Electroporation apparatus and method

Abstract

Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane. This allows chemicals, drugs, and / or macromolecules, such as proteins and nucleic acids, to be introduced into the cells. An embodiment relates to an electroporation device. The electroporation device includes: a plurality of chambers configured to store a plurality of cells during the electroporation process; a plurality of electrodes configured to generate a plurality of electric fields in the plurality of chambers during the electroporation process, each of the plurality of electric fields corresponding to one of the plurality of chambers; a flow channel configured to transport the plurality of cells during a cell collection process after the electroporation process; and a plurality of valves connecting the plurality of chambers to the flow channel.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 933,717, filed on November 11, 2019, under Section 119(e) of the U.S. Patent Act. This application also claims priority to U.S. Provisional Application No. 62 / 940,032, filed on November 25, 2019, under Section 119(e) of the U.S. Patent Act. U.S. Provisional Application No. 62 / 933,717 and U.S. Provisional Application No. 62 / 940,032 are incorporated herein by reference in their entirety. [Background technology]

[0002] Electroporation is a technique in which an electric field is applied to a cell to increase the permeability of the cell membrane. This allows drugs, chemicals, and / or macromolecules, such as proteins and nucleic acids (e.g., various forms of DNA and RNA), to be introduced into the cell. Electroporation may also be referred to as electrotransfer. [Overview of the Initiative] [Means for solving the problem]

[0003] Generally, in one embodiment, the embodiment relates to an electroporation apparatus. The electroporation apparatus includes a plurality of chambers configured to store a plurality of cells during an electroporation process, a plurality of electrodes configured to generate a plurality of electric fields in the plurality of chambers during the electroporation process, each of the multiple electric fields corresponding to one of the plurality of chambers, a flow channel configured to transport the plurality of cells during a cell collection process following the electroporation process, and a plurality of valves connecting the plurality of chambers to the flow channel.

[0004] Generally, in one aspect, an embodiment relates to a method. The method includes performing an electroporation process by generating a plurality of electric fields into a plurality of chambers using a plurality of electrodes, the plurality of chambers being configured to store a plurality of cells during the electroporation process. The method further includes performing a cell collection process by opening a plurality of valves connected to the plurality of chambers and transporting the plurality of cells to an outlet port using flow channels connected to the plurality of valves, the plurality of chambers, the plurality of electrodes, the plurality of valves, the outlet port, and the flow channels being positioned within an electroporation device.

[0005] Other aspects of the embodiments will become apparent from the following description and the appended claims.

Brief Description of the Drawings

[0006] [Figure 1] A perspective view of an electroporation device according to one or more embodiments. [Figure 2] A view showing a cross-section of an electroporation device according to one or more embodiments. [Figure 3] A top view of a chamber according to one or more embodiments. [Figure 4] A view showing a flowchart according to one or more embodiments. [Figure 5] A perspective view of a seal according to one or more embodiments. [Figure 6] A view showing a cross-section of a seal cap (also referred to as a chamber cap) according to one or more embodiments. [Figure 7] A side view of a single electroporation chamber according to one or more embodiments. [Figure 8] Another side view of a single electroporation chamber according to one or more embodiments. [Figure 9]A diagram showing a plurality of electroporation chambers in an electroporation device according to one or more embodiments. [Figure 10] A diagram showing an exemplary docking station according to one or more embodiments. [Figure 11] A cross-sectional view of a seal according to one or more embodiments. [Figure 12] A diagram showing a valve (i.e., a chamber valve) diagram according to one or more embodiments. [Figure 13] A front view of a lever portion of a chamber valve according to one or more embodiments. [Figure 14] A bottom view of an electroporation device according to one or more embodiments. [Figure 15] A cross-sectional view of an exemplary inlet pump and an exemplary outlet pump according to one or more embodiments. [Figure 16] An exploded view of an electroporation device according to one or more embodiments. [Figure 17] An assembly view of an electroporation device according to one or more embodiments. [Figure 18] A diagram showing an example of a single electroporation procedure according to one or more embodiments. [Figure 19] A diagram showing a flowchart for operating an electroporation docking station according to one or more embodiments.

Best Mode for Carrying Out the Invention

[0007] In the following detailed description of the embodiments, numerous specific details are set forth in order to provide a more thorough understanding of the disclosed technology. However, it will become apparent to those skilled in the art that the disclosed technology may be practiced without these specific details or may be practiced with equivalent alternatives in form and / or function.

[0008] Throughout this application, ordinal numbers (e.g., first, second, third, etc.) may be used as adjectives for elements (i.e., any noun in this application). The use of ordinal numbers is not intended to imply or generate a particular ordering of elements, nor to limit any element to being only a single element, unless explicitly disclosed by, for example, the use of terms such as “first,” “second,” “single,” and other such technical terms. Rather, the use of ordinal numbers is for distinguishing between elements. For example, the first element is different from the second element, and the first element can follow (or precede) the second element in the ordering of elements.

[0009] One or more embodiments relate to electroporation apparatus and methods for using / operating an electroporation apparatus. The electroporation apparatus enables the execution of large-scale electroporation processes.

[0010] Figure 1 shows an electroporation apparatus (100) according to one or more embodiments. The electroporation apparatus (100) may be referred to as a cartridge (or cassette). The electroporation apparatus can be sterile. The electroporation apparatus (100) may include a housing made of plastic (e.g., polycarbonate), glass, or other material suitable for biological and / or medical applications. As shown in Figure 1, the electroporation apparatus (100) has multiple components, including multiple openings (105), an inlet port (110), an outlet port (115), multiple electrodes (120), and multiple pump connectors (125). As further discussed and as shown in Figure 2, the electroporation apparatus (100) may additionally include pumps (e.g., diaphragm pumps; each pump equipped with two check valves (e.g., an inlet check valve and an outlet check valve that allows only unidirectional flow of the liquid)) to enable fluid movement throughout the electroporation apparatus (100). Each component is discussed below.

[0011] In one or more embodiments, multiple openings (105) are connected to chambers (further discussed below). Cells (with any accompanying suspension material) can be deposited into one or more of the chambers via the multiple openings (105). Chemicals, drugs, and / or macromolecules, such as proteins and nucleic acids (e.g., various forms of DNA and RNA), which will be introduced into the cells during electroporation, can also be deposited into the chambers via the multiple openings (105). Figure 1 shows eight openings (and thus eight chambers), but in other embodiments, a different number of openings (and thus a different number of chambers) may be present. For example, in some embodiments, the cartridge may have a number of chambers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc., in successive increments, depending on what may be required to increase the cell electroporation capacity (or batch electroporation capacity). In one or more embodiments, multiple chambers may share the same opening.

