Electroporation method, useful substance production method, and electroporation apparatus

Abstract

The present invention provides an electroporation method, a useful substance production method, and an electroporation apparatus that are capable of properly applying an electric field to a suspension. Provided is electroporation method in which a sheath liquid and a suspension that contains a biologically derived substance and a bioactive substance are continuously delivered through a main flow passage between a pair of electrodes in such a manner that the suspension is enclosed by the sheath liquid, and in which an electric field is applied to the suspension via the pair of electrodes, thereby introducing the bioactive substance into the biologically derived substance. The electroporation method comprises an application step for applying an electric field to the suspension after the flow of the suspension and the sheath liquid stabilizes between the pair of electrodes.

Description

Electroporation method, useful substance production method, and electroporation device

[0001] The present invention relates to an electroporation method, a method for producing a useful substance, and an electroporation device in which a suspension containing a biologically derived substance and a bioactive substance is sandwiched between a pair of electrodes by a sheath liquid and continuously fed between the electrodes, and in particular to an electroporation method, a method for producing a useful substance, and an electroporation device in which an electric field is applied to the suspension after the flow of the suspension and sheath liquid between the pair of electrodes has stabilized.

[0002] Electroporation, also known as electroporation, is a technique for introducing substances into cells by creating holes in the cell membrane using an electric pulse. For example, applying an electric field to a cell suspension using an electrode pair creates minute holes in the cell membrane, allowing DNA (deoxyribonucleic acid) to be introduced into the cells, thereby enabling cell transformation. Hereinafter, electroporation may be abbreviated as EP.

[0003] Batch electroporation is widely used as an EP method for introducing bioactive substances such as DNA, RNA (ribonucleic acid), and proteins into biologically derived materials such as cells, cell derivatives, organelles, intracellular granules, and vesicles. In batch electroporation, for example, a suspension containing a biologically derived material and a bioactive substance is placed in a container with an electrode pair installed on the inner surface, and an electric field is applied by the electrode pair. This creates micropores in the membrane covering the surface of the biologically derived material, increasing the membrane's permeability. Furthermore, the bioactive substance is introduced into the biologically derived material by passing through the membrane, whose permeability has been increased by diffusion or electrophoresis.

[0004] On the other hand, as shown in Patent Documents 1 and 2, for example, flow-type electroporation has also been developed in which a suspension containing a biologically derived substance and a bioactive substance is circulated through a flow path in which an electrode pair is installed.

[0005] Japanese Patent Application Laid-Open No. 2007-7430 U.S. Patent No. 1,1225,638

[0006] In Patent Documents 1 and 2, when performing electroporation on a suspension, an electric pulse is applied to two opposing electrodes to achieve a predetermined electric field strength. In flow-type electroporation, in which EP is performed while feeding a suspension, if the flow of the suspension is not stable, the introduction efficiency may decrease, resulting in low productivity. The object of the present invention is to provide an electroporation method, a method for producing a useful substance, and an electroporation device that can perform flow-type electroporation with high productivity.

[0007] The above-mentioned object can be achieved by the following configuration. Invention [1] is an EP method in which a suspension containing a biological substance and a bioactive substance and a sheath fluid are continuously fed into a main flow path between a pair of electrodes so that the suspension is sandwiched between the sheath fluid, and an electric field is applied to the suspension by the pair of electrodes, thereby introducing the bioactive substance into the biological substance. The EP method includes an application step of applying an electric field to the suspension after the flow of the suspension and the sheath fluid between the pair of electrodes has stabilized. Invention [2] is an EP method according to invention [1], in which, when feeding the sheath fluid into the main flow path so as to sandwich the suspension, a first tube is used to supply the suspension and a second tube is used to supply the sheath fluid, a standby position for the front of the suspension is predetermined in the first tube, a standby position for the front of the sheath fluid is predetermined in the second tube, and feeding of the suspension and sheath fluid begins after the fronts of the suspension and sheath fluid have all reached their standby positions.

[0008] Invention [3] is the EP method according to Invention [1] or [2], which includes a determination step of determining stability of the flows of the suspension and sheath fluid between the electrode pair before the application step, in which the determination step measures the electrical resistance of the flows of the suspension and sheath fluid between the electrode pair and determines the stability of the flows based on the electrical resistance, or the determination step detects the stability of the flow rate of the suspension in the first tube and the flow rate of the sheath fluid in the second tube to determine the stability of the flows. Invention [4] is the EP method according to Invention [3], in which the determination step measures the electrical resistance of the flows of the suspension and sheath fluid between the electrode pair on the upstream or downstream side of the main flow path of the electrode pair and determines the stability of the flows based on the electrical resistance.

[0009] Invention [5] is the EP method according to any one of Inventions [1] to [4], in which the suspension and the sheath fluid are delivered simultaneously. Invention [6] is the EP method according to any one of Inventions [1] to [4], in which the suspension and the sheath fluid are delivered prior to the delivery of the sheath fluid.

[0010] Invention [7] is an EP method in which a suspension containing a biological substance and a bioactive substance and a sheath fluid are continuously fed into a main flow path between a pair of electrodes so that the suspension is sandwiched between the sheath fluid, and an electric field is applied to the suspension by the pair of electrodes, thereby introducing the bioactive substance into the biological substance. When feeding the sheath fluid into the main flow path so that the suspension is sandwiched between the first and second tubes, a first tube is used to supply the suspension and a second tube is used to supply the sheath fluid, a standby position for the front of the suspension is predetermined in the first tube, and a standby position for the front of the sheath fluid is predetermined in the second tube, the feeding of the suspension and the sheath fluid is started after it is detected that the fronts of the suspension and the sheath fluid have all reached the standby positions, and after the feeding of the suspension and the sheath fluid, an electric field is applied to the suspension after the flows of the suspension and the sheath fluid between the pair of electrodes have stabilized. Invention [8] is the EP method according to any one of Inventions [1] to [7], in which, in the application step, an electric field is applied to the suspension after a preset time has elapsed since the start of the transfer of the suspension and sheath liquid. Invention [9] is the EP method according to Invention [7], in which the position of the front of the suspension in the first tube is detected by an ultrasonic sensor. Invention

[10] is a method for producing a useful substance, including the EP method according to any one of Inventions [1] to [9].

[0011] Invention

[11] is an EP device that introduces the bioactive substance into the biologically derived substance by continuously feeding a suspension containing the biologically derived substance and the bioactive substance and applying an electric field to the suspension. The EP device has a main flow path, an electrode pair having electrodes arranged opposite each other and applying an electric field to the suspension flowing through the main flow path, a suspension supply port that supplies the suspension to the main flow path, a sheath fluid supply port that is provided in the main flow path downstream of the suspension supply port and supplies sheath fluid to the main flow path, a pump that feeds the suspension and sheath fluid, and a power supply unit that applies an electric field to the suspension after the flows of the suspension and sheath fluid between the electrode pair have stabilized. Invention

[12] is the EP device according to invention

[11] , which has a sheath fluid flow path that is connected to the sheath fluid supply port and that merges the sheath flow with the suspension in the main flow path, a first tube that is connected to the suspension supply port and that supplies the suspension to the main flow path, and a second tube that is connected to the sheath fluid supply port and that supplies the sheath fluid into the main flow path, wherein a standby position for the head of the suspension is predetermined in the first tube, and a standby position for the head of the sheath fluid is predetermined in the second tube, and further comprises a control unit that starts the delivery of the suspension and the sheath fluid by a pump after all of the heads of the suspension and the sheath fluid have reached their standby positions. Invention

[13] is the EP device according to Invention

[12] , which has a determination unit that determines the stability of the flows of the suspension and sheath fluid between the electrode pair, and the determination unit measures the electrical resistance of the flows of the suspension and sheath fluid between the electrode pair and determines the stability of the flows based on the electrical resistance, or the determination unit detects the stability of the flow rate of the suspension in the first tube and the flow rate of the sheath fluid in the second tube and determines the stability of the flows. Invention

[14] is the EP device according to Invention

[13] , which has an auxiliary electrode provided upstream or downstream in the main flow path of the electrode pair, and the determination unit measures the electrical resistance in the thickness direction of the flows of the suspension and sheath fluid using the auxiliary electrode and determines the stability of the flows based on the electrical resistance.

[0012] Invention

[15] is an EP device according to any one of Inventions

[12] to

[14] , which has a sensor that detects the position of the head of the suspension in the first tube and the position of the head of the sheath fluid in the second tube, and the control unit controls the delivery of the suspension and sheath fluid by the pump based on the positions of the head of the suspension in the first tube and the head of the sheath fluid in the second tube detected by the sensor.

