0.02 mL / min, approximately 0.03 mL / min, approximately 0.04 mL / min, approximately 0.05 mL / min, approximately 0.06 mL / min, approximately 0.07 mL / min, approximately 0.08 mL / min, approximately 0.09 mL / min, approximately 0.1 mL / min, approximately 0.2 mL / min, approximately 0.3 mL / min, approx. 0.4mL / min, approx. 0.5mL / min, approx. 0.6mL / min, approx. 0.7mL / min, approx. 0.8mL / min, approx. 0.9mL / min, approx. 1mL / min, approx. 2mL / min, approx. 3mL / min, approx. 4mL / min, approx. 5mL / min, approx. 6mL / min, approx. 7mL / min, approx. 8mL / min 9 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 55 mL / min, 60 mL / min, 65 mL / min, 70 mL / min, It has a volumetric flow rate of approximately 75 mL / min, 80 mL / min, 85 mL / min, 90 mL / min, 95 mL / min, 100 mL / min, 150 mL / min, 200 mL / min, 250 mL / min, 300 mL / min, 350 mL / min, 400 mL / min, 450 mL / min, 500 mL / min, 550 mL / min, 600 mL / min, 650 mL / min, 700 mL / min, 750 mL / min, 800 mL / min, 850 mL / min, 900 mL / min, 950 mL / min, or 1,000 mL / min.In certain embodiments, the flow rate is 10 mL / min to about 100 mL / min, for example, about 10 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, or 100 mL / min. 【0167】 The residence time of cells in the electroporation zone of the device of the present invention is approximately 0.5 ms to approximately 50 ms, for example, approximately 0.5 ms to approximately 5 ms, approximately 1 ms to approximately 10 ms, approximately 5 ms to approximately 15 ms, approximately 10 ms to approximately 20 ms, approximately 15 ms to approximately 25 ms, approximately 20 ms to approximately 30 ms, approximately 25 ms to approximately 35 ms, approximately 30 ms to approximately 40 ms, approximately 35 ms to approximately 45 ms, or approximately 40 ms to approximately 50 ms, for example, approximately 0.5 ms, approximately 0.6 ms, approximately 0.7 ms, approximately 0.8 ms, approximately 0.9 ms, approximately 1 ms, approximately 1.5 ms, approximately 2 ms The residence time may be approximately 2.5ms, 3ms, 3.5ms, 4ms, 4.5ms, 5ms, 5.5ms, 6ms, 6.5ms, 7ms, 7.5ms, 8ms, 8.5ms, 9ms, 9.5ms, 10ms, 10.5ms, 11ms, 11.5ms, 12ms, 12.5ms, 13ms, 13.5ms, 14ms, 14.5ms, 15ms, 20ms, 25ms, 30ms, 35ms, 40ms, 45ms, or 50ms. In some embodiments, the residence time is 5–20ms (e.g., 6–18ms, 8–15ms, or 5–14ms). 【0168】 The system of the present invention typically features a housing that encloses and supports the device(s) of the present invention and any necessary electrical connections, such as electrode connections. The housing may be configured to hold and energize a single device of the present invention, or alternatively, to hold and energize multiple devices of the present invention simultaneously. For example, in the embodiment of the system of the present invention shown in Figures 2A-2B, the housing is configured as a rack capable of receiving and simultaneously energizing 96 individual devices of the present invention operating in parallel. The housing may include a thermal controller that can regulate the temperature of the device(s) of the present invention during electroporation, or thermally regulate a component of the system, such as a fluid, such as a buffer or a suspension containing cells. The thermal controller may be configured to heat the device(s) of the present invention or a component of the system containing these devices, or to cool the device(s) of the present invention or a component of the system containing these devices, or to perform both actions. When the devices of the present invention, or components of a system of such devices, are configured to heat, suitable thermal controllers include, but are not limited to, a heating block or mantle, liquid heating such as immersion or circulating fluid baths, battery-operated heaters, or resistive heaters such as thin-film heaters or heat tapes. When the devices of the present invention, or components of a system of such devices, are configured to cool, suitable thermal controllers include, but are not limited to, liquid cooling such as immersion or circulating fluid baths, evaporative coolers, or thermoelectric coolers such as Peltier coolers. For example, when implemented with liquid cooling, the devices of the present invention, or a housing configured to hold the devices of the present invention, may be in direct contact with tubes circulating a cooled fluid, or may be surrounded by a cooling jacket including tubes circulating a cooled fluid. Other heating and cooling elements are known in the art. 【0169】 The system of the present invention may include one or more outer structures configured to cover the electrodes of one or more devices of the present invention, for example, to reduce the end user's exposure to active electrical connections. Typically, a device of the present invention (e.g., a Flowfect® device) includes one outer structure covering the electrodes and electroporation zones of the device. The outer structure may be made of a non-conductive material, such as a non-conductive polymer, and may include structural features for electromechanically engaging a portion of the device, such as the electrodes or electroporation zones. The outer structure may include one or more recesses, notches, or similar openings within the structure for housing the device. The outer structure may be configured to be a component that can be removed from the device. For example, the outer structure may consist of two separate components connected by a hinge, such as a living hinge, so that the outer structure can be folded onto the device of the present invention. Alternatively, the outer structure may be one or more separate parts that can be joined together using suitable mating features to form a single structure. In these embodiments, the outer structure can be secured to the device of the present invention using any suitable fasteners, such as snaps, latches, buttons, or clips, which can be integrated into the outer structure or externally connected to the outer structure. Other suitable types of fasteners are known in the art. In some embodiments, the outer structure includes one or more alignment features, such as pins, divots, grooves, or tabs, to ensure proper alignment of one or more components of the outer structure. In some cases, the outer structure is configured to be permanently connected to the device of the present invention. 【0170】 In any of the embodiments of the outer structure described herein, the outer structure provides an electrical connection between an external potential source and the electrodes of the device of the present invention. For example, the outer structure may include one or more electrical inputs for electrical connections, such as spades, banana plugs, or bayonets, such as BNC connectors, which facilitate the electrical connection between the potential source and the electrodes of the device of the present invention inside the outer structure. 【0171】 The device and external structure of the present invention may be combined in a kit with additional external components such as reagents, buffers, transfection buffers or recovery buffers, and / or samples. In some cases, the transfection buffer contains a composition suitable for cell electroporation. In some cases, the transfection buffer contains a preferred concentration of one or more salts (e.g., potassium chloride, sodium chloride, potassium phosphate, potassium dihydrogen phosphate) or sugars (e.g., dextrose or myo-inositol), or any combination thereof, at a concentration of 0.1 to 200 mM (e.g., 0.1 to 1.0 mM, 1.0 mM to 10 mM, or 10 mM to 100 mM). 【0172】 method The present invention features a method for introducing a composition, such as a transfection, into at least a portion of multiple cells suspended in a fluid, using an electroporation device described herein. Using the method described herein, the throughput of composition delivery to cell types, which is often considered a bottleneck in the fields of genetic engineering research and genetically modified cell therapy, can be significantly increased. In particular, the method described herein significantly increased the number of cells recovered after transfection, transfection efficiency, and cell viability when applied to a greater number of cell types than typical transfection methods, such as lentiviral transfection, or commercially available cell transfection devices, such as the NEON® transfection system (Thermo Fisher, Carlsbad, CA) or NUCLEOFECTOR 4D (Lonza, Switzerland). 【0173】 As described herein, a composition is introduced into at least a portion of a plurality of cells suspended in a fluid by passing a fluid containing suspended cells, which also contains the composition to be introduced into the cells, through a device of the present invention, such as an electroporation device. The composition and cells suspended in the fluid can be delivered through the device of the present invention by applying positive pressure from, for example, a pump connected to a fluid source, such as a peristaltic pump, a digital pipette, or an automated liquid handling source. The composition and cells suspended in the fluid move from a first electrode, which includes, for example, an entry zone, to an electroporation zone fluid-connected to the first electrode, and then to a recovery zone fluid-connected to the electroporation zone. As the composition and cells suspended in the fluid flow through the first electrode to the electroporation zone, a potential difference is applied to the first and second electrodes, producing cells and thus exposing the cells to the electric field in the electroporation zone. Exposure of cells to the generated electric field increases the transient permeability of the plurality of cells, and thus introduces the composition into at least a portion of the plurality of cells. 【0174】 In some cases of the method, the phenotype of cells may or may not change relative to baseline measurements of the phenotype when they leave the electroporation zone of the device of the present invention. In some cases, the phenotype of cells changes by 0% to about 25% relative to the baseline measurement of cell phenotype when the cells leave the electroporation zone of the device of the present invention, for example, about 0% to about 2.5%, about 1% to about 5%, about 1% to about 10%, about 5% to about 15%, about 10% to about 20%, about 15% to about 25%, or about 20% to about 25%, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%. In certain cases, the phenotype of multiple cells does not change when the cells leave the electroporation zone. For example, baseline or control measurements for establishing the cellular phenotype may be measurements of the expression of cell surface markers on cells not transfected using the device of the present invention. Changes in the cellular phenotype can be assessed using corresponding identical measurements of the expression of the same cell markers on cells transfected using the device of the present invention. To ensure that the cellular phenotype changes minimally or not after electroporation, the cellular phenotype is assessed by flow cytometry analysis of cell surface marker expression. Examples of cell surface markers to be assessed include, but are not limited to, CD3, CD4, CD8, CD19, CD45RA, CD45RO, CD28, CD44, CD69, CD80, CD86, CD206, IL-2 receptor, CTLA4, OX40, PD-1, and TIM3. Cellular morphology is assessed using bright-field or fluorescence microscopy to confirm the absence of phenotypic changes after electroporation. 