[0012] In one or more embodiments, each of the plurality of electrodes (120) is associated with one of the chambers. Furthermore, each of the plurality of electrodes (120) has an internal portion and an external portion. The internal portion is inside the chamber and is in contact with the contents stored inside the chamber (e.g., cells). The external portion is outside the chamber and is exposed on the surface of the electroporation apparatus (100) and / or protrudes from the surface of the electroporation apparatus (100). The internal portion and / or external portion may include an elliptical (e.g., circular) surface. Other shapes are also possible (e.g., a rectangle). Each of the electrodes (120) may include a base made of one metal or alloy and a coating made of the same or a different metal or alloy. For example, each of the plurality of electrodes (120) may include a base made of aluminum and a gold coating. Also, other metals (e.g., copper, silver, etc.) may be used instead of or in addition to aluminum and / or gold. Metals and / or alloys may be selected based on their chemical inertness, and therefore less likely to chemically react with the contents of the chamber (e.g., cells) or leach into the chamber.

[0013] In one or more embodiments, electrodes are positioned on both side surfaces of the electroporation apparatus (100). In other words, multiple electrodes (120) can be replicated on opposite surfaces. As a result, each chamber can be associated with a pair of electrodes (one electrode from each surface) on its opposing side walls. The electroporation process can be carried out by applying a voltage across the pair of electrodes, resulting in an electric field within the chamber associated with the pair of electrodes.

[0014] In one or more embodiments, the inlet port (110) and outlet port (115) are located at opposing ends of the electroporation apparatus (100). The inlet port (110) and outlet port (115) may be located on the same surface or different surfaces of the electroporation apparatus (100) (e.g., the top surface or the bottom surface). The inlet port (110) acts as an input for the liquid medium during the cell collection process. The liquid medium acquired at the inlet port (110) may be used, for example, to rinse the chamber after the electroporation process. In one or more embodiments, the inlet port (110) is configured to connect to a bag (or other container) for storing the liquid medium via a male Luer lock fitting (not shown). The outlet port (115) acts as a collection point during the cell collection process. After the electroporated cells (in the liquid medium) and the cell-free liquid medium (for rinsing the chamber) have been transported through the flow channel, the outlet port (115) retrieves the cells from the chamber. In one or more embodiments, the outlet port (115) is configured to connect to a bag (or other container) for storing the collected cells and the collected liquid medium via a male Luer lock fitting (not shown).

[0015] In one or more embodiments, a hydrodynamic device (e.g., a pump) is connected to a pump connector (Figure 1(125)) for use during the cell harvesting process to propel the movement of fluid within the electroporation apparatus (100) (e.g., the movement of cells after the electroporation process for harvesting). The hydrodynamic device and the cell harvesting process are discussed below.

[0016] Figure 2 shows a linear cross-sectional diagram of an electroporation apparatus (100) according to one or more embodiments. As shown in Figure 2, the electroporation apparatus (100) includes a plurality of chambers (205), a plurality of valves (210), a flow channel (215), a plurality of flanking flow channels (e.g., flanking flow channel A (220A), flanking flow channel B (220B)), a plurality of pumps (e.g., pump A (225A), pump B (225B)), and an air flow channel (230) with a vent (235). Pumps A 225A and B 225B may be referred to as the inlet pump and the outlet pump, respectively. Each of these components is discussed below.

[0017] In one or more embodiments, the chamber (205) is configured to store cells along with chemicals, drugs, and / or macromolecules, such as proteins and nucleic acids, which will be introduced into the cells during the electroporation process. The chamber (205) may be formed from the housing of the electroporation apparatus (100) and therefore may be formed from plastic (e.g., polycarbonate). In one or more embodiments, the lower portion of each chamber (205) is teardrop-shaped, as discussed below with respect to Figure 7. In other words, the wall of the lower portion of the chamber slopes inward toward the bottom of the chamber (i.e., the chamber narrows). This can help to drain the chamber (205) (discussed below). The chambers (205) can be designed to store any desired volume for each chamber (e.g., including at least 250 microliters (uL), 300uL, 350uL, 400uL, 450uL, 500uL, 600uL, 640uL, 700uL, 750uL, 800uL, 900uL, 1 milliliter (mL), 2mL, etc.). Different chambers (205) can be of different sizes, and different chambers (205) can store different volumes. In one or more embodiments, the chambers are designed to store a range of 300uL to 640uL for electroporation (volume of cells in a liquid suspension). In one or more embodiments, the chambers are designed to store a maximum volume of 600uL of cells in a liquid suspension for electroporation. In one or more embodiments, the chambers are designed to store a maximum volume of 640uL of cells in a liquid suspension for electroporation.

[0018] As discussed above, the electroporation apparatus may have eight chambers (120). These eight chambers may be combined to store at least 2 mL (e.g., 250 μL × 8 chambers), at least 2.4 mL (e.g., 300 μL × 8 chambers), at least 3.2 mL (e.g., 400 μL × 8 chambers), at least 4 mL (e.g., 500 μL × 8 chambers), at least 4.8 mL (e.g., 600 μL × 8 chambers), at least 5.6 mL (e.g., 700 μL × 8 chambers), or at least 6.4 mL (e.g., 800 μL × 8 chambers) of cells in a liquid suspension for electroporation.

[0019] In one or more embodiments, valves (210) connect chambers (205) to flow channels (215). (See, for example, Figure 2.) It is possible that there is one valve for each chamber. Alternatively, multiple chambers can share a single valve. Each of the valves (210) can correspond to an umbrella valve, pinch valve, piston valve, gate valve, spring valve, lever valve, etc. The valves (210) can be "off-the-shelf" (i.e., commercially available) valves of the above types. Preferably, the choice of valves can reduce the possibility of leakage, reduce the possibility of clogging, and increase the number of cells collected during the cell collection process (discussed below). The default position for valves (210) is closed. Multiple valves (210) can be opened simultaneously. Alternatively, the valves (210) can be opened sequentially, such as one at a time.

[0020] In certain embodiments, each chamber valve is a pinch valve that is leak-free up to at least 35 pounds per square inch (PSI) and leaks down to a negative pressure of at least (-)10 (minus 10) PSI.

[0021] Figure 12 shows diagrams of valve (1200) according to one or more embodiments with respect to both the open and closed positions of the valve. Valve (1200) can correspond to any of the valves (210) discussed above with reference to Figure 2. Valve (1200) can include a lever portion (1201) and a spring (1210). The lever portion (1201) can include a spring connector (1206) to which the spring (1210) is attached. The lever portion (1201) can also include a hinge (1203), a dome (1205), and a force portion (1207).