[0013] Invention

[16] is an EP device that introduces the bioactive substance into the biologically derived substance by continuously feeding a suspension containing a biologically derived substance and a biologically active substance and applying an electric field to the suspension, the EP device comprising: a main flow path; an electrode pair having electrodes arranged opposite to each other and applying an electric field to the suspension flowing through the main flow path; a power supply unit that applies an electric field to the suspension; a suspension supply port that supplies the suspension to the main flow path; a sheath liquid supply port that is provided in the main flow path downstream of the suspension supply port and supplies sheath liquid to the main flow path; a pump that feeds the suspension and sheath liquid; a first tube that is connected to the suspension supply port and that supplies the suspension to the main flow path; and a second tube that is connected to the sheath liquid supply port and that supplies sheath liquid into the main flow path. and a control unit that controls the pump to feed the suspension and sheath fluid, wherein a standby position for the suspension's lead in the first tube is predetermined, and a standby position for the lead of the sheath fluid is predetermined in the second tube, and the control unit starts the pump to feed the suspension and sheath fluid after the sensor detects that the entire lead of the suspension and sheath fluid has reached the standby position, and after the suspension and sheath fluid have been fed, the power supply unit applies an electric field to the suspension after the flow of the suspension and sheath fluid between the electrode pair has stabilized. Invention

[17] is an EP device according to any one of inventions

[12] to

[16] , wherein the control unit starts the pump to feed the suspension and sheath fluid, and after a preset time has elapsed, the power supply unit applies an electric field to the suspension. Invention

[18] is the EP device according to invention

[15] or

[16] , wherein the sensor that detects the position of the front of the suspension in the first tube is an ultrasonic sensor.

[0014] According to the present invention, it is possible to provide an electroporation method capable of performing flow-type electroporation with high productivity, a method for producing a useful substance, and an electroporation apparatus.

[0015] FIG. 1 is a schematic diagram showing an example of an electroporation system having an electroporation apparatus according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view showing a first example of a flow channel device of an electroporation apparatus according to an embodiment of the present invention. FIG. 3 is a schematic cross-sectional view for explaining the operation of the flow channel device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 4 is a schematic cross-sectional view showing the stability of the flow of suspension and sheath fluid between the electrode pairs of the flow channel device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 5 is a schematic cross-sectional view showing an example of the instability of the flow of suspension and sheath fluid between the electrode pairs of the flow channel device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 6 is a schematic cross-sectional view showing another example of the instability of the flow of suspension and sheath fluid between the electrode pairs of the flow channel device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 7 is a graph showing the stability of the flow of suspension and sheath fluid between the electrode pairs of the flow channel device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 8 is a flowchart showing a first example of an electroporation method according to an embodiment of the present invention. FIG. 9 is a schematic cross-sectional view showing a second example of the flow channel device of an electroporation apparatus according to an embodiment of the present invention.

[0016] The electroporation method, useful substance manufacturing method, and electroporation apparatus of the present invention will be described in detail below based on the preferred embodiments shown in the accompanying drawings. Note that the drawings described below are illustrative for explaining the present invention, and are simplified for explaining the present invention, and the present invention is not limited to the drawings shown below. In the following, the symbol "to" indicating a numerical range includes the numerical values ​​written on both sides. For example, when ε is a numerical value ε α ~Number ε β That is, the range of ε is the number ε α and the number ε β The range includes ε α ≦ε≦ε βFurthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for specific angles, parallel and perpendicular directions, and vertical directions. Furthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for length, width, thickness, etc. Furthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for temperature, flow rate, electrical conductivity, electric conductivity, volume fraction, etc.

[0017] (First Example of Electroporation Apparatus) FIG. 1 is a schematic diagram showing an example of an electroporation system having an electroporation apparatus (EP apparatus) according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view showing a first example of a flow path device of an electroporation apparatus according to an embodiment of the present invention. FIG. 3 is a schematic cross-sectional view for explaining the operation of the flow path device of the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view taken along line A-A in FIG. 1. The EP apparatus 10 shown in FIGS. 1 and 2 is an apparatus that performs electroporation by continuously feeding a suspension Q (see FIG. 3) containing a biologically derived substance and a bioactive substance and a sheath liquid s (see FIG. 3) between an electrode pair 34 having opposing electrodes into a main flow path 30, with the suspension Q sandwiched between the sheath liquid s, and applying an electric field to the suspension Q by the electrode pair 34, thereby introducing the bioactive substance into the biologically derived substance. As shown in Figure 3, the electroporation device 10 is a flow-type EP device that uses a suspension Q and a sheath liquid s, and performs EP by continuously feeding the suspension Q between a pair of electrodes 34 with the suspension Q sandwiched between the sheath liquid s. Performing EP by continuously feeding the suspension Q and the sheath liquid s between a pair of electrodes 34 with the suspension Q sandwiched between the sheath liquid s is also called continuous electroporation. The suspension Q is fed so as to be sandwiched between the sheath liquid s, and there are two flows of sheath liquid s, i.e., two sheath liquid flows. A three-layered flow is formed: a sheath liquid flow, a suspension flow, and a sheath liquid flow.

[0018] As shown in FIGS. 1 and 2 , the EP device 10 includes a flow path device 11, pumps 12a and 12b that supply suspension Q to the flow path device 11, a power supply unit 14 that performs EP, a control unit 16, and a determination unit 18. The EP device 10 also includes pumps 20a and 20b that supply sheath fluid s to the flow path device 11, and a tank 22 that stores the sheath fluid s. The pump 12a of the EP device 10 is connected, for example, to a culture device 24, which serves as a suspension supply unit, via piping 15. The pump 12a is provided between the mixer 25 and the culture device 24. The culture device 24 does not constitute the EP device 10. The tank 23 and the mixer 25 that store the bioactive substance also do not constitute the EP device 10. While the culture device 24 is shown in FIG. 1 as the suspension supply unit, a tank that stores the suspension removed from the culture device 24 may also be used. The flow path device 11 is also connected by a third tube 28 to a waste tank 26 and a container 27 that store liquids such as the suspension Q and sheath fluid s discharged from the flow path device 11 to the outside. A valve 29 is provided in the third tube 28. The third tube 28 constitutes a flow path for liquids such as the suspension Q and sheath fluid s discharged from the flow path device 11 to the outside. The valve 29 switches the liquid destination between the waste tank 26 and the container 27. The liquid discharged from the flow path device 11 to the outside passes through the third tube 28 and is sent to the waste tank 26 or the container 27 by the valve 29. The waste tank 26, the container 27, and the valve 29 do not constitute the EP apparatus 10. The EP apparatus 10, including the culture apparatus 24, the tank 23 and mixer 25 described below, as well as the waste tank 26, the container 27, and the valve 29, is also referred to as an electroporation system 40.

[0019] 1 and 2, the flow channel device 11 has a main flow channel 30. The main flow channel 30 is a linear flow channel. The main flow channel 30 extends in an extending direction D ＬThe cross-sectional shape of the main channel 30 in a direction perpendicular to the axis of the flow channel 30 is, for example, a rectangle with all interior angles of 90°. The cross-sectional shape of the main channel 30 is not limited to a rectangle with all interior angles of 90°. As shown in FIG. 2 , an inlet 31 for supplying the suspension Q to the main channel 30 is provided at one end 30a of the main channel 30, for example, on the surface 11a of the flow channel device 11. An outlet 32 ​​for discharging liquids such as the suspension Q and the sheath fluid s to the outside is provided at the other end 30b of the main channel 30, for example, on the back surface 11b of the flow channel device 11. In the main channel 30, the inlet 31 side is the upstream side, and the outlet 32 ​​side is the downstream side. Furthermore, as shown in FIG. 2 , a first inlet 31a for supplying the sheath fluid s to the main channel 30 is provided at one end 30a of the main channel 30, for example, on the surface 11a of the flow channel device 11. A second inlet 31b for supplying the sheath fluid s to the main channel 30 is provided, for example, on the rear surface 11b of the channel device 11. For example, the first inlet 31a and the second inlet 31b are arranged in the extending direction D Ｌ The positions in are the same.

[0020] A first tube 13 is connected to the inlet 31. A pump 12a is connected to the inlet 31 via the first tube 13 connected to the inlet 31 and a pipe 15. The first tube 13 is used to supply the suspension Q when feeding the sheath fluid s to the main channel 30 so as to sandwich the suspension Q. The configurations of the first tube 13 and the pump 12a are not particularly limited, and those known in the technical field can be used as appropriate depending on the viscosity of the suspension Q, the amount of suspension Q to be fed, etc. The first tube 13 may be flexible, such as a silicone tube.

[0021] A second tube 21a is connected to the first inlet 31a. A pump 20a is connected to the first inlet 31a via the second tube 21a connected to the first inlet 31a. A tank 22 is connected to the pump 20a via piping 21c. A second tube 21b is connected to the second inlet 31b. A pump 20b is connected to the second inlet 31b via the second tube 21b connected to the second inlet 31b. The tank 22 is connected to the pump 20b via piping 21d. Since there are the first inlet 31a and the second inlet 31b for supplying the sheath fluid s, there are two second tubes 21a, 21b. The two second tubes 21a, 21b are used to supply the sheath fluid s when sending the sheath fluid s to the main channel 30 so as to sandwich the suspension Q therebetween. The configurations of the second tubes 21 a, 21 b and the pumps 20 a, 33 b are not particularly limited, and any known configurations in the art may be used as appropriate depending on the viscosity of the sheath fluid s, the amount of sheath fluid s to be delivered, etc. The second tubes 21 a, 21 b may be flexible, such as a silicone tube.