【0175】 In some cases, after introducing a composition into at least a portion of a group of cells, the cells are stored in a recovery buffer. The recovery buffer is configured to facilitate the final closure of the pores formed in the cells. The recovery buffer typically includes a cell culture medium, which may also contain other components for cell nutrition and growth, such as serum and minerals. Those skilled in the art will understand that the selection of the recovery buffer depends on the cell type undergoing electroporation. 【0176】 In some embodiments of the methods described herein, the volume of the fluid having suspended cells and a composition introduced to the cells flowing through the electroporation zone of the device of the present invention is about 0.001 mL to about 2000 mL, about 0.001 mL to about 1000 mL, for example, 0.001 mL to about 1000 mL, for example, about 0.001 mL to about 0.1 mL, about 0.01 mL to about 1 mL, about 0.1 mL to about 5 mL, about 1 mL to about 10 mL, about 2.5 mL to about 20 mL. Approximately 5 mL to 40 mL, approximately 10 mL to 60 mL, approximately 30 mL to 80 mL, approximately 50 mL to 200 mL, approximately 100 mL to 500 mL, or 250 mL to 750 mL, or approximately 500 mL to 1000 mL, for example, approximately 0.01 mL to 100 mL, approximately 0.1 mL to 99 mL, approximately 1 mL to 97 mL, or approximately 10 mL to 95 mL, for example, approximately 0.0025 mL to 10 mL, approximately 0.01 mL to 1 mL, or approximately 0.025 mL to 0.1 mL, e.g., about 0.001 mL, about 0.0025 mL, about 0.005 mL, about 0.0075 mL, about 0.01 mL, about 0.025 mL, about 0.05 mL, about 0.075 mL, about 0.1 mL, about 0.25 mL, about 0.5 mL, About 0.75mL, about 1mL, about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, about 15mL, about 20mL, about 25mL, about 30mL, about 35mL, about 40mL, about 45mL, It is not necessarily about 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, 650 mL, 700 mL, 750 mL, 800 mL, 850 mL, 900 mL, 950 mL, or 1000 mL. 【0177】 In certain embodiments, the conductivity of the fluid in which the cells are suspended can affect the electroporation of the cells in the suspension, and therefore the delivery of the composition to these cells. The conductivity of the fluid containing the suspended cells can range from about 0.001 mS to about 500 mS, for example, about 0.001 mS to about 0.1 mS, about 0.01 mS to about 1 mS, about 0.1 mS to about 10 mS, about 1 mS to about 50 mS, about 10 mS to about 100 mS, about 25 mS to about 200 mS, about 50 mS to about 400 mS, or about 100 mS to about 500 mS, for example, about 0.01 mS ~approximately 100 mS, approximately 0.1 mS to approximately 50 mS, or approximately 1 to 20 mS, for example, approximately 0.001 mS, approximately 0.002 mS, approximately 0.003 mS, approximately 0.004 mS, approximately 0.005 mS, approximately 0.006 mS, approximately 0.007 mS, approximately 0.008 mS, approximately 0.009 mS, approximately 0.01 mS, approximately 0.02 mS, approximately 0.03 mS, approximately 0.04 mS, approximately 0.05 mS, approximately 0 0.06mS, approx. 0.07mS, approx. 0.08mS, approx. 0.09mS, approx. 0.1mS, approx. 0.2mS, approx. 0.3mS, approx. 0.4mS, approx. 0.5mS, approx. 0.6mS, approx. 0.7mS, approx. 0.8mS, approx. 0.9mS, approx. 1mS, approx. 2mS, approx. 3mS, approx. 4mS, approx. 5mS, approx. 6mS, approx. 7mS, approx. 8mS, approx. 9mS, approx. 10mS, approx. 15mS, approx. 20mS, approx. 25 mS, approximately 30mS, approximately 35mS, approximately 40mS, approximately 45mS, approximately 50mS, approximately 55mS, approximately 60mS, approximately 65mS, approximately 70mS, approximately 75mS, approximately 80mS, approximately 85mS, approximately 90mS, approximately 95mS, approximately 100mS, approximately 150mS, approximately 200mS, approximately 250mS, approximately 300mS, approximately 350mS, approximately 400mS, approximately 450mS, or approximately 500mS, may be. 【0178】 The methods of the present invention can deliver compositions to a diverse range of cell types, including, but not limited to, mammalian cells, eukaryotes, prokaryotes, synthetic cells, human cells, animal cells, plant cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, or activated cell immune cells, stem cells (e.g., primary human induced pluripotent stem cells, e.g., iPSCs, embryonic stem cells, e.g., ESCs, mesenchymal stem cells, e.g., MSCs, or hematopoietic stem cells, e.g., HSCs), blood cells (e.g., erythrocytes), T cells (e.g., primary human T cells), B cells, antigen-presenting cells (APCs), natural killer (NK) cells (e.g., primary human NK cells), monocytes (e.g., primary human monocytes), macrophages (e.g., primary human macrophages), and peripheral blood mononuclear cells (PBMCs), neutrophils, dendritic cells, human embryonic kidney (e.g., HEK-293) cells, or Chinese hamster ovary (e.g., CHO-K1) cells. The typical number of cells that can be electroporated is about 10 4 cells ~ about 10 12 Cells, (for example, about 10 4 cells ~ about 10 5 cells, about 10 4 cells ~ about 10 6 cells, about 10 4 cells ~ about 10 7 cells, approximately 5 x 10 4 Cells ~ approx. 5 x 10 5 cells, about 10 5 cells ~ about 10 6 cells, about 10 5 cells ~ about 10 7 cells, approximately 2.5 x 10 5 cells ~ about 10 6 cells, approximately 5 x 10 5 Cells ~ approx. 5 x 10 6 cells, about 10 6 cells ~ about 10 7 cells, about 10 6 cells ~ about 10 8 cells, about 10 6 cells ~ about 10 12 cells, approximately 5 x 10 6 Cells ~ approx. 5 x 10 7 cells, about 10 7 cells ~ about 10 8 cells, about 10 7 cells ~ about 10 9 cells, about 107 cells ~ about 10 12 cells, approximately 5 x 10 7 Cells ~ approx. 5 x 10 8 cells, about 10 8 cells ~ about 10 9 cells, about 10 8 cells ~ about 10 10 cells, about 10 8 cells ~ about 10 12 cells, approximately 5 x 10 8 Cells ~ approx. 5 x 10 9 cells, about 10 9 cells ~ about 10 10 cells, about 10 9 cells ~ about 10 11 cells, about 10 10 cells ~ about 10 11 cells, about 10 10 cells ~ about 10 12 Cells, or about 10 11 cells ~ about 10 12 Cells, for example, about 10 4 cells, approximately 2.5 x 10 4 cells, approximately 5 x 10 4 cells, about 10 5 cells, approximately 2.5 x 10 5 cells, approximately 5 x 10 5 cells, about 10 6 cells, approximately 2.5 x 10 6 cells, approximately 5 x 10 6 cells, about 10 7 cells, approximately 2.5 x 10 7 cells, approximately 5 x 10 7 cells, about 10 8 cells, approximately 2.5 x 10 8 cells, approximately 5 x 10 8 cells, about 10 9 cells, approximately 2.5 x 10 9 cells, approximately 5 x 10 9 cells, about 10 10 cells, approximately 5 x 10 10 cells, about 10 11 Cells, or about 10 12 It could be a cell. 【0179】 about 10 4 cells ~ about 10 12The cell concentration required to achieve the number of cell perforations, i.e., the number of cells per mL of fluid, is typically about 10. 3 cells / mL~about 10 11 cells / mL, for example, about 10 3 cells / mL~about 10 4 cells / mL, approximately 5×10 3 cells / mL ~ approx. 5×10 4 cells / mL, approximately 10 5 cells / mL~about 10 5 cells / mL, approximately 5×10 5 cells / mL ~ approx. 5×10 6 cells / mL, approximately 10 6 cells / mL~about 10 7 cells / mL, approximately 5×10 6 cells / mL ~ approx. 5×10 7 cells / mL, approximately 10 7 cells / mL~about 10 8 cells / mL, approximately 5×10 7 cells / mL ~ approx. 5×10 8 cells / mL, approximately 10 8 cells / mL~about 10 9 cells / mL, approximately 5×10 8 cells / mL ~ approx. 5×10 9 cells / mL, approximately 10 9 cells / mL~about 10 9 cells / mL, approximately 5×10 9 cells / mL ~ approx. 5×10 10 cells / mL, or about 10 10 cells / mL~about 10 11 cells / mL, for example, about 10 3 cells / mL, approximately 5×10 3 cells / mL, approximately 10 4 cells / mL, approximately 5×10 4 cells / mL, approximately 10 5 cells / mL, approximately 5×10 5 cells / mL, approximately 10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 10 7 cells / mL, approximately 5×10 7 cells / mL, approximately 10 8 cells / mL, approximately 5×10 8 cells / mL, approximately 10 9 cells / mL, approximately 5×109 cells / mL, approximately 10 10 cells / mL, approximately 5×10 10 cells / mL, or about 10 11 This is in the range of cells / mL. 【0180】 The methods of the present invention described herein can deliver any composition to cells suspended in a fluid. Compositions that can be delivered to cells include, but are not limited to, therapeutic agents, vitamins, nanoparticles, charged molecules (e.g., ions in solution), uncharged molecules, nucleic acids (e.g., DNA or RNA), CRISPR-Cas complexes, proteins, polymers, ribonucleoproteins (RNPs), manipulated nucleases, activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing nucleases, meganucleases (MNs), megaTALs, enzymes, peptides, transposons, or polysaccharides (e.g., dextran, e.g., dextran sulfate). Exemplary compositions that can be delivered to cells in suspension include nucleic acids, oligonucleotides, antibodies (or antibody fragments, e.g., bispecificity fragments, tripspecificity fragments, Fab, F(ab')2, or single-strand variable fragments (scFv)), amino acids, peptides, proteins, gene therapies, genomically engineered therapies, epigenomically engineered therapies, carbohydrates, chemical drugs, contrast agents, magnetic particles, polymer beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotics, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, anti-inflammatory agents, antimicrobial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacterial phages, adjuvants, minerals, and combinations thereof. The delivered composition may comprise a single compound, such as those described herein. Alternatively, the delivered composition may comprise multiple compounds or components targeting different genes. 【0181】 Typical concentrations of the composition in the fluid range from approximately 0.0001 μg / mL to approximately 1000 μg / mL (for example, approximately 0.0001 μg / mL to approximately 0.001 μg / mL, approximately 0.001 μg / mL to approximately 0.01 μg / mL, approximately 0.001 μg / mL to approximately 5 μg / mL, approximately 0.005 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 0.1 μg / mL, approximately 0.01 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 1 μg / mL, approximately 0.1 μg / mL to approximately 5 μg / mL, approximately 1 μg / mL to approximately 10 μg / mL, approximately 1 μg / mL to approximately 50 μg / mL, approximately 1μg / mL to about 100μg / mL, about 2.5μg / mL to about 15μg / mL, about 5μg / mL to about 25μg / mL, about 5μg / mL to about 50μg / mL, about 5μg / mL to about 500μg / mL, about 7.5μg / mL to about 75μg / mL, about 10μg / mL to about 100μg / mL mL, about 10 μg / mL to about 1,000 μg / mL, about 25 μg / mL to about 50 μg / mL, about 25 μg / mL to about 250 μg / mL, about 25 μg / mL to about 500 μg / mL, about 50 μg / mL to about 100 μg / mL, about 50 μg / mL to about 250 μg / mL, about 50 μg / mL mL to approximately 750 μg / mL, approximately 100 μg / mL to approximately 300 μg / mL, approximately 100 μg / mL to approximately 1,000 μg / mL, approximately 200 μg / mL to approximately 400 μg / mL, approximately 250 μg / mL to approximately 500 μg / mL, approximately 350 μg / mL to approximately 500 μg / mL, approximately 400 μg / mL to approximately 1,000 μg / mL, approximately 500 μg / mL to approximately 750 μg / mL, approximately 650 μg / mL to approximately 1,000 μg / mL, or approximately 800 μg / mL to approximately 1,000 μg / mL, for example, approximately 0.0001 μg / mL, approximately 0.0005 μg / mL, approximately 0.001 μg / mL, approximately 0.005μg / mL, approximately 0.01μg / mL, approximately 0.02μg / mL, approximately 0.03μg / mL, approximately 0.04μg / mL, approximately 0.05μg / mL, approximately 0.06μg / mL, approximately 0.07μg / mL, approximately 0.08μg / mL, approximately 0.09μg / mL, approximately 0.1μg / mL, approximately 0. 