[0022] The valve (1200) is associated with one of the chambers (205). In one or more embodiments, when the valve (1200) is closed, the dome (1205) displaces and compresses a rubber layer between the outlet at the bottom of the chamber and the flow channel (215). This effectively seals the outlet at the bottom of the chamber, preventing the contents of the chamber from draining into the flow channel (215) and / or preventing the liquid in the flow channel (215) from rising into the chamber. In one or more embodiments, the rubber layer is a flexible portion of the flow channel (215). A spring (1210) maintains the valve (1200) in the closed position when not subjected to external force.

[0023] In one or more embodiments, a force is applied to the force portion (1207) of the lever portion (1201) to open the valve (1200). For example, the force may be applied by a valve actuator of a docking station (discussed below). In response to the force, the lever portion (1201) rotates around the hinge (1203). This movement of the lever portion (1201) also causes the dome (1205) to move, opening the outlet at the bottom of the chamber. Thus, when the outlet at the bottom of the chamber is open, the contents of the chamber can be drained into the flow channel (215), and / or the liquid in the flow channel (215) can rise into the chamber (for example, when subjected to a pumping force). When the force is removed from the force portion (1207), the spring (1210) causes the valve (1200) to return to the closed position. In other words, the spring (1210) causes the lever portion (1201) to rotate around the hinge (1203), which causes the dome (1205) to displace and compress the rubber layer, effectively sealing the outlet.

[0024] Figure 13 shows a front view of a lever portion (1201) according to one or more embodiments. As shown in Figure 13, the lever portion (1201) includes a hinge (1203), a dome (1205), and a spring connector (1206).

[0025] Figure 14 shows a bottom view of an electroporation device (100) according to one or more embodiments. In this bottom view, both the flow channel (215) and the chamber outlet of the chamber (205) (e.g., the chamber outlet (1405)) can be seen. When the valve (1200) is closed, the dome (1205) causes the chamber outlet (1405) to be sealed. As discussed above, this prevents the contents of the chamber from draining into the flow channel (215) and / or prevents the liquid in the flow channel (215) from rising into the chamber. When the valve (1200) is open, the dome (1205) no longer seals the chamber outlet (1405), and the contents of the chamber can drain into the flow channel (215). Similarly, if subjected to a pumping force or other force that can move the liquid (e.g., gravity (gravity flow), increased air pressure, etc.), the liquid in the flow channel (215) can rise into the chamber.

[0026] Figure 15 shows cross-sectional views of exemplary inlet pumps (225A) and exemplary outlet pumps (225B) according to one or more embodiments. The exemplary pumps (225A, 225B) in this figure are integrated inline with the flangen flow channels (220A, 220B). Each pump (225A, 225B) has a flexible (e.g., silicon) diaphragm (1508) adjacent to a fluid cavity which is juxtaposed with a “duckbill” check valve (1506) (for regulating unidirectional fluid flow), as shown herein. Each of the pumps (225A, 225B) is operated by repeatedly flattening the “dome” of the diaphragm (1508) (via a docking station actuator) to displace the fluid. In certain embodiments, each of the pumps (225A, 225B) has a normal operating flow of approximately 15 mL / min at 300 RPM (revolutions per minute) and a "fast flow" operation of approximately 30 mL / min at 600 RPM of the pump actuator. In certain embodiments, the pump flow is adjustable in 50 μL increments. In certain embodiments, each of the pumps (225A, 225B) can also act as a valve, being leak-free up to at least 35 pounds per square inch (PSI) and leak-free down to a negative pressure of at least (-)10 (minus 10) PSI.

[0027] Figure 15 also shows a male Luer lock fitting (1504) inserted into both the inlet port (110) and the outlet port (115). The male Luer lock fitting (1504) is covered by a Luer cap (1502).

[0028] Referring back to Figure 2, in one or more embodiments, the flanking flow channels (220A, 220B) connect the flow channel (215) to the inlet port (110) and the outlet port (115). Each of the channels (220A, 220B, 215) can be a tube, which is formed within a housing, or otherwise made of plastic (e.g., polycarbonate), glass, metal, etc. During the cell collection process, the contents of the chamber (210) (e.g., a liquid suspension of cells) can be drained into the flow channel (215) by opening the valve (210). The liquid culture medium (acquired at the inlet port (110)) can travel to the flow channel (215) via the flanking flow channel A (220A), and the drained contents (e.g., a liquid suspension of cells) can be pushed from the flow channel (215) to the outlet port (115) via the flanking flow channel B (220B). Furthermore, the liquid medium can enter the chamber through an open valve (i.e., the liquid medium enters the chamber through the flow channel (215)), and additional cells are collected (i.e., more cells are removed from the chamber) by rinsing the chamber before proceeding to the exit port (115) via the flanking flow channel B (220B). Thus, the flow channel (215) and at least one of the flanking flow channels (e.g., 220B) are configured to transport the electroporated cells during the cell collection process.

[0029] In one or more embodiments, one or more pumps (pump A(225A), pump B(225B)) are used to move the liquid culture medium, and thus to rinse the chamber (205) and push the cells toward the outlet port (115). As discussed above, pump A 225A and pump B 225B may be referred to as the inlet pump and the outlet pump, respectively. The number and volume of pump strokes required to rinse a given chamber and push the drained contents (i.e., liquid suspension of cells) toward the outlet port (115) depends, for example, on the distance of a given chamber from the inlet port (110).

[0030] In one or more embodiments, an air flow channel (230) connects airflow between multiple chambers (205) below a seal cap (500). The air flow channel (230) is connected to the outside of the electroporation device via a vent or filter (235) (e.g., a microbial air filter, e.g., a commercially available 0.2 micron filter) (e.g., to maintain atmospheric pressure). After the cells are deposited into the chambers (205), but before the electroporation process is performed, the opening (105) is closed (capped) by a seal (further discussed with respect to Figures 5 and 6) made of, for example, silicon (or other biocompatible material). This effectively creates a closed system. The external vent (235) or filter and air flow channel (230) reduce or eliminate the possibility of a partial vacuum forming (e.g., lower than atmospheric pressure inside the chamber), and thus assist the chamber in draining (into the flow channel (215)) while maintaining the sterile integrity of the chamber during the cell collection process. In one or more embodiments, pressurized air can be forced into the vent (235) and, consequently, into the air flow channel (230) to facilitate the draining of the chamber during the cell collection process (wherein such pressurized air is not large enough to lift or open the seal cap).

[0031] As discussed above, there is an electrode (120) associated with the chamber (205). As also discussed above, the internal portion of each electrode can have an elliptical (e.g., circular) surface. The elliptical surface of electrode (120) is shown in Figure 2. In one or more embodiments, the elliptical surface is circular with a diameter of 19.5 mm or approximately 19.5 mm. Other diameters and electrode shapes are also possible. In one or more embodiments, the elliptical (or circular) shape increases conductivity across the surface of the electrode.