[0022] Although the configuration in which the pumps 20a and 20b are provided is described above, the present invention is not limited thereto, and a configuration in which one pump is provided for the first inlet 31a and the second inlet 31b may also be used. That is, the sheath fluid s may be supplied to the flow path device 11 by one pump. The tank 22 stores the sheath fluid s. The configuration of the tank 22 is not particularly limited as long as it can store the sheath fluid s. As described above, the configuration in which the inlet 31 is provided on the front surface 11a of the flow path device 11 and the outlet 32 ​​is provided on the back surface 11b of the flow path device 11 is described above, but the present invention is not limited thereto. For example, a configuration in which one of the inlet 31 and the outlet 32 ​​is provided on the front surface 11a of the flow path device 11 and the other is provided on the back surface 11b may also be used.

[0023] The flow path device 11 shown in FIG. 2 is configured such that, for example, the extending direction D of the linear main flow path 30 is parallel to the horizontal plane Hp. Ｌ are arranged so as to be perpendicular to the extending direction D of the main flow path 30. ＬThe width direction of the main flow channel 30 is parallel to the direction x perpendicular to the vertical direction z, and the extension direction D of the main flow channel 30 is parallel to the direction x perpendicular to the vertical direction z. Ｌ With respect to the horizontal plane Hp, for example, the extending direction D of the linear main flow path 30 Ｌ is perpendicular to the horizontal plane Hp. Ｌ The angle is 90°, but ±10° is allowed for 90°. Therefore, the extension direction D of the main flow path 30 with respect to the horizontal plane Hp is Ｌ When the angle is 90°, the extension direction D of the main flow path 30 Ｌ is parallel to the vertical direction z, but is parallel to the extension direction D of the main flow path 30 Ｌ The extension direction D of the main flow path 30 is allowed to be inclined by ±10° with respect to the vertical direction z. Ｌ is the direction Df in which the suspension Q flows within the main flow channel 30. In the electroporation device 10, the liquid transfer direction Dd of the suspension Q between the electrode pair is a vertical direction from bottom to top. The suspension Q flows within the main flow channel 30 in a vertical direction from bottom to top. The suspension Q is supplied from the inlet 31, flows through the main flow channel 30 in the direction Df shown in FIG. 2, and is discharged from the outlet 32. The sheath fluid s is supplied from the first inlet 31a and the second inlet 31b, flows through the main flow channel 30 in the direction Df shown in FIG. 2, and is discharged from the outlet 32. The direction Df shown in FIGS. 1 and 2 is also a direction from upstream to downstream of the main flow channel 30.

[0024] The culture device 24 obtains a culture solution (not shown) containing a biologically derived substance and a culture medium. The culture device 24 obtains the culture solution containing a culture medium, for example, by cell culture.

[0025] A tank 23 is connected to the mixer 25. A pump 12b is provided between the mixer 25 and the tank 23. A bioactive substance is stored in the tank 23, for example, in a state dispersed in a liquid. The configuration of the tank 23 is not particularly limited as long as it is capable of storing the bioactive substance. The mixer 25 mixes the culture solution and the bioactive substance. The mixture of the culture solution and the bioactive substance is called suspension Q, and the suspension Q includes a culture medium. By using pump 12a to aspirate the culture solution from the culture device 24 and pump 12b to aspirate the bioactive substance from the tank 23, the culture solution from the culture device 24 and the bioactive substance from the tank 23 are mixed in the mixer 25 to form suspension Q without replacing the culture medium with an electroporation (EP) buffer. The configuration of the mixer 25 is not particularly limited as long as it is capable of mixing the culture solution and the bioactive substance, and a known mixer can be used. The suspension Q is supplied by the pumps 12a and 12b through the first tube 13 and the inlet 31 to the main flow channel 30. In this way, the suspension Q containing the culture medium is supplied to the main flow channel 30 without replacing the culture medium of the culture solution. The suspension Q is transported vertically from below to above between the electrode pair 34.

[0026] The culture device 24 is not necessarily required. Instead of the culture device 24, for example, a tank (not shown) in which the suspension Q is stored may be used. In this case, the tank is connected to the pump 12a. The above-mentioned pumps 12a and 12b are pumps that send the suspension from a suspension supply unit such as the culture device 24. Such pumps 12a and 12b are not particularly limited, and known pumps can be used as appropriate, such as syringe pumps.

[0027] A third tube 28 is connected to the outlet 32. The third tube 28 may be made of a flexible material such as a silicone tube. The waste tank 26 stores liquids, such as the suspension Q and sheath fluid s, that are discharged from the flow path device 11 to the outside, that are not suitable for post-culture. The container 27 stores liquids, such as the suspension Q and sheath fluid s, that are discharged from the flow path device 11 to the outside, that are to be used for post-culture. The configurations of the waste tank 26 and the container 27 are not particularly limited as long as they can store liquids, such as the suspension Q and sheath fluid s, that are discharged from the flow path device 11 to the outside. For example, the suspension Q and sheath fluid s discharged from the outlet 32 ​​are delivered and stored in the waste tank 26 from the start of delivery of the suspension Q and sheath fluid s until the application of an electric field. For example, the electroporated suspension Q is delivered and stored in the container 27 after a predetermined time has elapsed since the application of an electric field to the suspension Q. The configuration of the valve 29 is not particularly limited, and any known valve may be used as appropriate, as long as it can switch the liquid destination between the waste tank 26 and the container 27. The valve 29 may also be controllable by the control unit 16, in which case the liquid destination is switched between the waste tank 26 and the container 27 by the control unit 16.

[0028] In the main flow channel 30, an electrode pair 34 is provided downstream of a confluence 59 where the suspension flow and the sheath liquid flow converge, as shown in FIG. 3 . The electrode pair 34 includes electrodes arranged opposite each other and applies an electric field to the suspension Q flowing through the main flow channel 30. The electrode pair 34 that applies the electric field to the suspension Q includes a first electrode 35 and a second electrode 36 arranged opposite each other. The first electrode 35 has an electrode surface 35a, which is flat. The second electrode 36 has an electrode surface 36a, which is flat. The electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 are arranged opposite each other and parallel to each other. The electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 are each arranged facing the inner surface 30c of the main flow channel 30. The electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 each constitute the inner surface 30c of the main flow channel 30, and come into contact with the sheath fluid s when the suspension Q and the sheath fluid s are supplied to the main flow channel 30 during electroporation, for example. A space 37 between the opposing electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36, which constitute the electrode pair 34, is the space between the electrode pair 34.

[0029] As shown in FIG. 2 , the first electrode 35 and the second electrode 36 of the electrode pair 34 are electrically connected to the power supply unit 14. The power supply unit 14 applies a voltage to the electrode pair 34. That is, the power supply unit 14 applies a voltage to the opposing first electrode 35 and second electrode 36. The configuration of the power supply unit 14 is not particularly limited, as long as it can apply a pulse voltage to the electrode pair 34 and adjust the pulse width and pulse period. For example, a pulse power supply is used as the power supply unit 14. The determination unit 18 determines whether the flow of the suspension Q and the sheath fluid s between the electrode pair 34 is stable. The determination unit 18 outputs a determination signal containing information indicating that the flow has stabilized to the control unit 16, and the control unit 16 receives the determination signal. Based on the received determination signal, the control unit 16 controls various components, such as the power supply unit 14, to apply an electric field to the suspension Q. The determination of the stability of the flow of the suspension and the sheath fluid between the electrode pair 34 will be described later.

[0030] For example, a sensor 38 is provided near the inlet 31 of the first tube 13. Furthermore, for example, a sensor 39a is provided near the first inlet 31a of the second tube 21a, and a sensor 39b is provided near the second inlet 31b of the second tube 21b. The vicinity of the inlet 31 refers to a range within 10 cm, preferably within 5 cm, from the inlet 31. The vicinity of the first inlet 31a refers to a range within 10 cm, preferably within 5 cm, from the first inlet 31a. The vicinity of the second inlet 31b refers to a range within 10 cm, preferably within 5 cm, from the second inlet 31b.