2μg / mL, approximately 0.3μg / mL, approximately 0.4μg / mL, approximately 0.5μg / mL, approximately 0.6μg / mL, approximately 0.7μg / mL, approximately 0.8μg / mL, approximately 0.9μg / mL, approximately 1μg / mL, approximately 1.5μg / mL, approximately 2μg / mL, approximately 2.5μg / mL, approximately 3μg / mL, approximately 3.5μg / mL, approximately 4μg / mL, approximately 4.5μg / mL, approximately 5μg / mL, approximately 5.5μg / mL, approximately 6μg / mL, approximately 6.5μg / mL, approximately 7μg / mL, approximately 7.5μg / mL, approximately 8μg / mL, approximately 8.5μg / mL, approximately 9μg / mL, approximately 9.5μg / mL, approximately 1 0μg / mL, approximately 15μg / mL, approximately 20μg / mL, approximately 25μg / mL, approximately 30μg / mL, approximately 35μg / mL, approximately 40μg / mL, approximately 45μg / mL, approximately 50μg / mL, approximately 55μg / mL, approximately 60μg / mL, approximately 65μg / mL, approximately 70μg / mL, approximately 7 It may be approximately 5 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, or 1,000 μg / mL. 【0182】 In some cases, the temperature of the fluid containing the suspended cells and composition is controlled using a thermal controller incorporated into the housing supporting the device(s) of the present invention. Since excessively high temperatures can impair cell viability after electroporation, the fluid temperature is controlled to reduce the effects of Joule heating resulting from the electric field generated within the electroporation zone. The fluid temperature is approximately 0°C to approximately 50°C, for example, approximately 0°C to approximately 10°C, approximately 1°C to approximately 5°C, approximately 2°C to approximately 15°C, approximately 3°C to approximately 20°C, approximately 4°C to approximately 25°C, approximately 5°C to approximately 30°C, approximately 7°C to approximately 35°C, approximately 9°C to approximately 40°C, approximately 10°C to approximately 43°C, approximately 15°C to approximately 50°C, approximately 20°C to approximately 40°C, approximately 25°C to approximately 50°C, or approximately 35°C to approximately 45°C, for example. For example, approximately 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C. It could be approximately 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C. 【0183】 Cells transfected using the method of the present invention are more efficiently transfected and have a higher viability than those transfected using typical transfection methods, such as lentiviral transfection, or commercially available cell transfection equipment, such as the NEON® transfection system (Thermo Fisher, Carlsbad, CA) or NUCLEOFECTOR 4D (Lonza, Switzerland). For example, with respect to the methods described herein, the transfection efficiency, i.e., the efficiency of successfully delivering the composition to cells, is about 0.1% to about 99.9%, for example, about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, or about 50% to about 99.9%, for example, about 10% to about 90%, about 25% to about 85%, for example, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0. It could be 0.4%, approximately 0.45%, approximately 0.5%, approximately 0.55%, approximately 0.6%, approximately 0.65%, approximately 0.7%, approximately 0.75%, approximately 0.8%, approximately 0.85%, approximately 0.9%, approximately 0.95%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or approximately 99.9%. 【0184】 The cell viability of cells suspended in a fluid after introducing a composition using the method of the present invention described herein, i.e., the number or percentage of cells that survived electroporation, is about 0.1% to about 99.9%, for example, about 0.1% to about 5%, about 1% to about 10%, about 2.5% to about 20%, about 5% to about 40%, about 10% to about 60%, about 30% to about 80%, or about 50% to about 99.9%, for example, about 10% to about 90%, about 25% to about 85%, for example, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%. It could be approximately 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9%. 【0185】 The number of cells recovered, i.e., the number of viable cells recovered after electroporation, was approximately 10. 4 cells ~ about 10 12 Cells, for example, about 10 4 cells ~ about 10 5 cells, about 10 4 cells ~ about 10 6 cells, about 10 4 cells ~ about 10 7 cells, approximately 5 x 10 4 Cells ~ approx. 5 x 10 5 cells, about 10 5 cells ~ about 10 6 cells, about 10 5 cells ~ about 10 7 cells, approximately 2.5 x 10 5 cells ~ about 10 6 cells, approximately 5 x 10 5 Cells ~ approx. 5 x 10 6 cells, about 10 6 cells ~ about 10 7 cells, about 10 6 cells ~ about 10 8 cells, about 10 6 cells ~ about 10 12cells, approximately 5 x 10 6 Cells ~ approx. 5 x 10 7 cells, about 10 7 cells ~ about 10 8 cells, about 10 7 cells ~ about 10 9 cells, about 10 7 cells ~ about 10 12 cells, approximately 5 x 10 7 Cells ~ approx. 5 x 10 8 cells, about 10 8 cells ~ about 10 9 cells, about 10 8 cells ~ about 10 10 cells, about 10 8 cells ~ about 10 12 cells, approximately 5 x 10 8 Cells ~ approx. 5 x 10 9 cells, about 10 9 cells ~ about 10 10 cells, about 10 9 cells ~ about 10 11 cells, about 10 10 cells ~ about 10 11 cells, about 10 10 cells ~ about 10 12 Cells, or about 10 11 cells ~ about 10 12 Cells, for example, about 10 4 cells, approximately 2.5 x 10 4 cells, approximately 5 x 10 4 cells, about 10 5 cells, approximately 2.5 x 10 5 cells, approximately 5 x 10 5 cells, about 10 6 cells, approximately 2.5 x 10 6 cells, approximately 5 x 10 6 cells, about 10 7 cells, approximately 2.5 x 10 7 cells, approximately 5 x 10 7 cells, about 10 8 cells, approximately 2.5 x 10 8 cells, approximately 5 x 10 8 cells, about 10 9 cells, approximately 2.5 x 10 9 cells, approximately 5 x 10 9 cells, about 10 10 cells, approximately 5 x 10 10 cells, about 10 11 Cells, or about 1012 It could be a cell. 【0186】 The recovery yield, i.e., the percentage of live, manipulated cells collected after electroporation, ranges from approximately 0.1% to 500%, for example, approximately 0.1% to 5%, approximately 1% to 10%, approximately 2.5% to 20%, approximately 5% to 40%, approximately 10% to 60%, approximately 30% to 80%, approximately 50% to 99.9%, approximately 75% to 150%, approximately 100% to 200%, approximately 150% to 250%, approximately 200% to 300%, approximately 250% to 350%, and approximately 300%. %~approximately 400%, approximately 350%~approximately 450%, or approximately 400%~approximately 500%, for example, approximately 0.1%, approximately 0.15%, approximately 0.2%, approximately 0.25%, approximately 0.3%, approximately 0.35%, approximately 0.4%, approximately 0.45%, approximately 0.5%, approximately 0.55%, approximately 0.6%, approximately 0.65%, approximately 0.7%, approximately 0.75%, approximately 0.8%, approximately 0.85%, approximately 0.9%, approximately 0.95%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99.9%, approximately 100%, approximately 110%, approximately 120%, approximately 130%, approximately 140%, approximately 150%, approximately 160%, approximately 170%, approximately 180%, approximately 190%, approximately 200%, approximately 210%, approximately 220%, approximately 230%, approximately 240%, approximately 250%, approximately 260%, approximately 270%, approximately 280%, approximately 290%, approximately 300%, approximately 310%, approximately 320%, approximately 330%, approximately 340%, approximately 350%, approximately 360%, approximately 370%, approximately 380%, approximately 390%, approximately 400%, approximately 410%, approximately 420%, approximately 430%, approximately 440%, approximately 450%, approximately 460%, approximately 470%, approximately 480%, approximately 490%, or approximately 500%, possibly. 【0187】 Those skilled in the art will understand that the optimal conditions may vary depending on the cell type or other factors. For each new cell type, the following parameters, namely waveform, electric field, pulse duration, buffer exposure time, buffer temperature, and post-electroporation conditions, can be adjusted as needed. [Examples] 【0188】 Example 1 - Device and System Continuous flow electroporation devices and associated systems are designed and manufactured to enhance or maximize the throughput of cell engineering and / or accelerate biological discoveries by enabling the use of multiple devices in parallel to increase or maximize the number of cell electroporation events occurring within a fixed time window. The electroporation devices are configured to be compatible with current automated fluid handling systems, such as pipette tip-based dispensers and robotic fluid pumps. 【0189】 Figure 1A shows a schematic diagram of an exemplary embodiment of an electroporation device, referred to as a pipette tip in this configuration. Figure 1A shows a close-up view of the active area of ​​the device, including the electroporation zone. This device provides continuous flow gene manipulation of both eukaryotic and prokaryotic cells within a platform that can be easily automated through integration with a liquid handling robot. In the devices of Figures 1A–1C, the active area of ​​the device includes three distinct zones: an entry zone, an electroporation zone, and a recovery zone. In the embodiments shown in Figures 1A–1C, the composition to be introduced into the cells and the cells to be transfected are placed in the entry zone. The cells and composition are passed through the electroporation zone, and the transfected cells are dispensed into a buffer for storage in the recovery zone. Thus, the space between the entry zone and the recovery zone is the electroporation zone, and all three zones are fluid-connected (e.g., fluid-linked) so that there is a single flow path through the device. 【0190】 In the embodiment shown in Figure 1A, the entry zone and the recovery zone are manufactured from a suitable material, for example, a hollow electrode made of stainless steel, the entry zone electrode acts as the current-carrying electrode and the recovery zone electrode acts as the ground electrode, thus completing the circuit while allowing an electric field to be generated between the two electrodes (in combination with the conductivity of the fluid that transports the cells and composition). 【0191】 The electroporation device of the present invention is designed to meet the requirements of injection molding and insert molding manufacturing techniques, both of which are inherently scalable, as shown in Figures 1B and 1C. In Figures 1B and 1C, the device body is integrated with an electroporation zone located between commercial stainless steel electrodes in which the electric field is active. The electroporation zone geometry was modified to exhibit a substantially uniform cross-section, resulting in more predictable electric field exposure during the residence time of the electroporated sample. Using current production methods, e.g., 3D printing, approximately 100 devices can be manufactured per day, which is scalable to over 10,000 devices per day using more robust, large-scale production methods, e.g., injection molding and insert molding. 