[0032] Figure 3 shows a typical top view of a single chamber (305) according to one or more embodiments. Chamber (305) can correspond to any of the chambers (205) discussed above with reference to Figure 2. Chamber (305) has opposing edges (330A, 330B). As shown in Figure 3, chamber (305) is associated with a pair of electrodes (electrode A (320A), electrode B (320B)). The two electrodes (320A, 320B) can correspond to the electrodes (120) discussed above with reference to Figures 1 and 2. The pair of electrodes (320A, 320B) are positioned on both sides of chamber (305). In one or more embodiments, the inner surfaces of the electrodes (320A, 320B) form opposing side walls (340A, 340B) of chamber (305). In one or more other embodiments, the inner surfaces of each electrode (320A, 320B) are adjacent to an existing side wall of the chamber (305). As discussed above, during the electroporation process, a voltage is applied across the electrodes (320A, 320B) to generate an electric field within the chamber (305). Each electrode in the pair may be spaced apart from each other by a distance sufficient to reduce or eliminate arc discharge between the electrodes, but may also be spaced close enough to allow an electric field to be maintained between the electrodes. For example, the surface of electrode 320A in side wall 340A may be spaced approximately 4 millimeters (mm) apart from the surface of electrode 320B in side wall 340B. Other separation distances (e.g., approximately 1 mm, 3 mm, 5 mm, 7 mm, 10 mm, etc.) are also possible.

[0033] Figure 7 shows a side view of a single chamber (305) according to one or more embodiments, with electrodes not yet installed. In the embodiment of Figure 7, the chamber (305) has an inverted teardrop cross-section (i.e., the chamber is narrower at the bottom (or narrows towards the bottom)). This teardrop shape assists in draining the electroporated cells from the chamber (305) into the downward flow channel (215). While the embodiment of Figure 7 shows an inverted teardrop cross-section, those skilled in the art will recognize that other chamber shapes may also be used (including rounded, rectangular, triangular, diamond-shaped, tubular, etc.).

[0034] In one or more embodiments, a rim (702) surrounds the edge of the chamber (305). The rim (702) supports one of a pair of electrodes (320A, 320B). Similar rims are present on both sides of the chamber (305) to support the other of the pair of electrodes (320A, 320B). Figure 8 shows a side view of Figure 7 with electrode (320A) installed.

[0035] Returning to Figure 7, the edge surfaces (330A, 330B) of the chamber (305) (which may include the bottom surface) may be formed by the housing of the electroporation apparatus (100). The side walls of the chamber (305) (340A, 340B in Figure 3) may be formed when the electrodes (320A, 320B) are inserted into the rim (702).

[0036] Figure 9 shows a plurality of chambers (305) arranged adjacent to each other according to one or more embodiments. In one or more embodiments, the electrodes (320) are separated by a distance of 902 to eliminate interaction between adjacent electrodes (320).

[0037] As discussed above in relation to Figure 2, in one or more embodiments, the opening (105) at the top of the chamber (205) is sealed, and when the required materials (e.g., liquid suspension of cells and electroporation materials (e.g., nucleic acids)) are added to the chamber (205), it is possible to create a self-contained, biologically safe device. Figure 5 illustrates an exemplary seal 500 that may be used to seal multiple openings (105). Figure 5 includes multiple seal caps (502A-502H), each seal cap (502) corresponding to one of the openings (105). Figure 5 illustrates a plurality of seal caps (502) connected together via bridge portions (504A to 504G). A person skilled in the art who benefits from this detailed explanation will recognize that each individual seal cap (502) may be applied separately to each of the openings (105), or that smaller groups of seal caps (502) may be connected together to seal a subset of the openings (105).

[0038] Each seal cap (502) includes an upper portion (506) and a bottom lip (508). Figure 6 shows a cross-section of a seal cap (502) according to one or more embodiments. The upper portion (506) of the seal cap (502) is configured to seal the top of the corresponding chamber (205). The bottom lip (508) is configured to extend into the corresponding opening (105) of the corresponding chamber (205) to ensure a tight fit between the opening (105) and the seal cap (502). In one or more embodiments, the bottom lip (508) partially extends into the opening (105), but sufficient space is left between the bottom lip (508) and the corresponding chamber (205) to allow airflow between the chamber (205) and the airflow channel (230). Similarly, when the seal (500) is installed across multiple chambers (205), sufficient space is left beneath the bridge portion (504) to allow air to pass through the airflow channel (230). Since the airflow channel (230) is vented through an air filter or vent (235), the seal (500) creates a biologically closed, independent system that still allows the air pressure within the system to be maintained.

[0039] Figure 11 shows a cross-sectional view of a seal (500) inserted into the opening (105) of a corresponding chamber (205) according to one or more embodiments. As can be seen from Figure 11, the seal cap of the seal (500) fits into and interferes with the tapered chamber wall portion at the opening (105), thereby creating a seal. Double-headed arrows indicate the spacing between the internal electrode surfaces. A vent (or microbial air filter) is indicated by a circle to the left of the chamber cap (500).

[0040] Figure 16 shows an exploded view of an electroporation apparatus (100), which may also be referred to as a cartridge (as discussed above). In Figure 16, multiple electrodes (120), an inlet port (110), an output port (115), a male Luer lock fitting (1504), a Luer cap (1502), and a seal (500) are shown before the assembly of the electroporation apparatus (100). Figure 16 also shows pump housings A (1605A) and B (1605), which are configured to house the components of the inlet pump (225A) and the outlet pump (225B), respectively.

[0041] Figure 17 shows an assembly diagram of an electroporation apparatus (100), which may also be referred to as a cartridge (as discussed above). Figure 17 shows a plurality of electrodes 120, a pump housing A (1605A), a pump housing B (1605B), a plurality of chamber valves (210) each equipped with a spring (1210) and a lever (1201), a seal (500), a male Luer lock fitting (1504), and a Luer cap (1502).

[0042] Figure 4 shows a flowchart according to one or more embodiments of the present invention. The flowchart in Figure 4 shows the process for using / operating the electroporation apparatus (100) described above. In one or more embodiments, one or more of the steps shown in Figure 4 may be omitted, repeated, and / or performed in an order different from the order shown in Figure 4. Therefore, the scope of the present invention should not be considered to be limited to a specific arrangement of the steps shown in Figure 4.

[0043] In step 407, cells, as well as chemicals, drugs, and / or macromolecules to be introduced into the cells, such as proteins and nucleic acids, are loaded into the chamber (205) of the electroporation apparatus (100). This loading may be done, for example, through the opening (105). The opening (105) can then be closed using a seal (for example, a seal (500) and / or a seal cap (502)). The electroporation apparatus (100) has multiple chambers, but some chambers may not be used (i.e., liquid suspensions of cells may not be deposited into some chambers).