[0031] The leading position of the suspension Q refers to the liquid level in the first tube 13 at a position closest to the flow path device 11 for the suspension Q. The leading position of the sheath fluid s refers to the liquid level in the second tube 21a at a position closest to the flow path device 11 for the sheath fluid s, and refers to the liquid level in the second tube 21b at a position closest to the flow path device 11 for the sheath fluid s. The sensor 38 detects the leading position of the suspension Q in the first tube 13. For example, an ultrasonic sensor is used as the sensor 38. With ultrasonic sensors, ultrasonic waves tend to propagate more easily through liquids and solids than through gases, so false detection is less likely when a biological substance is used. Furthermore, as described above, ultrasonic waves tend to propagate more easily through liquids and solids than through gases, so a decrease in detection accuracy is suppressed even when the concentration of the biological substance in the suspension Q is high. For these reasons, the sensor 38 used to detect the suspension Q is preferably an ultrasonic sensor. The sensors 39a and 39b detect the leading positions of the sheath fluid s in the second tubes 21a and 21b. The configuration of sensors 39a and 39b is not particularly limited as long as they can detect the position of the head of sheath fluid s in second tubes 21a and 21b. Furthermore, when sensor 38 detects the head of suspension Q in first tube 13, it is preferable that sensor 38 transmits a detection signal to control unit 16. When sensors 39a and 39b detect the head of sheath fluid s in second tubes 21a and 21b, it is preferable that sensor 39a and 39b transmit a detection signal to control unit 16. The control unit 16 receives the detection signals from sensors 38, 39a, and 39b.

[0032] In the electroporation apparatus 10, a standby position for the head of the suspension Q in the first tube 13 is predetermined, and a standby position for the head of the sheath fluid s in the second tubes 21a and 21b is predetermined. Therefore, it is preferable to dispose the sensor 38 at the predetermined standby position for the head of the suspension Q in the first tube 13. The standby position for the first tube 13 is, for example, near the inlet 31 of the first tube 13. It is also preferable to dispose the sensors 39a and 39b at the predetermined standby positions for the head of the sheath fluid s in the second tubes 21a and 21b. The standby position for the second tube 21a is, for example, near the first inlet 31a of the second tube 21a. The standby position for the second tube 21b is, for example, near the second inlet 31b of the second tube 21b.

[0033] The control unit 16 of the EP device 10 is connected to the pump 12, the power supply unit 14, the determination unit 18, the pumps 20a and 20b, and the sensors 38, 39a, and 39b. The control unit 16 controls the operation of the pumps 12a, 12b and 20a, 20b, and controls the timing of starting and stopping the delivery of the suspension Q and sheath fluid s. The control unit 16 also adjusts the magnitude, pulse width, and pulse period of the voltage that the power supply unit 14 applies to the electrode pair 34. The control unit 16 also adjusts the timing of the operation of the pumps 12a, 12b and 20a, 20b, i.e., the timing of delivery of the suspension Q and sheath fluid s and the timing of application of voltage to the electrode pair 34 by the power supply unit 14. The control unit 16 can also adjust the operation timing of the pumps 12a, 12b and 20a, 20b, i.e., the timing of the delivery of the suspension Q and sheath fluid s and the timing of the application of voltage to the electrode pair 34 by the power supply unit 14, based on the determination unit 18's determination of the stability of the flow of the suspension Q and sheath fluid s between the electrode pair 34. The control unit 16 and the determination unit 18 also have a time measurement function, such as a clock (not shown) for measuring time. The control unit 16 can adjust the operation timing of the pumps 12a, 12b, 20a, 20b using the clock, and can operate the pumps 12a, 12b, 20a, 20b with a predetermined time difference. This allows the control of the delivery timing of the suspension Q and sheath fluid s. The determination unit 18 can also use the clock to determine whether the flow of the suspension Q and sheath fluid s between the electrode pair is stable.

[0034] The control unit 16 and the determination unit 18 may be configured, for example, by a computer that functions by executing a program, or may be a dedicated device configured with a dedicated circuit. The control unit 16 and the determination unit 18 may be configured as a virtual configuration provided, for example, on the cloud, separate from the flow path device 11, or may be configured as a server that runs on the cloud. When the control unit 16 and the determination unit 18 are configured as virtual configurations as described above, the control unit 16 is connected to the pumps 12a, 12b, pumps 20a, 20b, and power supply unit 14 via the Internet or a local area network (LAN). The above-mentioned computer, dedicated device, and server each have, for example, a processor. The processor may be configured with one or more pieces of hardware, and the type of hardware is not limited. For example, the processor may be configured with hardware such as a programmable logic device such as a CPU (Central Processing Unit), an MPU (Micro Processing Unit), or an FPGA (Field Programmable Gate Array), a dedicated circuit for executing specific processing such as an ASIC (Application Specific Integrated Circuit), a GPU (Graphic Processing Unit), or an NPU (Neural Processing Unit). The processor also has various units or means for executing various processes in this embodiment. The type of hardware may also be a combination of different types of hardware. When multiple pieces of hardware are configured to execute one or more processes of a certain processor, the multiple pieces of hardware may be located in devices physically separated from each other, or may be located in the same device. Furthermore, in any embodiment, the order of the processes performed by the processor is not limited to the order described above and may be changed as appropriate. The hardware is configured by an electric circuit (circuitry) or the like that combines circuit elements such as semiconductor elements.

[0035] 2 , the flow path device 11 in the EP apparatus 10 includes a first substrate 52, a second substrate 54, a first inlet portion 56, a second inlet portion 58, and a flow path substrate 55 provided between the first substrate 52 and the second substrate 54. The first inlet portion 56, the first substrate 52, the flow path substrate 55, the second substrate 54, and the second inlet portion 58 are stacked in this order. A first electrode 35 is incorporated into the first substrate 52, and a second electrode 36 is incorporated into the second substrate 54, thereby constituting an electrode pair 34. As described above, the electrode surface 35 a of the first electrode 35 and the electrode surface 36 a of the second electrode 36 each constitute the inner surface 30 c of the main flow path 30. As described above, the flow path device 11 has the inlet 31 for supplying the suspension Q, the main flow path 30, the outlet 32 ​​for discharging the processed suspension that has undergone flow-type EP, and the first inlet 31 a and second inlet 31 b for allowing the sheath fluid s to flow in. The main flow path 30 is formed in a substantially linear shape between the first substrate 52 and the second substrate 54.

[0036] A through hole 56b extending in the stacking direction Ds of the first introduction portion 56, first substrate 52, flow path substrate 55, second substrate 54, and second introduction portion 58 is provided in the first introduction portion 56. Furthermore, a through hole 52b communicating with the through hole 56b of the first introduction portion 56 and extending in the stacking direction Ds is provided in the first substrate 52. The through hole 52b communicates with the most upstream portion of the main flow path 30. The opening of the through hole 56b of the first introduction portion 56 on the opposite side of the first substrate 52 is the inlet 31. The inlet 31 communicates with the most upstream portion of the main flow path 30 via the through hole 56b of the first introduction portion 56 and the through hole 52b of the first substrate 52. On the inner surface 30c of the main flow path 30 (flow path substrate 55), protrusions 55a and 55b are provided from the most upstream portion toward downstream, protruding in the opposing direction of the electrodes in the electrode pair 34, i.e., the opposing direction of the first electrode 35 and the second electrode 36. The stacking direction Ds is parallel to the direction y perpendicular to the vertical direction z. The opposing direction of the electrodes in the electrode pair 34 is parallel to the direction y perpendicular to the vertical direction z.

[0037] Furthermore, the first substrate 52 is provided with a through hole 52a communicating with the main flow path 30. The second substrate 54 is provided with a through hole 54a communicating with the main flow path 30. The through holes 52a and 54a are provided at an angle with respect to the main flow path 30. The first introduction section 56 is provided with a through hole 56a communicating with the through hole 52a of the first substrate 52. Furthermore, the second introduction section 58 is provided with a through hole 58a communicating with the through hole 54a of the second substrate 54. The through hole 56a communicates with the first inlet 31a and the through hole 52a of the first substrate 52, extends from the first inlet 31a in the stacking direction Ds, and is inclined at the boundary with the first substrate 52 in the extension direction Ds of the main flow path 30. Ｌ The through-hole 56a having such a shape is conveniently referred to as a substantially L-shaped through-hole 56a. The through-hole 58a communicates with the second inlet 31b and the through-hole 54a of the second substrate 54, extends from the second inlet 31b in the stacking direction Ds, and is bent in the extension direction Ds of the main flow path 30 at the boundary with the second substrate 54. Ｌ The through hole 58a of this shape is conveniently referred to as a substantially L-shaped through hole 58a. The through hole 56a of the first introduction part 56 and the through hole 52a of the first substrate 52, and the through hole 58a of the second introduction part 58 and the through hole 54a of the second substrate 54 are arranged symmetrically with respect to the main flow path 30 extending in the vertical direction z.

[0038] The first inlet 31a communicates with the main channel 30 via a substantially L-shaped through-hole 56a provided in the first introduction part 56 and a through-hole 52a provided in the first substrate 52. Here, the through-hole 52a is oriented in the extension direction D of the main channel 30. Ｌ 55a of the main flow passage 30. The main flow passage 30 is inclined toward the downstream direction and opens immediately downstream of the convex portion 55a of the main flow passage 30.