【0192】 The housing can be configured to energize multiple electroporation devices in parallel within an industry-standard 96-well pipette tip tray having grid electrodes for 96 electroporation devices, so that all of the electroporation devices are energized simultaneously with the same applied voltage pulse, ensuring that the electric field within each electroporation device is identical. Electrical energy can be delivered using a single power supply. Therefore, a mechanism for distributing power to each electroporation device may be required. One method for implementing this is shown in Figure 2A, with an exploded view in Figure 2B. This design features spring-loaded electrodes into which 96 individual electroporation devices enter the housing, with the first and second electrodes of each electroporation device physically contacting the electrical grid of the housing. Each spring-loaded electrode is connected in parallel to the electrical grid of the housing, which is connected to a power supply by a single set of leads. The housing is reusable once connected to the power supply, allowing for simultaneous genetic modification of up to 96 discrete samples. The power supply may include additional circuitry or programming configured to modulate pulse delivery so that each of the individual devices of the present invention, for example, 96 individual devices, receives a different voltage or a different waveform. 【0193】 Example 2 - Initial Development of Experimental Parameters for Optimal Transfection Experiments have been conducted using the device of the present invention to study the physical and biological parameters affecting electroporation in Jurkat immortalized T cell lines. Using industry-standard flow cytometry methods, both cell viability (measured by 7AAD dye exclusion) and transfection efficiency (measured by GFP expression) of genetically engineered Jurkat cells were evaluated using the device of the present invention. Both of these are common measures of electroporation success in the field of gene delivery. 【0194】 Unless otherwise specified, the experimental results shown below are for 1 × 10⁶ times in 100 μL of buffer using 5 μg of plasmid (e.g., GFP expression plasmid). 6 Jurkat cells were generated by electroporation of a population at a cellular concentration. Electroporation experiments were performed at 100 Hz with a square waveform and a pulse duration of 9.5 ms. After 24 hours of incubation, cells were stained with 7-AAD and analyzed by flow cytometry to identify viable cells and live GFP-expressing cells. Three experiments were performed, with error bars representing the mean standard error (SEM). Table 1 below presents a summary of the parameters used for transfection using the device of the present invention. [Table 1] 【0195】 Example 3 - Transfection data using the device of the present invention The device of the present invention demonstrates peak transfection performance when the flow rate is maximized through the electroporation channel (Figures 3A and 3B). Using a pipette with a controlled dispensing rate, the desired flow rate was achieved to increase both viability and efficiency, corresponding to a residence time of approximately 6.5 ms for the cell sample in the electric field. A peak cell viability of 54% was achieved with a transfection efficiency of 65%, demonstrating a significant advance in the transfection of human immune cells using the device of the present invention. 【0196】 Figures 4A–4D illustrate flow simulations along the exemplary active zone of the device (i.e., from the first electrode lumen, through the electroporation zone, into the second electrode lumen). In this embodiment, the medium contains flowing biological cells. From the simulated fluid flows at 10 mL / min and 100 mL / min, the average linear velocity of the sample passing through the electroporation zone is determined. The lower flow rate of 10 mL / min results in an average linear velocity of 324 mm / s. The higher flow rate of 100 mL / min results in an average linear velocity of 2,990 mm / s. The two linear velocities are used to determine estimated residence times (τ) of 12.35 ms and 1.34 ms, respectively. res ) may correlate with these. These devices provided a flow rate of 16 mL per minute. Notably, commercial systems require exposure of approximately 30 ms or more under similar electric field exposure to yield comparable transfection efficiency. This demonstrates that the combination of high flow rate and electric field results in improved delivery of genetic material to living cells using the devices of the present invention. 【0197】 The transfection efficiency using the device of the present invention is affected by the electric field strength. Figures 5A and 5B show the resulting cell viability and transfection efficiency at various electric field strengths, respectively. A transfection efficiency of 86% and a viability of 77% were achieved. 【0198】 The device of the present invention showed an increase of approximately 20% in both cell viability and transfection efficiency by cooling the sample on ice to minimize any potential harmful thermal effects that could affect cell viability due to the rise in temperature during electroporation (Figures 6A and 6B). Numerical modeling in COMSOL Multiphysics, which couples the electric field, fluid flow, and thermal effects, was also developed to better understand the effect of sample temperature in the device of the present invention, using applied voltages of 225V or 275V in this model. The results shown in Figures 7A to 7D show a substantially uniform electric field in the electroporation zone. Figures 8A to 8D show the temperature distribution within the device over time. 【0199】 Electroporation using the device of the present invention showed no significant change in performance when electroporation was performed over a range of pulse durations at matching frequencies (Figures 9A and 9B). Varying the number of pulses within a 9.5 ms duration from 1 to 5 did not result in any significant change in viability or efficiency, demonstrating the waveform flexibility for electroporation using the device of the present invention. In this experiment, a peak cell viability of 83% was achieved with a transfection efficiency of 88%. 【0200】 Electroporation using the device of the present invention did not show significant changes in performance when electroporation was performed over a range of volume and cell density (Figures 10A and 10B). By varying the cell number over a volume range of 25–100 μL, no significant changes were observed in either viability or efficiency, demonstrating the flexibility of the physical response to electroporation using the device of the present invention. In this experiment, a peak cell viability of 83% was achieved with a transfection efficiency of 86%. 【0201】 Electroporation using the device of the present invention showed no significant change in performance when electroporation was performed across a range of cross-sectional dimensions in the electroporation zone (Figures 11A and 11B). Similar viability rates were observed by varying the cross-sectional dimensions of the electroporation zone from 500 to 900 μm, and there was no significant change in efficiency when the flow rate was modified to match the total residence time in the electroporation zone, demonstrating the flexibility of cross-sectional dimensions for electroporation using the device of the present invention. In this experiment, a peak cell viability of 51% was achieved with a transfection efficiency of 67%. 【0202】 Survival rates and efficiency depended on the voltage pulse waveform shape, as shown in Figures 12A and 12B. By changing the waveform shape, the duration and intensity of the current to which each Jurkat cell was exposed were adjusted, thereby altering the survival rate or efficiency. In this experiment, high cell viability was observed using square, sinusoidal, and ramp waveform shapes, combined with high transfection efficiency (higher than 50%). Exemplary waveforms useful for the device of the present invention are shown in Figures 12C to 12L. 【0203】 Figures 13A and 13B show the viability and efficiency of the device of the present invention using a flow rate of 10–25 mL / min with an electric field of 400–700 V / cm under cooled conditions. All optimizations performed enable nucleic acid delivery with higher efficiency compared to state-of-the-art commercially available NEON® transfection systems in multiple independent experiments (Figures 13A and 13B). 【0204】 Example 4 - Application of the present invention device to genetic engineering The therapeutic applications of primary human T cells represent significant advances in the field of immuno-oncology by targeting a patient's immune system to be effective in fighting cancer. Several technologies, including chimeric antigen receptors and engineered T cell receptors, have shown clinical success in recent years. However, the application of genetically modifying a patient's immune system remains somewhat limited to the treatment of hematological malignancies, as the tumor microenvironment of solid tumors inhibits T cell function at the tumor site. To overcome some of the biological challenges of suppressing the tumor microenvironment, it is desirable to further modify T cells to be more effective by knockout genes that express regulatory ligands on the T cell surface. The identification of these genes is often achieved through CRISPR screening, in which Cas9 and guide RNA libraries are delivered into T cells to knock out a wide range of endogenous genes to achieve enhancement of function against specific tumors. However, the delivery of these libraries remains a hurdle for identifying genes in "difficult-to-transfect" cell types such as primary T cells and natural killer cells. Typically, in these cases, CRISPR libraries are delivered as lentiviral particles that infect cells and transduce the Cas9 / guide RNA sequence into the cellular genome, subsequently knocking out the gene of interest in a sequence-specific manner. These libraries are highly labor-intensive to produce and require the cloning of viral expression plasmids and purification of viral particles for delivery. Additionally, this methodology leaves undesirable "baggage" of gene-integrated Cas9 / guide RNA sequences at random genomic insertion sites that may disrupt other functional genes. The use of non-viral delivery to the Cas9 ribonucleoprotein complex is an attractive way to overcome these hurdles, allowing researchers to screen for multiple knockouts in the absence of viral integration using transient delivery of the Cas9 protein complexed with the guide RNA molecule. 【0205】 Figure 13C is a flowchart of a method for delivering the Cas9 ribonucleoprotein complex to cells using the device of the present invention. Delivery of the Cas9 ribonucleoprotein complex to electroporating cells enables highly efficient high-throughput analysis of targeted CRISPR knockouts and transforms the process of discovering novel gene targets for therapeutic applications. The study utilizes over 200–1,000 gene subsets, e.g., 25,000, from commercially available cell surface receptor libraries to identify genes that inhibit T cell survival and sustained tumor microenvironment suppression. 【0206】 Example 5 - Electroporation of human cells Figures 14A and 14B show the viability and efficiency data of electroporation of primary human T cells using two different molecular weights of fluorescent dextran molecules at an electric field strength of 700 V / cm. In this experiment, a peak cell viability of 30% was achieved with a transfection efficiency of 67%, demonstrating a significant improvement in the transfection of primary human immune cells using the device of the present invention. 