[0044] In step 409, the electroporation apparatus (100) is loaded into the docking station. Figure 10 illustrates an exemplary docking station (1000) according to one or more embodiments. The docking station (1000) includes a receiving section (1002), a valve actuator (1004), electrical contacts (1006), and a pump actuator (1008). The receiving section (1002) is sized and shaped to receive the electroporation apparatus (100) and to maintain the electroporation apparatus (100) in a safe, upright position. The valve actuator (1004) is configured to engage with a valve (210) on the electroporation apparatus (100). For example, if the valve (210) on the electroporation apparatus (100) is a spring-type valve, the valve actuator (1004) will include a component that applies force to the valve (210) to cause the valve (210) to open (e.g., presses the valve (210)). In one or more embodiments, each valve actuator (1004) has a one-to-one correspondence with the valve (210), so that each valve (210) can be controlled individually by the corresponding valve actuator (1004).

[0045] The electrical contact (1006) of the docking station (1000) engages with the electrode (120) of the electroporation device (100). As shown in Figure 10, the electrical contact (1006) may be linearly aligned along the length of the receiving portion (1002). Furthermore, the electrical contact (1006) is positioned on the opposing side of the receiving portion (1002). For the purpose of clarity, only one side (1002) is shown in Figure 10. The electrical contact (1006) can be, for example, a high-voltage contact. The pump actuator (1008) of the docking station (1000) engages with the fluid component of the electroporation apparatus (100), such as the pumps (pump A (225A), pump B (225B)). Each of the valve actuators (1004), electrical contacts (1006), and pump actuators (1008) may be controlled by one or more control boards or devices (not shown) that are operably linked to the docking station.

[0046] Returning to Figure 4, in step 409, as a result of loading the electroporation apparatus (100) into the docking station, each external portion of the electrode (120) is in contact with one or more electrical circuits of the docking station (e.g., the electrical contacts (1006) of the docking station (1000)). Thus, the electrode (120) becomes an element of one or more electrical circuits after loading the electroporation apparatus (100) into the docking station. Furthermore, as a result of loading the electroporation apparatus (100) into the docking station, one or more pumps (225A, 225B) and valves (210) or valve lever portions (1201) can be in operable contact with actuators of the docking station. A bag (or other container) containing the liquid culture medium may be attached to the inlet port (110), and a collection bag (or other container) may be attached to the outlet port (115) of the electroporation apparatus (100).

[0047] In step 412, an electric field may be generated in one or more of the chambers (205) using an electrode (120). For example, the docking station may generate an electric field by applying one or more voltage pulses to the electrode (120) using a circuit controlled by software (e.g., via a linked computer device) (e.g., via an electrical contact 1006, etc.). The electric field may be generated in all the chambers (205) simultaneously. Alternatively, the electric field may be generated one at a time for each chamber (205), or for a subset of the chambers (205) at a time. These applied electric fields increase the permeability of the cell membrane and thus allow chemicals, drugs, and / or macromolecules (e.g., proteins and nucleic acids, etc.) to be introduced into the cell.

[0048] In step 414, the valves (210) of the electroporation apparatus (100) are opened. For example, the valve actuator (1004) of the docking station (1000) can open the valves (210) of the docked electroporation apparatus (100). The docking station can open all the valves (210) simultaneously. Alternatively, the docking station can open one valve (210) at a time, or it can open a subset of the valves (210) at a time. Depending on the type of valve, the actuator may need to operate a piston, lever, spring, etc., to open the valve (210). In other words, the valves (210) can be operated using spring motion, lever motion, piston motion, etc. Opening one of the valves (210) causes the contents of the chamber connected to the valve to drain into the flow channel (215). Such drain discharge may result from one or more of the following: fluid pressure generated by the operation of one or more pumps, gravity (depending on the orientation of the valve (210) relative to the chamber (205)), pressure difference between the chamber (205) and the flow channel (215), increased air pressure, capillary effect, etc. In one or more embodiments, a vented air flow channel (230), located below the opening (105) and running between the chambers (205), can assist the drain discharge process by preventing the creation of a partial vacuum. In one embodiment, pressurized air can be forced into an air filter or vent (235) connecting the air flow channel (230) to the outside of the electroporation apparatus (100), thereby facilitating the drain discharge process.

[0049] In step 416, the liquid culture medium is pumped from the inlet port (110) into the chamber (205) of the electroporation apparatus (100), and the electroporated cells are collected at the outlet port (115). For example, the pump actuator (1008) of the docking station (1000) can operate one or more pumps (225A, 225B) to pump the liquid culture medium from a bag (or other container) attached to the inlet port (110) into the electroporation apparatus (100). Operating the pumps (225A, 225B) causes the liquid culture medium to travel through various channels (220A, 215, 220B) and also causes the liquid suspension of drained cells in the flow channel (215) to be transported toward the outlet port (115) into a collection bag (or other container) attached to the outlet port (115). Furthermore, operating the pumps (225A, 225B) forces the liquid medium to enter the chambers (205) from the flow channels (215) (through the open valves), and the liquid medium rinses the chambers (205) of any residual / lingering cells still in the chambers (205) before transporting the cells through the flow channels (215) toward the outlet port (115). The chambers (205) may be rinsed simultaneously. Alternatively, the chambers (205) may be rinsed one at a time, or a subset of the chambers (205) may be rinsed together. Moreover, each chamber may be rinsed immediately after it has been drained.

[0050] In one or more embodiments, step 412 corresponds to an electroporation process, while steps 414 and 416 correspond to a cell harvesting process performed after the electroporation process.

[0051] Figure 19 shows a flowchart of one or more embodiments. The flowchart in Figure 19 shows the process for using / operating the docking station (1000) described above with reference to Figure 10. In one or more embodiments, one or more of the steps shown in Figure 19 may be omitted, repeated, and / or performed in an order different from the order shown in Figure 19. Therefore, the scope of the invention should not be considered to be limited to a particular arrangement of the steps shown in Figure 19. The process shown in Figure 19 relates to the process shown in Figure 4 (discussed above).

[0052] In step 1907, the electroporation apparatus (100) (which, in one embodiment, is preloaded with cells in a liquid suspension) is fixed into the receiving section (1002) of the docking station (1000). The receiving section (1002) includes an opening for inserting the electroporation apparatus (100) and for fixing the electroporation apparatus (100) in an upright position. After the electroporation apparatus (100) is fixed into the receiving section, the electrical contacts (1006) of the docking station (1000) are brought into contact with the electrodes (120) of the electroporation apparatus (100). As discussed above, the electrical contacts (1006) are located on the opposing sides of the receiving section (1002).

[0053] Similarly, after fixing the electroporation apparatus (100), the valve actuator (1004) of the docking station (1000) can engage with the valve (210) of the electroporation apparatus (100), and the pump actuator (1008) of the docking station (1000) can engage with the pumps (225A, 225B) of the electroporation apparatus (100).