[0039] The through-hole 56a (part of the sheath liquid flow path) may widen the flow path in the width direction, for example, from the bent position of the approximately L-shaped flow path toward downstream, so that the flow velocity of the sheath liquid in the width direction becomes uniform. This also applies to the through-hole 58a described later. The through-hole 56a may be provided across the first introduction part 56 and the first substrate 52. On the other hand, the second inlet 31b communicates with the main flow path 30 via the approximately L-shaped through-hole 58a provided in the second introduction part 58 and the through-hole 54a provided in the second substrate 54. Here, the through-hole 54a is oriented in the extension direction D of the main flow path 30. Ｌ The through-hole 58a is inclined toward the downstream direction with respect to the second introduction part 58 and opens immediately downstream of the convex part 55b of the main flow channel 30. The through-hole 58a may be provided across the second introduction part 58 and the second substrate 54. The downstream end parts 55c of the convex parts 55a and 55b form a suspension supply port 55d that supplies the suspension Q to the main flow channel 30 in the flow channel device 11. The first tube 13 is connected to this downstream end part 55c, i.e., the suspension supply port 55d, via the through-hole 52b, the through-hole 56b, and the inlet 31. The opening of the through-hole 52a to the main flow channel 30 and the opening of the through-hole 54a to the main flow channel 30 form sheath fluid supply ports 52c, 54c for the sheath fluid s to the main flow channel 30 in the flow channel device 11. It is preferable that the widths of the suspension supply port for the suspension Q and the sheath fluid supply ports 52c, 54c for the sheath fluid s are approximately the same. In addition, in the flow path device 11, the downstream end 55c serves as a confluence 59 where the suspension flow and the sheath liquid flow join together.

[0040] A sheath fluid supply port 52c, which forms a sheath fluid flow in the main flow path 30 that contacts the electrodes of the electrode pair 34, is provided downstream of the suspension supply port 55d in the main flow path 30. The through-holes 52a and 56a form a sheath fluid flow path 64 in the main flow path 30 that merges the sheath fluid flow with the suspension Q at a junction 59. A second tube 21a is connected to the sheath fluid flow path 64. The second tube 21a is connected to the sheath fluid supply port 52c via the first inlet 31a, the through-hole 56a, and the through-hole 52a. A sheath fluid supply port 54c, which forms a sheath fluid flow in the main flow path 30 that contacts the electrodes of the electrode pair 34, is provided downstream of the suspension supply port 55d in the main flow path 30. The through-holes 54a and 58a form a sheath fluid flow path 65 in the main flow path 30 that merges the sheath fluid flow with the suspension Q at a junction 59. The second tube 21b is connected to the sheath liquid flow path 65. The second tube 21b is connected to the sheath liquid supply port 54c via the second inlet 31b, the through-hole 58a, and the through-hole 54a. The sheath liquid flow paths 64, 65 are inclined downstream with respect to the flow direction of the liquid in the main flow path 30, and open downstream of the suspension Q supply port and upstream of the electrode pair 34 so that the sheath liquid S flows therethrough. The region upstream of the main flow path 30 through which only the suspension Q flows is sometimes referred to as the suspension flow path, and the region downstream of the suspension flow path and sandwiched between the convex portions 55a and 55b is the restriction region. Only the sheath liquid flows through the sheath liquid flow paths 64, 65. The sheath liquid flow paths 64, 65 preferably have a region in which the flow path widthwise widens downstream. As a result, even if the main flow path 30 is wide, the flow velocity distribution downstream is uniform, enabling uniform EP for the suspension Q. In this case, it is preferable that the suspension flow path also has a region where the flow path widens in the width direction toward the downstream, so that the flow velocity distribution in the suspension flow path also becomes uniform.

[0041] In the flow channel device 11, the flow channel substrate 55 may be formed by any of various known methods. Alternatively, the flow channel substrate 55 may be formed using a plurality of flow channel plates. As an example, the flow channel substrate 55 may be formed using three flow channel plates: one having a portion (non-opening) corresponding to the convex portion 55a and an opening corresponding to the main flow channel 30, another having an opening corresponding to the entire main flow channel 30, and another having a portion (non-opening) corresponding to the convex portion 55b and an opening corresponding to the main flow channel 30.

[0042] In the flow path device 11, the suspension Q is supplied from the first tube 13 to the inlet 31, flows into the main flow path 30 from the most upstream portion, and then reaches the outlet 32 ​​from the main flow path 30. The sheath fluid s is supplied from the second tube 21a to the first inlet 31a and from the second tube 21b to the second inlet 31b. The sheath fluid s that flows into the first inlet 31a passes through the through-hole 56a in the first introduction part 56 and the through-hole 52a in the first substrate 52, i.e., through the sheath fluid flow path 64, to the main flow path 30 and reaches the outlet 32 ​​from the main flow path 30. The sheath fluid s that flows into the second inlet 31b passes through the through-hole 58a in the second introduction part 58 and the through-hole 54a in the second substrate 54, i.e., through the sheath fluid flow path 65, to the main flow path 30 and reaches the outlet 32 ​​from the main flow path 30.

[0043] As described above, the main flow channel 30 (flow channel substrate 55) is provided with convex portions 55a and 55b extending from the most upstream portion toward downstream, protruding in the opposing direction of the first electrode 35 and the second electrode 36 of the electrode pair 34. In the region having the convex portions 55a and 55b, the thickness Dm of the suspension Q is the distance d between the convex portions 55a and 55b. As described above, the through-hole 52a is provided so as to incline toward the downstream direction with respect to the flow direction of the liquid in the main flow channel 30, and opens immediately downstream of the convex portion 55a of the main flow channel 30 (the region of the suspension flow channel sandwiched between the convex portions 55a and 55b is sometimes referred to as the restriction region). As described above, the region sandwiched between the convex portions 55a and 55b is the restriction region. Meanwhile, the through-hole 54a is provided so as to incline toward the downstream direction with respect to the flow direction of the liquid in the main flow channel 30, and opens immediately downstream of the convex portion 55b of the main flow channel 30. 2, the sheath fluid flow path is composed of the through-hole 56a (58a) and the through-hole 52a (54a), but the sheath fluid flow path only requires the through-hole 52a (54a), and the flow path configuration is appropriately designed depending on the device configuration. In the sheath fluid flow path 64 composed of the through-hole 56a and the through-hole 52a, the sheath fluid flow path 64 only requires the through-hole 52a, and the configuration of the sheath fluid flow path is appropriately determined depending on the configuration of the flow path device.

[0044] Furthermore, the sheath liquid flow path 65, which is composed of the through-holes 54a and 58a, only needs to have the through-hole 54a, and the configuration of the sheath liquid flow path is determined appropriately depending on the configuration of the flow path device. Therefore, upstream of the electrode pair 34, as conceptually shown in FIG. 3 , sheath liquid flows composed of the sheath liquid s are formed on both sides of the suspension flow of the suspension Q in opposing directions, forming a three-layer laminated flow of sheath liquid flow / suspension flow / sheath liquid flow, and the three-layer laminated flow flows between the electrode pair 34. In the EP device 10, the suspension Q and the sheath liquid s are continuously delivered between the electrode pair 34, i.e., into the above-mentioned space 37, in a state where they form a laminated flow of sheath liquid flow / suspension flow / sheath liquid flow. A pulsed electric field, for example, is applied to the suspension Q in the three-layer laminated flow state by the first electrode 35 and the second electrode 36, thereby enabling flow-type EP treatment using the sheath liquid flow as described above. In this case, the thickness of the suspension flow becomes approximately equal to the thickness of the regulated region, and the thickness Dm of the suspension flow is maintained at 1 to 10 mm at least between the electrode pair 34 and at the upstream end of the electrode pair 34. Note that Fig. 3 does not illustrate the first tube 13, second tubes 21a and 21b, pumps 12a, 12b, 20a, and 20b, tank 22, culture device 24 and tank 23, power supply unit 14, control unit 16, determination unit 18, and mixer 25 shown in Fig. 2. Sensors 38, 39a, and 39b are also not illustrated in Fig. 3.

[0045] As described above, the sheath fluid s supplied from the first inlet 31a flows into the main flow channel 30 immediately downstream of the convex portion 55a, sloping downward. On the other hand, the sheath fluid s supplied from the second inlet 31b flows into the main flow channel 30 immediately downstream of the convex portion 55b, sloping downward. Furthermore, convex portions 55a and 55b are formed in the main flow channel 30 upstream of the inlet portion for the sheath fluid s, and the thickness of the main flow channel 30 in the opposing direction is thinner than that downstream of the inlet position of the sheath fluid s. Furthermore, the thickness of the main flow channel 30 in this region in the opposing direction, i.e., the thickness Dm of the suspension flow, is determined by the distance between the convex portions 55a and 55b.