【0207】 In related experiments, electroporation using the device of the present invention showed significantly improved performance compared to NEON® in the THP-1 monocyte cell line (ATCC number TIB-202) using the published NEON® transfection system monocyte electroporation protocol (Figures 15A and 15B). In this experiment, a 56.4% increase in cell viability was observed using the device of the present invention compared to 23.4% with the NEON® transfection system, while transfection efficiency was maintained at 6%. 【0208】 Electroporation using the device of the present invention demonstrated improved performance compared to the NEON® transfection system in primary human monocytes using the published NEON® transfection system monocyte electroporation protocol (Figures 16A and 16B). In this experiment, a 22.3% increase in cell viability was observed using the device of the present invention compared to 16.6% observed with the NEON® transfection system, and a 21.6% increase in transfection efficiency was observed using the device of the present invention compared to 4.7% observed with the NEON® transfection system. 【0209】 Electroporation using the device of the present invention demonstrated improved performance compared to the NEON® transfection system in independent experiments, for the successful delivery of 40 kDa dextran molecules into natural killer cell lines of the NK-92(ATCC) (Figures 17A and 17B) and NK-92MI(ATCC) (Figures 18A and 18B) lineages. These results confirm the ability of the device of the present invention to deliver molecules outside the nucleic acid space with comparable cell viability and improved transfection efficiency compared to non-scalable commercial platforms. 【0210】 SIRPα mRNA delivery to primary monocytes In another study, transient expression of SIRPα in primary human monocytes was achieved using the device of the present invention (Figures 19A–19F). This delivery of non-GFP mRNA in primary human monocytes further demonstrates the device's ability to function in this historically "difficult to transfect" immune cell population. Primary T cells, which are primarily SIRPα-negative, were used as a control for this overexpression demonstration (only 3.4% of live T cells were positive for the surface marker, Figure 19B). After transfection, 86.9% of live T cells were positive for the SIRPα surface marker (Figure 19B). In primary monocytes with a high baseline (86.5% positive for the surface marker (Figure 19A)), mean fluorescence intensity (MFI) was quantified to determine whether receptor expression density increased after transfection. A 1.8-fold increase in SIRPα expression compared to the control cell baseline was observed 24 hours after mRNA delivery (Figure 19F). 【0211】 CXCR4-targeted Cas9-RNP delivery to primary macrophages eGFP-labeled Cas9-RNP was successfully delivered to monocyte-derived human macrophages using the device of the present invention. Delivery of eGFP-labeled Cas9-RNP to the nucleus was confirmed by microscopy and flow cytometry. eGFP expression was observed in up to 21.4% of differentiated macrophages 24 hours after transfection and decreased to 5.1% within 5 days. No gene editing was observed at 24 hours, but a knockout efficiency of 13.9% was observed by 48 hours. Subsequently, the knockout efficiency, as determined by flow cytometry, increased to 16.5% by day 5. 【0212】 Naive T cell manipulation by mRNA delivery Isolated naive T cells (CD45RA) + / CD45RO -)Cells were electroporated with GFP-encoding mRNA using the device of the present invention. After 24 hours, the cells were analyzed for viability and efficiency indicators. The naive cell count and viability of the electroporated cells were comparable to those of untreated cells, and a delivery efficiency of approximately 40% was observed (Figures 20A-20D). Additionally, the cells were stained for the naive T cell markers CD45RA and CD45RO. This staining did not alter the phenotype of the electroporated cells, and the cells were not affected by their "naive" CD45RA + / CD45RO - We demonstrated that the state could be maintained (Figures 21A and 21B). Finally, naive T cells were grown with a CD3 / CD28 activating reagent. In this experiment, the growth rate of electroporated cells was equivalent to that of untreated cells until 6 days after activation (Figure 22). 【0213】 Example 6 - Device for energizing multiple devices of the present invention Figures 23A to 23F show exemplary embodiments of the electroporation device of the present invention integrated into an external device, which can be further integrated into a liquid handling system for energizing the device of the present invention, allowing the electroporation process to be completed on an automated liquid handling platform. An external device called an electronic discharge machine (EDM) is used to energize the device of the present invention during the electroporation process. In the devices shown in Figures 23B, 23C, and 23E, 23.1 is a parallel beam integrated with a support rail. These beams are interchangeable, allowing for changes in the electrical contact style / mechanism. Furthermore, the beams allow for the final positioning of the electrical contacts. 23.2 is a mechanically retractable electrical contact. The electrodes use a spring-like mechanism to allow different areas of the device to slide throughout the EDM while maintaining contact with the body of the electroporation device. This element can be switched for other more flexible electrical contacts, e.g., leaf springs or wire brush type electrodes as shown in Figure 23E. 23.3 is a reservoir for the electroporation device of the present invention. 23.4 is a rocking support rail that allows for additional deflection of the electrodes as needed. This rail feature uses a spring-like mechanism to rotate and allow for more deflection of the electrical contacts while the electroporation device is positioned in place by an operator or an automated system, such as a robotic arm. 23.5 is a sliding rail that allows for the linear movement of a sample holding plate, such as the sample plate shown in 23.6. 23.7 is an alignment system that provides correct positioning of the electroporation device on the sample plate. The alignment system is used as a visual indicator when there is no automated alignment feature, such as robotic control applied to the EDM. When any form of linear device is applied, the system has the ability to complete one or more samples in any array form. 23.8 is the electroporation zone of the device of the present invention, which is fluidly connected to both the entry zone 23.9 and the recovery zone 23.10. 23.11 is a support rail that supports mechanically retractable electrical contacts (23.2).The support rail 23.11 may be electrically conductive so that all mechanically retractable electrical contacts (23.2) can be energized for simultaneous electroporation experiments. Alternatively, the support rail 23.11 may be a non-conductive material that insulates the mechanically retractable electrical contacts (23.2) so that individual electroporation experiments can be performed. 【0214】 When configured as an automated system, a sample of the specimen of interest is aspirated by the device of the present invention at another location on a liquid handling platform. The sample is then transported to an EDM, whose electrode contacts are suspended above the surface of the sample plate. The device of the present invention is then lowered into the device to establish contact with the electrode contacts of the EDM. The mechanisms depicted in Figures 23A–23C use pogo pin connections to close the circuit, while embodiments in Figures 23D–23F use flexible spring electrodes, such as leaf springs, to close the circuit. Alternative methods for connecting the circuit include the use of a conductive fluid or electrolyte, a conductive diaphragm inflated to make contact, or other conductive flexible material having a spring constant sufficient to deflect during the insertion process. This allows the EDM to be adapted to the use of a variety of different sizes of devices of the present invention. Using this system, one or more samples can be electroporated independently or simultaneously, depending on the experimental purpose. This technique can be scaled up to improve in all respects. For example, the EDM can be used with multiple electroporation devices of the present invention, or alternatively, with a single device of the present invention in single-sample or multi-sample experiments by adding two linear mechanisms. 【0215】 Figures 24A and 24B provide exemplary embodiments of a housing configured to energize the conductive device of the present invention in a temperature-controlled manner. In the device of Figure 24A, 24.1 is a hollow electrode configured to connect to a liquid handling manifold. The electrode may further incorporate an interaction collar to reduce stress on the electrode material induced by friction generated by the connection to the liquid handling manifold. 24.2 is a connection channel fluidly connected to the hollow electrode and configured to amplify the electric field generated when the electrode is energized. The connection channel further acts as a barrier to localize the fluid flow in order to enhance and control the electrical pulses received by the sample. 24.3 is a conductive base electrode connected to the connection channel 24.2. 24.4 is a support base configured to hold the hollow electrode 24.1, the connection channel 24.2, and the conductive base electrode 24.3. 24.5 is a conductive base that supports the hollow electrode 24.1, the connection channel 24.2, the conductive base electrode 24.3, and the support base 24.4, and is electrically connected to the conductive base electrode 24.3 to complete the electroporation circuit. The conductive base 24.5 includes a fluid connection section 24.6 for flowing a heating or cooling fluid through the conductive base 24.5 to regulate the temperature of the electroporation process. In Figure 24B, 24.7 is an outer frame that supports the other components. 【0216】 In the devices of Figures 24A and 24B, as the fluid flows from the hollow electrode 24.1, the conductivity of the sample fluid interacts with the surface of the base electrode 24.3 to form a closed circuit. The base electrode 24.3 can be of any shape to allow for systematic and controllable exposure of cells to an electric field that induces electroporation. The position of the hollow electrode 24.1 can be manipulated within the Z coordinate from the support base 24.4 to limit the cell exposure to the electric field. In this configuration, the base electrode 24.3 is raised from the bottom of the support base 24.4 to a position beyond a specified volume collection limit. The electroporated cells will receive a finite electric field throughout the sample (except to close the electroporation circuit). This design reduces the shear effect on the sample cells and increases the uniformity of the flow in the region where electroporation occurs. In addition, a connection channel 24.2 is added to the end of the hollow electrode 24.1 to create a stable electric field or to further manipulate the electric field, allowing the operator to amplify and control the electrical pulses, and therefore the electric field, received by the specimen. In addition, the electrode configuration of this system uses a non-parallel electrode configuration in which the cannula is circular and parallel to the axis of the flow sample, but the surface of the base electrode 24.3 is at some angle greater than 0 degrees with respect to the axis of the cannula. A variation of this design is the use of a floating electrode that floats above the well plate. As the sample flows across the surface of the base electrode 24.3 and is electroperforated, the sample falls into the well. In this configuration, the electrode is not physically attached to the well plate. 