[0054] One or more chambers (205) of the electroporation apparatus (100) can be used to introduce cells, as well as chemicals, drugs, and / or macromolecules (e.g., proteins and nucleic acids) to be introduced into the cells (via a liquid suspension deposit) before the electroporation apparatus (100) is fixed into the receiving section (1002). Furthermore, the seal (500) can be positioned appropriately above the opening (105) of the electroporation apparatus (100) before the electroporation apparatus (100) is fixed into the receiving section (1002). Before or after the electroporation device (100) is fixed inside the receiving section (1002), a bag (or other container) containing a liquid culture medium may be attached to the inlet port (110) (via a male Luer lock fitting 1504), and a collection bag (or other container) may be attached to the outlet port (115) of the electroporation device (100) (via a male Luer lock fitting 1504).

[0055] In step 1909, the electroporation docking station (1000) generates an electric field between a pair of electrodes (120) in the chambers (205) of the electroporation apparatus (100) using electrical contacts (1006). The electrical contacts (1006) are elements in the circuit of the docking station (1000). The electric field can be generated by driving the electrical contacts (1006) with one or more signals using a pulse generator. The electric field can be generated in all chambers (205) simultaneously. Alternatively, the electric field can be generated one at a time for each chamber (205), or for a subset of chambers (205) at a time. These applied electric fields increase the permeability of the cell membrane, thus allowing chemicals, drugs, and / or macromolecules (e.g., proteins and nucleic acids) to be introduced into the cell.

[0056] In step 1912, the valve actuator (1004) of the docking station (1000) is operated to open the valves (210) of the docked electroporation apparatus (100). The docking station (1000) is capable of opening all the valves (210) simultaneously. Alternatively, the docking station (1000) is capable of opening one valve (210) at a time, or opening a subset of the valves (210) at a time. Depending on the type of valve, the actuator may need to operate a piston, lever, spring, etc., to open the valve (210). Opening one of the valves (210) causes the contents of the chamber connected to the valve to drain into the flow channel (215) of the electroporation apparatus (100).

[0057] In step 1914, the pump actuator (1008) of the docking station (1000) is operated to activate the pumps (225A, 225B). This may include repeatedly flattening the diaphragms (1508) of each pump (225A, 225B). As a result, the liquid culture medium is pumped from the bag (or other container) attached to the inlet port (110) into the electroporation apparatus (100). Specifically, operating the pump actuator (1008) causes the pumps (225A, 225B) to pump the liquid culture medium through the various channels (220A, 215, 220B), and also causes the liquid suspension of drained cells in the flow channel (215) to be transported toward the outlet port (115) into the collection bag (or other container) attached to the outlet port (115). Furthermore, operating the pumps (225A, 225B) forces the liquid medium to enter the chambers (205) from the flow channels (215) (through the open valves), and the liquid medium rinses the chambers (205) of any residual / lingering cells still in the chambers (205) before transporting the cells through the flow channels (215) toward the outlet port (115). The chambers (205) may be rinsed simultaneously. Alternatively, the chambers (205) may be rinsed one at a time, or a subset of the chambers (205) may be rinsed together. Moreover, each chamber may be rinsed immediately after it has been drained.

[0058] In one or more embodiments, step 1909 corresponds to an electroporation process, while steps 1912 and 1914 correspond to a cell harvesting process performed after the electroporation process.

[0059] In one or more embodiments, the electroporation apparatus (100) is sterilized. In one or more embodiments, the electroporation apparatus (100) is sterilized by exposure to gamma rays at a dose of 50 kilogray (kGy) or more. In one or more embodiments, the electroporation apparatus (100) is sterilized by exposure to gamma rays at a dose of 50 to 70 kilogray (kGy). In one or more embodiments, the electroporation apparatus (100) is fully functional following the sterilization procedure. In one or more embodiments, the electroporation apparatus (100) is fully functional following exposure to gamma rays at a dose of 50 to 70 kilogray (kGy).

[0060] In one or more embodiments, the electroporation apparatus (100) is disposable. In one or more other embodiments, the electroporation apparatus (100) may be reused. In other words, the process shown in Figure 4 and / or Figure 19 may be repeated multiple times with respect to a single electroporation apparatus.

[0061] Conventional electroporation systems require the use of multiple cuvettes to electroporate a large number of cells. Furthermore, even if a biological safety cabinet (BSC) can be used to provide sterile conditions in such a process (i.e., pipetting cells into multiple cuvettes), the nature of handling a very large number of cuvettes inevitably increases the possibility of introducing microbial contamination (i.e., loss of sterile conditions). In addition, the nature of handling such a large number of cuvettes not only increases handling / processing time but also introduces inevitable variability in conditions and / or process consistency.

[0062] As an important improvement over previous systems, the electroporation device (100) and the docking station (1000) are useful for electroporating a large number of cells in a single electroporation procedure (i.e., in a single electroporation "run").

[0063] In one or more embodiments, the electroporation device (100) and the docking station (1000) are configured to electroporate, in a single electroporation procedure (i.e., in a single "run"), for example, but not limited to, at least 1×10 8 cells, at least 2×10 8 cells, at least 3×10 8 cells, at least 4×10 8 cells, at least 5×10 8 cells, at least 6×10 8 cells, at least 7×10 8 cells, at least 8×10 8 cells, at least 9×10 8 cells, at least 1×10 9 cells, at least 2×10 9 cells, at least 3×10 9 cells, at least 4×10 9 cells, at least 5×10 9 cells, at least 6×10 9 cells, at least 7×10 9 cells, at least 8×10 9 cells, at least 9×10 9 cells, at least 1×10 10 cells, at least 2×10 10 cells, at least 3×10 10 cells, at least 4×10 10 cells, at least 5×10 10 cells, at least 6×10 10 cells, at least 7×10 10 cells, at least 8×10 10 cells, at least 9×10 10 cells, at least 1×1011 Cells, at least 2 × 10 11 Cells, at least 3 × 10 11 Cells, at least 4 × 10 11 Cells, at least 5 × 10 11 Cells, at least 6 × 10 11 Cells, at least 7 × 10 11 Cells, at least 8 × 10 11 Cells, at least 9 × 10 11 Cells, at least 1 × 10 12 Cells, at least 2 × 10 12 Cells, at least 3 × 10 12 Cells, at least 4 × 10 12 Cells, at least 5 × 10 12 Cells, at least 6 × 10 12 Cells, at least 7 × 10 12 Cells, at least 8 × 10 12 cells, and at least 9 × 10 12 It is useful for electroporating cells.

[0064] In one or more embodiments, the electroporation apparatus (100) and docking station (1000) are useful for electroporating any type of eukaryotic or prokaryotic cell (e.g., non-adherent cells, such as immune cells, NK cells, T cells, etc.).