[0046] In this way, the convex portions 55a and 55b function to form a sheath liquid flow and to regulate the thickness Dm of the suspension flow. In the flow path device 11, the region in the main flow path 30 where the convex portions 55a and 55b are formed serves as a regulated region that regulates the thickness of the suspension flow before merging with the sheath liquid flow. The regulated region that regulates the thickness of the suspension flow before merging with the sheath liquid flow is sometimes referred to as the suspension flow path. Therefore, in the flow path device 11, by setting the distance d between the convex portions 55a and 55b to 1 to 10 mm, the thickness Dm of the suspension flow flowing through the regulated region is set to 1 to 10 mm. As a result, in the flow path device 11, the thickness Dm of the suspension flow (see FIG. 3) can be controlled to 1 to 10 mm in the three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow in the above-mentioned flow-type EP. Note that it is sufficient that only a portion of the suspension flow path serves as the regulated region, and only a portion downstream serves as the regulated region and the distance d is set to 1 to 10 mm. The thickness Dm of the suspension flow in this restriction region, i.e., the distance d (see FIG. 2), is preferably 1 to 8 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. The lower limit of the thickness of the restriction region is preferably 2 mm, more preferably 3 mm. The distance d (thickness Dm of the suspension flow) may be 2 to 10 mm, or may be 3 to 10 mm. Furthermore, by making the total thickness of the protrusions 55a and 55b, i.e., the total height of the electrodes in the opposing direction, equal to or less than the distance d between the protrusions 55a and 55b, the total thickness of the sheath liquid flow can be made equal to or less than the thickness of the suspension flow, and the dilution rate of the suspension after flow-type EP can be made twofold or less.

[0047] Furthermore, although there is no limitation on the thickness ds of the sheath liquid flow produced by the sheath liquid s (see FIG. 3 ), the thickness of the sheath liquid flow at the time of merging with the suspension flow is preferably 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.5 mm or more. By setting the total thickness of the two sheath liquids to be equal to or less than the thickness Dm of the suspension flow, the total thickness of the sheath liquid flow can be set to be equal to or less than the thickness Dm of the suspension flow, thereby enabling the dilution rate of the suspension after flow-type EP to be two times or less. For this reason, it is preferable that the total thickness ds of the two sheath liquid flows (see FIG. 3 ) be equal to or less than the thickness Dm of the suspension flow. In other words, it is preferable that the total thickness of the sheath liquid flow at the time of merging with the suspension flow be equal to or less than the thickness of the suspension flow in the regulated region. The thickness Dm of the suspension flow is determined by the distance d between the convex portions 55 a and 55 b (see FIG. 2 ), as described above. The convex portions 55 a and 55 b function as members for regulating the thickness Dm of the suspension flow. Therefore, the thickness Dm of the suspension flow can be adjusted by changing the thickness of the convex portions 55a and 55b in the opposing direction, and in this case, the thickness ds of the sheath liquid flow (see FIG. 3) is also adjusted.

[0048] In such a flow channel device 11, the angle formed by the suspension flow and the sheath liquid flow when the suspension flow and the sheath liquid flow are joined, i.e., the angle formed by the main flow channel 30 and the through-holes 52 a and 54 a, is not limited, but is preferably small. Ｌ The angle formed by the direction of extension of through hole 52a and the direction of extension of through hole 54a is preferably 90° or less, more preferably 60° or less, even more preferably 45° or less, and even more preferably 30° or less. Furthermore, it is preferable that the difference in flow velocity between the suspension flow and the sheath liquid flow when they are joined together is small. Specifically, the flow velocity of the sheath liquid flow when they are joined together is preferably ±10% or less of the flow velocity of the suspension flow, more preferably ±5% or less, even more preferably ±3% or less, and most preferably ±0%, i.e., the suspension flow and the sheath liquid flow are equal in speed.

[0049] In the flow path device 11 of Figure 2, it is preferable to have O-rings as sealing members (not shown) to prevent leakage of the suspension Q between the first introduction section 56 and the first substrate 52, between the first substrate 52 and the flow path substrate 55, between the flow path substrate 55 and the second substrate 54, and between the second substrate 54 and the second introduction section 58.

[0050] In the EP device 10, the pumps 12a, 12b, 20a, and 20b pump the suspension Q and the sheath fluid s between the electrode pair 34, i.e., in the space 37, such that the liquid flow direction Dd of the suspension Q and the sheath fluid s is from bottom to top in the vertical direction z. When an electric field is applied to the suspension Q using the power supply unit 14 to perform EP and bubbles are generated between the opposing first and second electrodes 35 and 36, the generated bubbles have a smaller specific gravity than the suspension Q and the sheath fluid s and therefore are more likely to move in the vertical direction z than the suspension Q and the sheath fluid s. Therefore, bubbles generated between the electrode pair 34 are removed from between the electrode pair 34 by moving in the vertical direction z, without remaining between the electrode pair 34 due to the flow of the suspension Q and the sheath fluid s, thereby preventing the bubbles from interfering with the application of an electric field to the suspension Q. This allows the EP device 10 to properly apply an electric field to the suspension Q, thereby ensuring proper introduction efficiency and utilization efficiency of the bioactive substance.

[0051] As shown in FIG. 3 , the EP device 10 performs flow-type EP by forming a sheath liquid flow (s) composed of a sheath liquid (s) separate from the suspension between the suspension flow and the first electrode 35 and the second electrode 36, along with the suspension flow. The use of the sheath liquid (s) prevents the flow of biologically derived substances from stagnating near the wall of the main channel 30, even when the concentration of biologically derived substances in the suspension Q is high, enabling highly efficient flow-type EP. According to the inventors' studies, when the concentration of biologically derived substances in the suspension Q is high but the sheath liquid (s) is absent, the flow of biologically derived substances stagnates very close to the wall of the main channel 30. The retention layer where the flow of biologically derived substances stagnates has higher electrical resistance than the region where the biologically derived substances are flowing because the biologically derived substances are in close contact with the retention layer. Therefore, this retention layer consumes a large amount of the electric field applied by the electrode pair 34. As a result, the electric field applied to the flow section, where an electric field should be applied, becomes weaker, preventing sufficient electric field from being applied, resulting in reduced EP introduction efficiency. Furthermore, in the absence of sheath liquid s, the biological material remains as a retention layer near the electrode, and an electric field is repeatedly applied to the biological material. As a result, the biological material burns near the electrode, and the burnt biological material adheres to the surface of the electrode. When such foreign matter adheres to the electrode surface, it blocks the electric field applied by the electrode, thereby reducing the efficiency of introducing EP. For this reason, the presence of sheath liquid s is preferable.

[0052] Furthermore, the inventors have found, through their investigations, that in flow-type EP using such sheath fluid s, highly efficient flow-type EP can be achieved by increasing the thickness Dm of the suspension flow in the opposing direction of the electrodes constituting the electrode pair. Specifically, highly efficient flow-type EP can be achieved by setting the thickness Dm of the suspension flow in the opposing direction of the electrodes constituting the electrode pair to 1 to 10 mm. It is sufficient that the thickness Dm of the suspension flow is 1 to 10 mm in a portion between the electrode pair, and that the thickness Dm of the suspension flow is 1 to 10 mm between the electrode pair and at the upstream ends of the electrodes. By performing flow-type EP with such a thick suspension flow Dm, the EP method enables more efficient flow-type EP. Specifically, more efficient flow-type EP can be achieved, with high throughput, introduction efficiency, and bioactive substance utilization efficiency. Throughput refers to the number of cells processed per unit time.

[0053] Furthermore, by performing flow-type EP with a thick suspension flow (Dm), the EP method can efficiently perform large-volume flow-type EP on highly concentrated suspensions while increasing the processing volume, thereby achieving high productivity. Conventional flow-type EP involves reducing the suspension flow thickness as much as possible. For example, Patent Document 2 exemplifies a preferred embodiment in which flow-type EP is performed with the suspension flow thickness, i.e., the spacing between sheath liquid flows, set to 50 to 100 μm. Patent Document 2 states that this allows for a monolayer flow in which biologically derived materials (cells) passing between the electrode pair do not overlap in the direction of the electrodes. Conventional flow-type EP is thought to enable highly efficient processing by reducing the suspension flow thickness in this way, thereby uniforming the flow of biologically derived materials between the electrode pair and uniforming the electric field applied to the biologically derived materials.

[0054] However, after further investigation, the inventors discovered that flow-type EP can be performed more efficiently by significantly increasing the thickness of the suspension flow. In flow-type EP, the diffusion of biologically derived substances and bioactive substances contained in the suspension forms a diffusion region at the interface between the sheath liquid flow and the suspension flow, where the biologically derived substances and bioactive substances are diffused. When the suspension flow thickness is set to 50 to 100 μm, as in Patent Document 2, the diffusion region becomes thicker relative to the thickness of the suspension flow. As a result, the diffusion of biologically derived substances and bioactive substances has a significant impact on flow-type EP, resulting in reduced introduction efficiency and bioactive substance utilization efficiency. On the other hand, when the suspension flow thickness is set to approximately 500 μm, the thickness of the diffusion region becomes smaller relative to the suspension flow thickness, reducing the impact of diffusion but resulting in poorer uniformity of the electric field in the suspension flow. As a result, the introduction efficiency and bioactive substance utilization efficiency are lower than when the suspension flow thickness is set to 50 to 100 μm, as in Patent Document 2.