【0217】 Example 7 - Fluid Chip-Based Electroporation Device Figures 25A and 25B show exemplary embodiments of a fluid tip-based electroporation device configured to accept industry-standard 1–5,000 μL conventional pipette tips for introducing samples into the device. In the device of Figure 25A, 25.1 and 25.2 are electrodes that are fluid-connected and electrically connected by the electroporation zone. 25.3 is a pipette tip insertion area fluid-connected to the electroporation zone, and 25.4 is a collection reservoir. Electrodes 25.1 and 25.2 of the fluid tip-based electroporation device are energized by an external power supply. In the exploded view of Figure 25B, 25.5 is a pipette tip, 25.6 is the fluid tip-based electroporation device of Figure 25A, and 25.7 shows a collection plate for holding seeds after electroporation. 【0218】 The pipette tip 25.5 floats above the surface of the fluid tip-based electroporation device 25.6. The fluid tip-based electroporation device includes two components: an electroporation plate containing encapsulated electrodes, and a cover plate having embedded microfluidic channels that allow the user to adjust the pulses of the electric field delivered to the cells. The electroporation plate allows multiple samples to flow through the electroporation simultaneously or individually, as needed. After electroporation of the specimen is performed within the electroporation plate, the sample flows towards the bottom of the collection plate 25.7. This system uses industry-standard liquid handling components, e.g., 1-5,000 μL pipette tips, and facilitates integration into industry-standard liquid handling manifolds. 【0219】 Example 8 - High-capacity (scalable) continuous-flow electroporation device Figures 26A and 26B show exemplary embodiments of a continuous flow electroporation device designed for use in large-volume cell production. In the embodiment shown in Figure 26A, 26.1 and 26.2 are inlets and outlets for circulating a fluid, such as a buffer solution, respectively. 26.3 is an outer housing that holds the electroporation device. 26.4 is the electroporation zone, which is fluid-connected to the fluid inlet 26.5 and the fluid outlet 26.9. Behind the inlet 26.5 and before the outlet 26.9 are cylindrical electrodes 26.7 and 26.8, having holes 26.6 on their surfaces. 26.10 is a reservoir for holding a fluid, such as a growth medium. 【0220】 In this embodiment, the cylindrical electrodes 26.7 and 26.8 are made of a conductive porous material, which allows fluid to move into the device cavity through the pores 26.6 of the conductive porous material. The pores 26.6 of the cylindrical electrodes 26.7 and 26.8 allow the buffer solution to stabilize chemical reactions on the surface of the cylindrical electrodes 26.7 and 26.8 and minimize pH transitions observed due to the application of potential during the electroporation process. The buffer introduced by the porous cylindrical electrodes 26.7 and 26.8 allows for changes in fluid flow to create a "lubricating" or sheath flow on the inner surface of the cylindrical electrodes 26.7 and 26.8, or to induce other hydrodynamic elements in the electroporation process when cells are electroporated (such as rotation of the suspension containing cells). The reduction in pH transitions mitigates the negative effects of high pH fluctuations of the suspension specimen used during electroporation. Cylindrical electrodes 26.7 and 26.8 satisfy external circuit requirements and allow the system to be energized using an external power supply. In an alternative embodiment, the outlet 26.2 of the electroporation device can be used to remove a highly conductive buffer, such as growth medium or PBS, and the inlet 26.1 can be used to introduce a low-conductivity buffer to minimize heating of the liquid sample as it flows through the electroporation zone 26.4. This buffer exchange will result in higher cell viability and higher transfection efficiency, ultimately leading to the generation of a larger number of successfully operated cells. The low-conductivity buffer can then be extracted in the outlet after the electroporation zone and replenished with growth medium when it comes into contact with the inlet after the electroporation zone. 【0221】 Example 9 - Modeling of electric field in a novel helical electrode Flowfect devices with specific electrode configurations that help improve the transformation / transfection efficiency of flow cells have been designed and computationally modeled. Figure 27A shows the helical nature of the electrode configuration, which is responsible for rotating the electric field as the battery flows through the electroporation region. Without being constrained by theory, this configuration allows for a lower electric field to be required to achieve an equivalent effect by enabling a larger portion of the cell surface to be electroporated. Figures 27B–27F show the cross-sectional area of ​​the electroporation region viewed from different axes. The energized and grounded electrodes are perpendicular to the flow direction, rather than parallel, as shown in Figures 1A–1C, for example. This design allows for desirable lower sample volume and reduced applied voltage in applications such as electroporation of primary human cells (e.g., immune cells or stem cells) where the number of cells is limited. In another embodiment, the helical electrodes are not in fluid contact with the electroporation zone, and the use of high-frequency pulses can induce an electric field inside the electroporation zone (e.g., through an intermediate medium) to deliver the composition into the cell. 【0222】 Example 10 - Two-part device of the present invention for creating scalability Figures 28A–28C illustrate embodiments of the device of the present invention, which are configured to be manufactured from two separate components that can be integrally fitted together to form a complete device that can be used with commercially available liquid handling systems. In this configuration, the insert-molded electrodes, shown as small dots near the joint of the two components in Figures 28A–28B, are then integrally welded by established industrial processes (e.g., spin welding, sonic welding, e.g., ultrasonic welding, thermal welding, e.g., hot plate welding, or laser welding). In this design, the fluid flow of a sample passing through the device, e.g., a cell-DNA sample, is isolated from the electric field exposure required for electroporation. 【0223】 Figures 29A and 29B show the device depicted in Figures 28A to 28C, which has identical internal dimensions, for example, having a distance of 4 mm between the upper and lower insert electrodes of an electroporation zone with a diameter of 700 μm. The difference between this embodiment of the device of the present invention and the embodiment shown in Figures 28A to 28C is that, in this concept, fluid flow control is coupled with electric field exposure. Specifically, a cannula (shown at the top of the device in Figures 29A to 29B) is the interface between the fluid handling system and the electroporation device of the present invention. When the electroporation device of the present invention is connected to the cannula, the implanted electrode (shown in red in the device in Figures 29A and 29B) is electrically connected to a power source for voltage pulse delivery. In the embodiments shown in Figures 29A to 29B, a single cannula is shown, but in the system of the present invention, it can be scaled up to include a system encompassing multiple electroporation devices of the present invention, for example, a system comprising 96 or 384 electroporation devices of the present invention configured to electroporate cells suspended in a fluid in parallel. 【0224】 Example 11 - Housing and Interface Examples Figures 30A and 30B provide exemplary embodiments of the device of the present invention, showing an outer housing that includes a user interface (Figure 30A) and a plurality of the devices of the present invention fluidly connected to a liquid dispensing manifold and sample plate (Figure 30B). 【0225】 Figure 30A shows an embodiment of a continuous flow transfection / transformation system. The 3D model shows a standalone electroporation system that includes a touchscreen user interface (30.1) or another alternative user interface(s)(if any) that allows the user to select parameters such as flow rate, waveform, applied potential, volume to be electroporated, time delay, cooling characteristics, heating characteristics, electroporation status, progress, and other parameters used to optimize the electroporation protocol. The interface also includes pre-defined parameter selections that allow the user to operate the system under standard conditions, such as those previously validated by the user or recommended by the manufacturer. The interface may be connected to programming that enables automated execution of the system and / or execution of algorithms that optimize the electroporation of a given sample of known cell types. The device also includes a cartridge (30.2) that encapsulates one or more electroporation devices from the aforementioned inventions or other electroporation devices used for continuous flow electroporation. The device also includes a cooling / heating area / enclosure (30.3) for cell / buffer storage during, before, and after electroporation of the specimen. The system is powered externally. The system also includes an algorithm that, if the user selects this functionality, has the ability to independently / autonomously adjust parameters. This allows for continuous adjustment of parameters used in the electroporation process, which may depend on cell type, conductivity, suspension volume, viscosity, electroporation cartridge lifespan, physical state of the suspension, or state of the electroporation device. 【0226】 Figure 30B shows an array of electroporation devices previously described herein. 30.4 is a liquid handling manifold that transports the present invention across a liquid handling platform and allows the device to aspirate fluid. 30.5 is the device shown in Figures 1A–1C. 30.6 is a well plate used to store samples before, during, and / or after specimen transfer. 【0227】 Example 12 - Flow cytometry gating strategy for optimizing electroporation parameters Figure 31 provides an example comparing two gating strategies. Historically, developers of electroporation technology have used regular “lymphocyte” pre-gating, which ignores cells that are not in the “lymphocyte” population, such as those whose morphology has been altered or who have undergone apoptosis. As shown in Figure 31, this artificially increases viability metrics by selecting a specific subpopulation of cells for analysis. “Whole cell” pre-gating is a more accurate depiction of experimental outcomes from electroporation. Thus, the reported viability shown in the table below may appear lower than expected in situ, but the data has been processed to focus on performance metrics that depict the impact of the electroporation device of the present invention on all input cells. In Figure 31, FSC represents forward scattering and SSC represents side scattering, illustrating how cell morphology data are collected during flow cytometry analysis. 