[0065] Figure 18 shows an example of a "closed" (i.e., sterile or sterile) configuration of electroporation apparatus components used in a single electroporation procedure (electroporation "run"). A run may include one or more of the following steps: Under sterile conditions (e.g., in a biosafety cabinet), a container (e.g., an input culture bag (1805)) is connected to the inlet port (e.g., via a sterile inlet tubing (1815)), and another container (e.g., an output cell culture bag (1810) for cell collection following electroporation) is connected to the outlet port (e.g., via a sterile outlet tubing (1820)). Next, the electroporation apparatus or cartridge (100) (now a closed system) is placed inside a docking station or “nest” (1000), and the remainder of the electroporation process may be controlled by a computer (e.g., a laptop computer or tablet computer) operably linked to the “nest,” along with an electroporation pulse generator (for the delivery of electrical signals). One or more electric fields are generated between the electrode pairs in the chamber. The electroporated cells are collected into a collection container (e.g., an output cell culture bag (1810), etc.) through an outlet port (115) (e.g., via pumping cell culture medium through the electroporation apparatus or cartridge (100)). The collection container may be pre-filled with a predetermined volume of culture medium. After the completion of electroporation, the output cell culture bag (1810) may be aseptically removed from the electroporation apparatus / cartridge (for example, by using a tubing heat sealer to seal / close the connection between the output cell culture bag (1810) and the outlet port (115)) and placed in an incubator.

[0066] The entire electroporation process using one or more of the disclosed embodiments is carried out in substantially less time than that required for systems requiring the use of multiple individual cuvettes. Therefore, the examples of electroporation apparatus or cartridges (100) described herein can be used to automatically carry out electroporation in a closed manner, thereby enabling the effective and consistent delivery of transfected cells (e.g., transfected immune cells / T cells) in higher yields than other available systems. Thus, the electroporation apparatus (100) described herein provides the ability to electroporate large numbers of cells in a closed system and in a highly automated manner (therefore providing the ability to rapidly and efficiently produce large numbers of transfected cells in sterile and / or cGMP manufacturing environments).

[0067] The containers or bags used in the electroporation process (1805, 1810) may be, for example, cell culture bags made of fluorinated ethylene propylene (FEP) material, which remain impermeable to water while providing high permeability to oxygen and carbon dioxide for improved culture and growth.

[0068] The components of the electroporation apparatus or cartridge (100) may include gold-coated electrodes. Gold may be selected due to its biocompatibility and favorable electrical properties. The electroporation apparatus or cartridge (100) may be assembled in a controlled cleanroom environment. The electroporation apparatus or cartridge (100) may be cleaned and sterilized by gamma irradiation before distribution and / or use.

[0069] As described above, the electroporation cartridge described herein may be used in a system which also includes a computer (e.g., including a laptop computer or tablet), an electric pulse generator, and a docking station or “nest” (1000), enabling the electroporation cartridge process (e.g., applying an electric field to cells in an electroporation chamber, pumping the culture medium and cells through the cartridge (i.e., flow channels and chamber), opening and closing the cartridge valve (210)) to be fixed (e.g., held) and operated automatically. In this type of system, the computer (or laptop computer / tablet) acts as a user interface and is also operablely connected to control the electric pulse generator. The generator supplies electroporation pulses through contact with the cartridge electrodes and through connections in the nest. Thus, the docking station or “nest” (1000) holds the cartridge and provides both mechanical and electrical contacts with the cartridge.

[0070] An exemplary cartridge may include eight chambers and caps for covering and sealing the chambers after they have been filled with cell suspension material (e.g., cells, culture medium, nucleic acids, proteins, small molecules). The cartridge may have two fittings (e.g., Luer-type fittings) (1502, 1504) that allow an input culture medium bag (1805) and an output cell culture bag (1810) to be aseptically mounted in a biosafety cabinet. The input culture medium bag (1805) is filled with an appropriate amount of recovery medium and mounted on the input fitting on the cartridge by the user in the biosafety cabinet (before electroporation). The output cell culture bag (1810) is filled with a predetermined volume of recovery medium and can be mounted on the output on the cartridge by the user in the biosafety cabinet (likewise before electroporation).

[0071] Each chamber (205) is normally closed to prevent the sample from draining into the manifold channel before electroporation. These valves (210) can be opened when activated by the docking station or "nest" (1000). Diaphragm pumps (225A, 225B) are located just below the inlet port (110) and outlet port (115) and fittings (1504). A motor in the docking station or "nest" (1000) can pump fluid through a check valve incorporated into the pump stack (i.e., within the pump stack). Such a system configuration ensures that the culture fluid flows in only one direction, through the chambers and manifold to the output cell culture bag (1810). The diaphragm pumps (225A, 225B) can also act as valves when closed.

[0072] For electroporation, the user can aseptically transfer the cell / nucleic acid mixture into the cartridge chambers (205) and cap the cartridge while it is in the biosafety cabinet. The valve (210) inside the cartridge can remain closed until opened by the actuator inside the nest. Each chamber (205) can be electroporated and then drained (by opening the valve (210)) and the diaphragm pumps (225A, 225B) can be activated until the sample reaches the output cell culture bag (1810). This process can be repeated until all chambers (205) have been electroporated, drained, and pumped to the output cell culture bag (1810). After electroporation, the recovered cell culture medium from the input medium bag (1805) can be pumped through the cartridge to flush the chambers (205) and cartridge flow channels (215, 220A, 220B). Once the flash cycle is complete, the output cell culture bag (1810) can be aseptically removed from the cartridge by heat-sealing the sterile exit tubing (1820) and placed in the cell culture incubator.

[0073] In short, the electroporation apparatus (100) described herein represents a significant improvement for the large-scale electroporation of cells (e.g., immune cells / T cells) and for the production of genetically modified cell products. The electroporation cartridge provides the ability to electroporate large quantities of cells in a closed system and in a short period of time with minimal manual operation (i.e., in a mostly automated manner), thereby dramatically reducing the probability of microbial contamination and enhancing the consistency of cell products.

[0074] The embodiments and examples described herein are presented to best illustrate various embodiments and their specific uses, and to enable those skilled in the art to construct and use the embodiments. However, those skilled in the art will recognize that the foregoing descriptions and examples are presented for illustrative purposes only. Such descriptions are not intended to be exhaustive or to limit the precise forms disclosed.