[0055] In contrast, by significantly increasing the thickness Dm of the suspension flow to 1 mm or more, the effects of diffusion of biologically derived substances and the like can be suppressed. In other words, because the diffusion region is formed by diffusion, the thickness of the diffusion region is not affected by the thickness Dm of the suspension flow. Therefore, by increasing the thickness Dm of the suspension flow to 1 mm or more, the thickness of the suspension Q can be sufficiently increased, making it possible to process a large amount of suspension Q, with a uniform thickness that is not affected by diffusion relative to the thickness of the diffusion region. In addition, by increasing the thickness Dm of the suspension flow to 1 mm or more, good electric field uniformity can be achieved. In other words, in EP, the strength of the electric field applied to the biologically derived substances varies depending on the number of biologically derived substances present in the direction between the electrode pairs. For example, as in Patent Document 2, when the thickness of the suspension flow is sufficiently narrow, such as approximately 50 μm, the probability of biologically derived substances overlapping in the direction between the electrode pairs is low, and the electric field applied to each biologically derived substance is approximately constant, resulting in good electric field uniformity. On the other hand, when the thickness of the suspension flow is about 500 μm, the number of biologically derived substances present in the direction between the electrode pair varies depending on the position in the flow direction, resulting in differences in the electric field applied to each biologically derived substance and poor electric field uniformity. In contrast, by making the thickness Dm of the suspension flow sufficiently thick, at 1 mm or more, the number of biologically derived substances present in the direction between the electrode pair at each position in the flow direction can be made uniform, resulting in a nearly constant electric field applied to each biologically derived substance and good electric field uniformity. As a result, this EP method makes it possible to perform flow-type EP with higher efficiency than conventional flow-type EP, which uses a thinner suspension flow.

[0056] <Stabilizing the Flow of Suspension and Sheath Fluid Between the Electrode Pair> In the EP device 10 and the EP method described below, after the suspension Q and sheath fluid s are delivered and the flows of the suspension Q and sheath fluid s stabilize between the electrode pair, an electric field is applied to the suspension Q by the power supply unit 14. The stabilization of the flow of the suspension and sheath fluid between the electrode pair will now be described. FIG. 4 is a schematic cross-sectional view illustrating the stability of the flow of the suspension and sheath fluid between the electrode pair of a flow path device in a first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 5 is a schematic cross-sectional view illustrating an example of an unstable flow of the suspension and sheath fluid between the electrode pair of a flow path device in a first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 6 is a schematic cross-sectional view illustrating another example of an unstable flow of the suspension and sheath fluid between the electrode pair of a flow path device in a first example of an electroporation apparatus according to an embodiment of the present invention. Note that in FIGS. 4-6, components identical to those in the EP device 10 shown in FIGS. 1 and 2 are designated by the same reference numerals, and detailed descriptions thereof will be omitted.

[0057] A stable flow of the suspension Q and sheath fluid s between the electrode pair 34 refers to a state in which the suspension flows smoothly without forming a retention layer near the wall of the flow channel where the flow of biologically derived substances would otherwise stagnate. Preferably, this refers to a state in which the flow velocity distribution of the suspension flow is approximately uniform. More preferably, as shown in FIG. 4 , the thickness Dm of the suspension flow and the thickness ds of the two sheath fluid flows are within a predetermined range, and the interface Bs between the suspension flow and the sheath fluid flow is flat and not wavy. The EP device 10 performs EP with a stable flow of the suspension Q and sheath fluid s between the electrode pair 34. This allows for proper application of an electric field to the suspension Q, resulting in higher introduction efficiency and utilization efficiency of the bioactive substance, reduced suspension Q waste, and high-productivity EP. As shown in Figure 5, when the thickness ds of the two sheath liquid flows is thicker and the thickness Dm of the suspension flow is thinner than the predetermined range between the electrode pair 34, even if the interface Bs between the suspension flow and the sheath liquid flow is flat and not wavy, the flows of the suspension Q and sheath liquid s between the electrode pair are not stable. When the flows of the suspension Q and sheath liquid s between the electrode pair 34 are in the state shown in Figure 5, the sheath liquid flow is thick and it is not possible to properly apply an electric field to the suspension Q, resulting in low introduction efficiency and utilization efficiency of the bioactive substance, and low productivity. Furthermore, as shown in Figure 6, when the interface Bs between the suspension flow and the sheath liquid flow between the electrode pair 34 is Ｌ If the interface Bs between the suspension flow and the sheath fluid s fluctuates, for example, in a wavy manner along the interface Bs in the direction opposite the first electrode 35 and the second electrode 36, the flow of the suspension Q and the sheath fluid s between the electrode pair 34 is not stable. The wavy fluctuation of the interface Bs described above is caused by pressure fluctuations, or so-called pulsation, during the delivery of the suspension Q or the sheath fluid s. If the interface Bs between the suspension flow and the sheath fluid flow fluctuates in a wavy manner as shown in Figure 6, the thickness of the sheath fluid flow between the electrode pair 34 is uneven, making it impossible to properly apply an electric field to the suspension Q. As a result, the introduction efficiency and utilization efficiency of the bioactive substance are low, and productivity is low. For these reasons, it is necessary to perform EP while avoiding the unstable flow of the suspension Q and the sheath fluid s between the electrode pair 34 as shown in Figures 5 and 6.

[0058] 4, 5, and 6, the thickness ds of the sheath fluid and the thickness Dm of the suspension Q are different between the electrode pair 34, resulting in different electrical resistances between the electrode pair 34. The electrical resistance between the electrode pair 34 exhibits a value corresponding to the thickness ds of the sheath fluid and the thickness Dm of the suspension Q, and reflects the electrical resistance in the thickness direction of the three-layer laminated flow of sheath fluid flow / suspension flow / sheath fluid flow. Utilizing this, the electrical resistance between the electrode pair 34 is measured by a measuring unit 19 (see FIGS. 1 and 2, etc.), which will be described later, to obtain the electrical resistance of the flow of the suspension Q and sheath fluid s between the electrode pair 34. Based on this electrical resistance of the flow, the determining unit 18 determines whether the flow is stable and outputs a determination signal to the control unit 16. For example, impedance may be measured as the electrical resistance, and based on this impedance, it may be possible to determine whether the flow of the suspension Q and sheath fluid s between the electrode pair 34 is stable. 4 , the electrical resistance or impedance between the electrode pair 34 when the flow of the suspension Q and the sheath fluid s is stable is measured in advance, and a range of the electrical resistance or impedance between the electrode pair 34 that indicates that the flow is stable is set in advance and stored in the determination unit 18. If the measured value of the electrical resistance or impedance between the electrode pair 34 is within the preset range, the determination unit 18 determines that the flow is stable and outputs a determination signal to the control unit 16.

[0059] FIG. 7 is a graph illustrating the stabilization of the flow of the suspension and sheath fluid between the electrode pair of the flow path device of the first example of the electroporation apparatus according to the embodiment of the present invention. In the graph of FIG. 7 , the vertical axis represents flow rate, and the horizontal axis represents time. In FIG. 7 , line 70 represents the change in flow rate of one of the two sheath fluids, sheath fluid s. Line 71 represents the change in flow rate of suspension Q. Line 72 represents the change in flow rate of the other of the two sheath fluids, sheath fluid s. For example, as shown in FIG. 7 , after a predetermined time has elapsed since the start of the delivery of the suspension Q and sheath fluid s, the flow rates of the suspension Q and sheath fluid s each reach the set flow rates and stabilize. In this case, the flow of the suspension Q and sheath fluid s between the electrode pair becomes stable. By utilizing the stabilization of the flow rates over time, the stable flow of the suspension Q and sheath fluid s between the electrode pair can be achieved by time control. In FIG. 7 , the start time of delivery of the suspension Q and sheath fluid s is set to t ０ When the time when the flow of the suspension Q and the sheath liquid s between the electrode pair becomes stable is ts, the start time t ０ The stability of the flow of the suspension Q and the sheath liquid s between the electrode pair is controlled by the time from the start time t ０ The time from the start time t to the time ts is preferably 5 seconds or more, more preferably 10 seconds or more, and even more preferably 15 seconds or more. ０ If the time from the start time t to the time ts is long, the amount of suspension Q and sheath liquid s to be discarded increases. ０ The upper limit of the time from the start time t ０ If the time from the start time t to the time ts is too short, the flow of the suspension Q and the sheath liquid s will not be stable, and if it is too long, the amount of waste of the suspension Q and the sheath liquid s will increase. ０ The time from the time ts to the time ts is preferably 5 to 60 seconds, more preferably 10 to 45 seconds, and even more preferably 15 to 30 seconds.