【0228】 Using the gating strategies described herein, performance data for Jurkat cells, activated primary human T cells, THP-1 monocytes, primary human monocytes, and differentiated primary human macrophages are shown in Table 2 below. In Table 2, yield represents the ratio of the number of viable cells expressing the payload of interest to the number of cells entered into the process. For example, a yield of 0.5 means that half of the entered cells are viable and express the desired payload at the time of analysis. For reference, if the yield with viral delivery is greater than approximately 0.1 at collection, the cell therapy product is administered to the patient. [Table 2] 【0229】 Example 13 - Electroporation of Chinese hamster ovary (CHO-K1) cells and human embryonic kidney (HEK-293T) cells Electroporation was performed on CHO-K1 (Chinese hamster ovary cells) and HEK-293T (human fetal kidney cells) cell lines. The device of the present invention can be used for electroporation of adherent cells that have been lifted and resuspended in electroporation buffer. CHO-K1 (Figures 32A and 32B) cells and HEK-293T (Figures 33A-33D) cells can be successfully transfected with GFP plasmid DNA using the device of the present invention. Peak transfection efficiency of HEK-293T cells was observed after 48 hours of culture following electroporation. Without being constrained by theory, reduced cell viability may be attributable to lifting adherent cells and resuspending them for flow cytometry analysis, whereas microscopy showed healthy GFP+ cells with normal morphology (Figures 34A, 34B, 35A, and 35B). 【0230】 Example 14 - Transfection of primary T cells Studies have been conducted on primary T cells. Fluorescent reporters primarily used for analyzing electroporation efficiency include small fluorescent molecules (e.g., FITC-labeled dextran), genes expressed from plasmid DNA (e.g., GFP), and genes expressed from mRNA (e.g., GFP). Delivery and expression of these reporters are determined using flow cytometry, in which case live cells are pre-gated using a gating strategy as described herein to determine fluorescence detection on a single-cell basis. These assays demonstrate intercellular detection of fluorescent reporters and, in some cases, direct nuclear delivery. Due to the gentle nature of electroporation performed with the device of the present invention, higher cell numbers are achieved after transfection compared to commercial systems, such as the Lonza NUCLEOFECTOR 4D® system or the NEON® transfection system (Thermo Fisher, Carlsbad, CA). 【0231】 a. Demonstration of proliferated T cells Transfection (10) using the device of the present invention to deliver fluorescently labeled (FITC) dextran molecules (40 kDa) to primary human T cells. 6 Cell / experimental conditions were used to start the transfection process, and four metrics were analyzed for a commercially available benchtop electroporation device (e.g., Thermo Fisher NEON® transfection system): total cell count (post-EP), cell viability, transfection efficiency, and total number of live transfected cells. The results are shown in Figures 36A-36D. In addition to the data shown in Figures 36A-36D using fluorescently labeled molecules, the delivery of plasmid DNA encoding GFP (3.5kB) to primary human T cells (10 6 Cells (at the cell density of the experimental conditions) were tested using the device of the present invention. These experiments again demonstrated superiority over the NEON® transfection system, as shown in Figure 37 as the total number of GFP-expressing T cells after 24 hours of incubation. Importantly, GFP expression from DNA plasmids also demonstrates effective delivery of genetic information (i.e., nucleic acids) into the nucleus, where the DNA is transcribed to RNA before translation into the final GFP protein. 【0232】 b. mRNA delivery by platform comparison The delivery of mRNA to cells was also demonstrated using the device of the present invention. These experiments were performed at two operational cell densities using commercially available mRNA. The experiments were then completed with two commercially available systems (Lonza NUCLEOFECTOR 4D® and Thermo Fisher NEON® transfection systems) and the device of the present invention for comparison, as shown in Figures 38A–38D. The device of the present invention outperformed the commercially available systems in terms of viability, efficiency, and yield. In addition, the performance of the device of the present invention was independent of cell concentration, unlike the commercially available systems, as shown by the experimental results in Figures 38A–38D. 【0233】 Example 15 - Delivery of non-transient payload Each of the payloads described in Examples 13 and 14 is transient upon delivery. To demonstrate the stable delivery of the reagent for genomic modification (i.e., CRISPR gene knockout), experiments were performed using Cas9 ribonucleoprotein complexes (RNPs) for CRISPR knockout in primary cells. As shown in Figures 39A–39D, knockout of endogenous genes in primary T cells, as confirmed by single-cell-based surface receptor staining, was successfully achieved using the device of the present invention and confirmed by flow cytometry. The device of the present invention can also be used for simultaneous CRISPR integration of exogenous genes to demonstrate stable genomic integration of Cas9 RNPs via electroporation. 【0234】 Example 16 - Transfection of monocyte (THP-1) and natural killer (NK-92MI) cell lines Figures 40A and 40B show bar graphs comparing the delivery of GFP plasmid and FITC-labeled dextran to THP-1 cells and NK-92MI cells using the device of the present invention and the commercial NEON® transfection system. As seen in Figures 40A and 40B, electroporation using the device of the present invention is consistently more efficient than NEON® in producing viable transfected cells of any type with any payload. As an additional comparative example, Figures 41A and 41B show increased cell viability and transfection efficiency in a sample containing THP-1 monocytes, where GFP mRNA was delivered using the device of the present invention compared to the NEON® transfection system. 【0235】 The immortalized monocyte cell line THP-1 was further used in comparative studies with both monocytes and macrophages. Activation of THP-1 cells with LPS (lipopolysaccharide) endotoxin induces macrophage-like THP1-Mac immortalized cells. As shown in Figures 42A-42C and 43A and 43B, both THP-1 (Figures 42A-42C) cells and THP1-Mac (Figures 43A and 43B) cells were successfully transfected with GFP mRNA using the device of the present invention. 【0236】 Example 17 - Transfection of primary monocytes and differentiated macrophages Primary human monocytes, a cell type notoriously difficult to transfect using conventional methods, were successfully transfected using the device of the present invention. As shown in Figures 44A to 44D, primary human monocytes isolated from peripheral blood were successfully transfected with FITC-labeled dextran molecules and GFP mRNA using the device of the present invention. 【0237】 Figures 45A and 45B show the expression of specific markers in primary peripheral blood monocytes transfected with GFP mRNA using the device of the present invention. As shown in Figures 45A and 45B, CD86+ monocytes (gated onto viable GFP+ cells) maintained their ability to be activated (represented here as CD80 expression) after LPS stimulation for more than 96 hours. This indicates that electroporation does not adversely affect the expression of the activation marker CD80 (Figure 45A) or the lineage marker CD86 (Figure 45B). 【0238】 Furthermore, primary monocytes electroporated using the device of the present invention retained the ability to differentiate into macrophages, as shown in Figures 46A–46C, indicating that cells retain their function after electroporation. Differentiated human macrophages were successfully transfected with FITC-labeled dextran molecules (Figures 47A–47B) and GFP mRNA (Figures 47C–47D) using the device of the present invention, as shown in Figures 47A–47D. Macrophages electroporated using the device of the present invention (as shown in Figures 48A–48B), polarized to either the M1 or M2 phenotype, suggest that cellular health and function are retained after electroporation using the device of the present invention. Electroporated macrophages, polarized to either the M1 (Figure 48A) or M2 (Figure 48B) phenotype, retained GFP mRNA expression up to 72 hours after electroporation using the device of the present invention. 【0239】 The device of the present invention is more efficient than commercial transfection systems for electroporation of primary monocytes. As shown in Figures 49A–49C, delivery of FITC-labeled dextran into primary monocytes using the device of the present invention surpasses the performance of the NEON® transfection system for primary human cells, resulting in a significant increase in the total number of successfully transfected live output cells. 【0240】 Example 18 - Continuous flow device of the present invention: High-volume and high-cell-number cell production The device of the present invention can be used for electroporation of large volumes and high cell count suspensions in a truly continuous flow manner. Existing technologies such as the Lonza 4D-NUCLEOFECTOR® LV unit and the Maxcyte Scalable Transfection System (STX, VLX, or GT) rely on fluid flow to fill the sample into a NUCLEOCUVETTE® cartridge or processing assembly, respectively. However, during electrical pulse delivery, the cell and payload suspensions are stationary. Commercial electroporation systems process static or stationary cell suspensions, which is a key difference from the device of the present invention. The device of the present invention allows for a continuous flow of the cell and payload suspensions during exposure to an electric field. Specifically, rapidly flowing cells are exposed to an electric field sufficient to disrupt the cell membrane and internalize the gene payload of interest, but are immediately dispensed into the growth medium for cell recovery. Additionally, any heat generated during the electroporation process is dissipated due to convective heat transfer facilitated by the sample flowing directly into the recovery medium. This study provides a significantly more detailed account of the data obtained in terms of both cell type and electroporation scale. 【0241】 a. Initial demonstration in Jurkat cells The scalability of a continuous flow platform over a single device platform using the device of the present invention was demonstrated using a range of cell density and electroporation volume. These experiments demonstrate that the scalable platform of the present invention operates across a wide range of Jurkat cell densities, as shown in Figures 50A–50D. 【0242】 b. Comparability study between platforms of the present invention Follow-up experiments were conducted to compare the electroporation performance of the device of the present invention with that of the continuous flow electroporation platform of the present invention, using the same delivery conditions for both Jurkat cells and primary T cells. In these comparative experiments, 5 million cells were processed through the continuous flow platform, and the results for Jurkat cells and primary T cells were comparable to those of the single-channel device of the present invention, as shown in Figures 51A and 51B. 