[0075] While many embodiments have been described, those skilled in the art who benefit from this disclosure will understand that other embodiments not departing from the scope can be devised. Therefore, the scope of the present invention should be limited only to the appended claims. [Explanation of Symbols]

[0076] 100 Electroporation device 105 Opening 110 Entrance Port 115 Exit Port 120 electrodes 125 Pump Connector 205 Chamber 210 valves 215 Flow Channels 220A Flanking Flow Channel A 220B Flanking Flow Channel B 225A Pump A 225B Pump B 230 Air Flow Channels 235 Bent 305 Chamber 320A Electrode A 320B Electrode B 330A Edge surface 330B Edge surface 340A side wall section 340B Side wall section 500 stickers 502 Seal Cap 502A~502H Seal Cap 504A~504G Bridge section 506 Upper part 508 Bottom lip 702 Rim 902 distance 1000 docking stations 1002 Receptor part 1004 Valve Actuator 1006 Electrical contacts 1008 Pump Actuator 1200 valve 1201 Valve lever part 1203 Hinge 1205 Dome 1206 Spring Connector 1207 Force section 1210 Spring 1405 Chamber outlet 1502 Lure Cap 1504 Male Lure Lock Fitting 1506 Duckbill Check Valve 1508 Diaphragm 1605A Pump Housing A 1605B Pump Housing B 1805 Input culture medium bag 1810 Output Cell Culture Bag 1815 Sterilization Inlet Tubing 1820 Sterilized Outlet Tubing

Claims

1. (a) Two or more chambers configured to contain cells during an electroporation process, each chamber having an inlet and an outlet, (b) During the electroporation process, two or more electrodes configured to generate an electric field in each of the two or more chambers, (c) Two or more valves, each valve connected to the outlet of one of the two or more chambers, and one flow channel configured to connect the chambers and move cells during the post-electroporation collection process. (e) Inlet port and outlet port, (f) First and second flanking flow channels connecting the inlet port and the outlet port to the flow channel, respectively, (i) During the post-electroporation collection process, a pump and a device are configured to pump the liquid medium from the flow channel into the chamber, and the liquid medium enters the flow channel through the inlet port. including, Electroporation device.

2. The electroporation apparatus according to claim 1, further comprising a surface that defines an opening connected to the two or more chambers, and an air flow channel provided below the opening, wherein the air flow channel connects the two or more chambers.

3. The electroporation apparatus according to claim 2, further comprising a vent or air filter connecting the air flow channel to the outside of the electroporation apparatus.

4. The electroporation apparatus according to claim 2, further comprising a seal configured to cover the aforementioned opening.

5. The electroporation apparatus according to claim 1, wherein each of the two or more chambers has a shape that narrows toward the valve connected to the flow channel.

6. The electroporation apparatus according to claim 1, wherein each of the two or more chambers includes a pair of electrodes, and each electrode of the pair of electrodes is positioned on the opposite surface of each chamber.

7. Each electrode in each pair of electrodes is, An internal portion provided within the chamber, Including an external portion provided outside the chamber, Each pair of electrodes is configured to connect to an electrical circuit. The electroporation apparatus according to claim 6.

8. The electroporation apparatus according to claim 7, wherein the internal portion has an elliptical surface including a gold coating.

9. The electroporation apparatus according to claim 1, wherein each of the two or more chambers has a volume of at least 250 μL.

10. The electroporation apparatus according to claim 1, wherein each of the two or more chambers has a volume of at least 500 μL.

11. The electroporation apparatus according to claim 1, wherein the pump further includes a valve that allows only unidirectional flow of the liquid culture medium.

12. The electroporation apparatus according to claim 1, wherein each valve connecting the outlet of the chamber to the flow channel is a pinch valve or a pinch-type valve.

13. The electroporation apparatus according to claim 1, wherein the two or more chambers are combined to contain at least 2 mL of cells in a liquid suspension for electroporation.

14. An electroporation method comprising the step of generating an electric field in each of the two or more chambers of the electroporation apparatus according to any one of claims 1 to 13, wherein the chambers contain cells.

15. The method of claim 14, further comprising the step of opening the valves connecting the outlets of the two or more chambers to the flow channel, thereby allowing the cells to flow through the flow channel to the outlet ports.

16. The method according to claim 15, wherein the valve can be opened one at a time.

17. The method according to claim 14, wherein a liquid culture medium enters the flow channel through the inlet port, and the method further comprises the step of pumping the liquid culture medium from the flow channel into at least one of the two or more chambers using a pump.

18. The method according to claim 14, further comprising the step of performing the cell collection process, the step of draining the chamber into the flow channel, wherein the pressure inside the chamber is maintained by a vent or air filter connected to an air flow channel running between the chambers.

19. The method according to claim 14, wherein the cells are deposited into the two or more chambers through an opening connected to the chamber.

20. The method according to claim 14, further comprising the step of applying a seal to the opening.

21. The method according to claim 14, further comprising the step of inserting the device into a docking station so that the electrodes of the device are connected to an electrical circuit.

22. (a) A receiving part configured to fix any one of the electroporation devices from claims 1 to 13 in an upright position, (b) When the device is fixed in the receiving portion, an electrical contact is provided which is arranged to match the corresponding electrode of the electroporation device, (c) When the device is fixed in the receiving portion, a valve actuator is arranged to engage with the valve that connects the chamber of the electroporation device to the flow channel. An electroporation docking station, including one.

23. The electroporation docking station according to claim 22, further comprising a pump actuator arranged to engage with the pump of the electroporation apparatus when the apparatus is fixed in the receiving portion.

24. The electroporation docking station according to claim 23, further comprising an electrical circuit configured to generate an electric field using the aforementioned electrical contacts.

25. (a) A container containing a liquid culture medium, the container being connected to the inlet port of the electroporation device when the device is fixed in the receiving part, (b) When the device is fixed inside the receiving part, a container connected to the outlet port of the electroporation device and The electroporation docking station according to claim 22, further comprising:

26. The electroporation docking station according to claim 22, wherein the receiving portion includes an opening, and the electrical contacts are linearly aligned along the length of the receiving portion.

27. The electroporation docking station according to claim 22, wherein the electrical contacts are located on opposite sides of the receiving portion.

28. The electroporation docking station according to claim 22, wherein each valve actuator is configured to open a spring-type valve by applying force to the valve.

29. The electroporation docking station according to claim 28, wherein the valve actuator is configured to open the valves simultaneously.

30. The electroporation docking station according to claim 28, wherein the valve actuator is configured to open one valve at a time.

31. (a) The step of fixing one of the electroporation devices according to any one of claims 1 to 13 in an upright position within the receiving portion of the electroporation docking station (EDS), (b) A step of generating an electric field in the electroporation apparatus using the electrical contacts of the EDS, wherein the electrical contacts are aligned with the corresponding electrodes of the electroporation apparatus when the apparatus is fixed in the receiving portion. An electroporation method including

32. The method according to claim 31, further comprising the step of pumping a liquid culture medium through the valve of the electroporation apparatus connecting the chamber to the flow channel during the cell collection process, the pumping step being performed by operating a pump actuator of the EDS engaged with the pump of the electroporation apparatus when the apparatus is fixed in the receiving section.

Images