【0243】 Increasing scale of cT cell electroporation To test whether electroporation is dependent on cell density, the electroporation experiments described in Figures 51A and 51B were extended to cell suspensions containing up to 100 million primary T cells. In the first experiment, the number of T cells was increased at the same cell density, and the scale was increased from 5 million (as shown in Figure 51B) to 100 million (as shown in Figures 52A-52D) without loss of yield. The desired cell density was then evaluated, and the T cells were increased to a maximum of 100 × 10⁶ cells, as shown in Figures 53A-53D. 6 We demonstrated that cells / mL can be processed through the scalable platform of the present invention. Importantly, processing 100 million T cells was successful with 5 times lower mRNA levels compared to T cells processed at the lowest cell density, demonstrating potential material cost savings for payloads delivered at high cell densities. The total processing time for 100 million T cells in this experiment ranged from 2.4 to 24 seconds. 【0244】 d. Comparative study with the Lonza High-Capacity (LV) system We compared the scalable platform of the present invention with the Lonza 4D LV system using primary T cells with both FITC-dextran and EGFP mRNA payloads. The experiment was performed with 50 million T cells. At 24 hours, cell staining revealed that the morphology and phenotype of Lonza-treated cells were significantly different from those of untreated cells (shown in the flow cytometry plot in Figure 54). Additionally, a significant population of dead cells was observed in Lonza LV-treated cells. These outcomes did not occur in T cells electroporated with the continuous flow platform of the present invention, indicating that the continuous flow platform of the present invention maintained T cell morphology throughout the electroporation process. As shown in Figure 55, the total cell yield using the continuous flow platform of the present invention is higher than that of the Lonza 4D LV system, regardless of the delivered payload, e.g., FITC-labeled dextran or GFP mRNA. 【0245】 The continuous flow platform of the present invention demonstrates the successful electroporation of a payload into a suspension of very high density cells, for example, 1 billion cells. As shown in Figures 56A and 56B, 1 billion THP-1 cells (100 × 10¹³) in a volume of 10 mL. 6 Cells (at a concentration of cells / mL) were successfully transfected with 40 kDa FITC-labeled dextran molecules using the continuous flow platform of the present invention. Figure 57 shows the yield measured up to 72 hours after electroporation, expressed as the live FITC cell count for the experiments shown in Figures 56A and 56B. At this point, the number of FITC-positive cells was approximately 500 million, resulting from a count of 1 billion input cells, demonstrating the ability of the continuous flow platform of the present invention to deliver one out of two input cells as a modified cell product at 72 hours. 【0246】 Example 19 - Pulse waveform, DC voltage, high-voltage-low-voltage combinations, and combinations thereof The device of the present invention was tested with both pulsed and direct current (DC) power supplies, as shown in Figures 58A–58D. At the higher voltages tested, both power supplies showed similar delivery efficiencies for FITC-dextran in Jurkat cells. Additionally, initial electroporation combining high and low voltages was tested with the same system. As shown in Figures 59A–59D, we analyzed the use of modified waveforms for enhancing electroporation using the device of the present invention with combined high and low voltages, initially with FITC-dextran, for optimization of primary human T cell delivery. The experiments in Figures 59A–59D were repeated for delivery of a commercially available mRNA payload encoding the eGFP fluorescent reporter protein, as shown in Figures 60A–60D. 【0247】 Example 20 - Dynabead electroporation To demonstrate the compatibility of the device of the present invention with a specific T cell proliferation protocol, T cells grown with CD3 / CD28 Dynabeads were electroporated using the device of the present invention. Electroporation of Dynabead-grown samples was performed e...

Claims

[Claim 1] A method for introducing a composition into multiple cells suspended in a fluid, the method comprising the following steps (a), (b), and (c): Step (a) is a step of providing a device, and the device is (i) A first hollow cylindrical electrode including a first outlet, a first inlet, and a first lumen having a diameter, (ii) A second hollow cylindrical electrode including a second outlet, a second inlet, and a second lumen having a diameter, (iii) A cylindrical electroporation zone disposed between the first outlet and the second inlet and The electroporation zone has a minimum cross-sectional dimension greater than 500 μm, and the electroporation zone has a substantially uniform cross-sectional area. The first outlet, the electroporation zone, and the second inlet are in fluid communication, the cross-sectional dimensions of the electroporation zone are such that there are no contraction portions that come into contact with cells and deform the cell membrane, and the first hollow cylindrical electrode and / or the second hollow cylindrical electrode contain conductive carbon. Step (b) is a step of generating an electric field within the electroporation zone by applying a potential difference between the first hollow cylindrical electrode and the second hollow cylindrical electrode, The method is a step of increasing the permeability of the plurality of cells and the composition by passing the plurality of cells and the composition through the first hollow cylindrical electrode, the electroporation zone, and the second hollow cylindrical electrode, thereby introducing the composition into the plurality of cells. [Claim 2] The method according to claim 1, wherein the plurality of cells are in a separate liquid other than the composition prior to step (b), and / or step (b) comprises applying positive pressure by the fluid. [Claim 3] The method according to claim 1 or 2, wherein the diameter of the first lumen, the diameter of the second lumen, or the minimum cross-sectional dimension of the electroporation zone does not temporarily compress the cross-sectional dimension of any of the plurality of cells suspended in the liquid. [Claim 4] The method according to any one of claims 1 to 3, wherein the device further comprises a fluid delivery source in fluid communication with the first inlet, the flow rate of the plurality of cells in the liquid and / or suspension delivered from the fluid delivery source from the first lumen to the electroporation zone is 0.001 mL / min to 1,000 mL / min, the fluid delivery source is configured to deliver the plurality of cells in the liquid and / or suspension through the first lumen to the second outlet, and / or the residence time of the plurality of cells suspended in the liquid in the electroporation zone is 0.5 ms to 50 ms. [Claim 5] The method according to any one of claims 1 to 4, wherein the electric field is generated by a voltage pulse. [Claim 6] The method according to claim 5, wherein the voltage pulse energizes the first hollow cylindrical electrode with a specific applied voltage while the second hollow cylindrical electrode is energized with a specific applied voltage, and thus applies a potential difference between the first hollow cylindrical electrode and the second hollow cylindrical electrode. [Claim 7] The method according to claim 5 or 6, i) each of the voltage pulses has an amplitude of -3 kV to 3 kV, and / or ii) the voltage pulses have a duration of 0.01 ms to 1,000 ms, and / or iii) the voltage pulses are applied to the first hollow cylindrical electrode and the second hollow cylindrical electrode at a frequency of 1 Hz to 50,000 Hz, and / or iv) the voltage pulses include a waveform selected from the group consisting of DC, square, pulse, bipolar, sinusoidal, ramp, asymmetric bipolar, and superposition or combination thereof, and / or v) the electric field generated from the voltage pulses has a magnitude of 1 V / cm to 50,000 V / cm, and / or vi) the duty cycle of the voltage pulses is 0.001% to 100%. [Claim 8] The method according to any one of claims 1 to 7, wherein the liquid has an electrical conductivity of 0.001 mS / cm to 500 mS / cm, and / or the temperature of the plurality of cells suspended in the liquid is 0°C to 50°C, and / or the concentration of the composition in the liquid is 0.0001 μg / mL to 1000 μg / mL. [Claim 9] The method according to any one of claims 1 to 8, wherein the composition comprises at least one compound selected from the group consisting of therapeutic agents, vitamins, nanoparticles, charged molecules, uncharged molecules, DNA, RNA, CRISPR-Cas complexes, proteins, enzymes, peptides, viruses, polymers, ribonucleoproteins, polysaccharides, manipulated nucleases, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), homing nucleases, meganucleases (MN), megaTAL, and transposons. [Claim 10] The method according to any one of claims 1 to 9, wherein the plurality of cells suspended in the liquid are selected from the group consisting of eukaryotic cells, prokaryotic cells, plant cells, mammalian cells, animal cells, erythrocytes, human cells, primary cells, cell lines, cells in suspension, adherent cells, stem cells, blood cells, Chinese hamster ovary cells, immune cells, human embryonic kidney cells, unstimulated cells, stimulated cells, activated cells, induced pluripotent stem cells, primary human induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, and synthetic cells. [Claim 11] The method according to claim 10, wherein the immune cells include peripheral blood mononuclear cells, adaptive immune cells, innate immune cells, antigen-presenting cells, monocytes, T cells, B cells, dendritic cells, macrophages, neutrophils, natural killer cells, Jurkat cells, primary human T cells, THP-1 cells, primary human macrophages, primary human monocytes, primary human natural killer cells, unstimulated cells, activated cells, or stimulated cells. [Claim 12] The method according to any one of claims 1 to 11, further comprising storing the plurality of cells suspended in the liquid in a recovery buffer after perforation. [Claim 13] The method according to any one of claims 1 to 12, wherein the cross-section of the electroporation zone is a shape selected from the group consisting of circular, disc-shaped, elliptical, and curved shapes. [Claim 14] The electroporation zone has a minimum cross-sectional dimension of 0.5 mm to 5 mm and / or approximately 7850 μm. ２ ~Approx. 2000mm ２ The method according to any one of claims 1 to 13, wherein the cross-sectional area is and / or the electroporation zone has a length of 0.1 mm to 50 mm, and / or the lumen of either the first hollow cylindrical electrode and / or the second hollow cylindrical electrode has a minimum cross-sectional dimension of 0.01 mm to 500 mm. [Claim 15] The method according to any one of claims 1 to 14, wherein the ratio of the diameter of the lumen of either the first hollow cylindrical electrode or the second hollow cylindrical electrode to the minimum cross-sectional dimension of the electroporation zone is 1:10 to 10:1, and / or the ratio of the minimum cross-sectional dimension of the electroporation zone to the length of the electroporation zone is 1:100 to 100:1, and / or the ratio of the cross-sectional area of ​​the lumen of either the first hollow cylindrical electrode or the second hollow cylindrical electrode to the cross-sectional area of ​​the electroporation zone is 1:10 to 10:1.

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