[0060] As shown in FIG. 7, after the suspension Q and sheath liquid s are stabilised, that is, after time ts, １ From time t, the application of an electric field to the suspension Q by the electrode pair is started, and the flow-type EP is performed. １ After that, at time t ２ The liquid is sent to the container 27 (see FIG. 1) from the start time t ０ From time t ２ The suspension Q and sheath fluid s are sent to the waste tank 26 (see FIG. 1) until the time ts. The switching between the waste tank 26 (see FIG. 1) and the container 27 (see FIG. 1) is performed by a valve 29. At the time t ３ At this time, the application of the electric field to the suspension Q by the electrode pair is stopped. The feeding of the suspension Q and the sheath liquid s is also stopped. ３ The state up to this point corresponds to a state in which the flow of the suspension Q and the sheath liquid s between the electrode pair is stable.

[0061] In the EP device 10, a standby position for the head of the suspension Q is predetermined in the first tube 13, and a standby position for the head of the sheath fluid s is predetermined in the second tubes 21a and 21b. The control unit 16 controls the pumps 12a, 12b, 20a, and 20b to start feeding the suspension Q and the sheath fluid s after the heads of the suspension Q and the sheath fluid s have all reached their standby positions. The sensor 38 is disposed at the standby position for the head of the suspension Q in the first tube 13, thereby detecting the standby position for the head of the suspension Q. When the sensor 38 detects the head of the suspension Q in the first tube 13, a detection signal is transmitted from the sensor 38 to the control unit 16, and the control unit 16 receives the detection signal. The sensors 39a and 39b are disposed at the standby positions for the head of the sheath fluid s in the second tubes 21a and 21b, thereby detecting the standby position for the head of the sheath fluid s. When the sensors 39a, 39b detect the leading edge of the sheath fluid s in the second tubes 21a, 21b, the sensors 39a, 39b transmit detection signals to the control unit 16, which receives the detection signals. In this manner, the control unit 16 can control the pumps 12a, 12b to deliver the suspension Q and the pumps 20a, 20b to deliver the sheath fluid s based on the detection signals from the sensors 39a, 39b. For example, when the leading edges of the suspension Q and the sheath fluid s have reached their standby positions, the control unit 16 stops the pumps 12a, 12b, 20a, and 20b to stop the delivery of the suspension Q and the sheath fluid s. In this state, the control unit 16 then starts the delivery of the suspension Q and the sheath fluid s by the pumps 12a, 12b, 20a, and 20b. After the flow of the suspension Q and the sheath liquid s between the electrode pair has stabilized, the control unit 16 causes the power supply unit 14 to apply an electric field to the suspension Q.

[0062] The timing of feeding the suspension Q and the sheath fluid s may be simultaneous, or the sheath fluid s may be fed before the suspension Q. Feeding the suspension Q and the sheath fluid s simultaneously means that the pumps 12a and 12b that feed the suspension Q and the pumps 20a and 20b that feed the sheath fluid s are driven at the same time. That is, at the start time t ０7, the pumps 12a, 12b and the pumps 20a, 20b are driven to deliver the suspension Q and the sheath fluid s. The timing of delivery of the suspension Q and the sheath fluid s being earlier than the timing of delivery of the sheath fluid s means that the drive timing of the pumps 20a, 20b that deliver the sheath fluid s is earlier than the drive timing of the pumps 12a, 12b that deliver the suspension Q. That is, at the start time t ０ The pumps 20a and 20b are driven to deliver the sheath fluid s, and then the pump 12 is driven to deliver the suspension Q. The timing of delivering the sheath fluid s before the suspension Q means that the sheath fluid s fills the flow path between the electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36. If the sheath fluid s is delivered before the suspension Q, the electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 are covered with the sheath fluid s, thereby preventing the suspension Q from coming into contact with the electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36. This prevents the electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 from becoming contaminated.

[0063] The determination unit 18 determines the stability of the flows of the suspension Q and the sheath fluid s between the electrode pair. The determination unit 18 measures, for example, the electrical resistance of the flows of the suspension Q and the sheath fluid s between the electrode pair 34 and determines the stability of the flows based on the electrical resistance. In this case, the determination unit 18 is provided with a measurement unit 19 (see FIGS. 1 and 2 , etc.). When electrical resistance is used to determine the stability of the flows, the measurement unit 19 is electrically connected to the first electrode 35 of the electrode pair 34 by a wiring 19 a and to the second electrode 36 by a wiring 19 b. The measurement unit 19 measures the electrical resistance between the electrode pair 34 to measure the electrical resistance of the flows of the suspension Q and the sheath fluid s between the electrode pair 34. The configuration of the measurement unit 19 is not particularly limited as long as it can measure the electrical resistance between the electrode pair 34. Furthermore, if the determination unit 18 includes the measurement unit 19, the power supply unit 14 and the determination unit 18 are configured to be electrically insulated from each other so that they do not affect each other during operation. For example, the power supply unit 14 and the determination unit 18 are each provided with a circuit breaker (not shown), such as a switch. The circuit breaker can place the power supply unit 14 and the electrode pair 34 in a conductive state or an electrically insulated state. The circuit breaker can also place the determination unit 18 and the electrode pair 34 in a conductive state or an electrically insulated state. When the power supply unit 14 and the electrode pair 34 are in a conductive state, the circuit breaker electrically insulates the determination unit 18 from the electrode pair 34, thereby electrically insulating the power supply unit 14 from the determination unit 18. On the other hand, when the determination unit 18 and the electrode pair 34 are in a conductive state, the circuit breaker electrically insulates the power supply unit 14 from the electrode pair 34, thereby electrically insulating the power supply unit 14 from the determination unit 18. As a result, no voltage is applied to the determination unit 18 when the power supply unit 14 is operating. Furthermore, when the measurement unit 19 of the determination unit 18 measures the electrical resistance, the electrical resistance including the power supply unit 14 is not measured. Note that if the electrical resistance is not used to determine the stability of the flow as described above, there is no need to provide the measurement unit 19 in the determination unit 18. The determination unit 18 is used to measure, for example, impedance as the electrical resistance of the flow of the suspension Q and sheath fluid s between the electrode pair 34. The electrical resistance in the state shown in FIG. 4 is measured in advance, and a range of electrical resistance indicating that the flow is stable is set in advance and stored in the determination unit 18.The determination unit 18 determines that the flow is stable when the electrical resistance between the electrode pair 34 measured using the measurement unit 19 is within a preset range. In this case, the determination unit 18 outputs a determination signal to the control unit 16.

[0064] The determination unit 18 can also determine the stability of the flows of the suspension Q and the sheath fluid s by detecting the stability of the flow rate of the suspension Q in the first tube 13 and the flow rate of the sheath fluid s in the second tubes 21a and 21b. In this case, a flow meter (not shown) is provided in the first tube 13 downstream of the pump 12a and in a region up to the inlet 31. Another flow meter (not shown) is provided in the second tube 21a downstream of the pump 20a and in a region up to the first inlet 31a. Another flow meter (not shown) is provided in the second tube 21b downstream of the pump 20b and in a region up to the second inlet 31b. Data on the flow rates measured by each flow meter is output to the determination unit 18. The determination unit 18 determines the stability of the flows based on fluctuations in the flow rates measured by each flow meter. The determination unit 18, for example, compares the set flow rate with the measured flow rate, and determines that the flows of the suspension Q and the sheath fluid s are stable when the set flow rate is maintained for a preset time after reaching the set flow rate, as shown in Fig. 7. In this way, the stability of the flow rate of the suspension Q in the first tube 13 and the flow rate of the sheath fluid s in the second tubes 21a, 21b can be detected, and the stability of the flows of the suspension Q and the sheath fluid s can be determined. The above-mentioned preset time is stored in the determination unit 18.

[0065] In determining the stability of the flow rates, the flow rate of the suspension Q is compared with a set flow rate, and there are two sheath liquid flows. For example, the flow rates of the two sheath liquid flows are each compared with a set flow rate, and if both of them reach their set flow rates and then the set flow rates are maintained for a preset time, it is determined that the flows of the suspension Q and the sheath liquid s are stable. The flow meter is not particularly limited as long as it can measure the flow rates of the suspension Q and the sheath liquid s. For example, an ultrasonic, Coriolis, or thermal MEMS (Micro Electro Mechanical Systems) type flow meter can be used.

[0066] Furthermore, the determination unit 18 may determine the stability of the flow of the suspension Q and the sheath fluid s between the electrode pair by the above-mentioned time control. In this case, the drive start time (start time t ０ ) is measured by, for example, the determination unit 18. When a preset time has elapsed since the start of the feeding of the suspension Q and the sheath fluid s, the determination unit 18 determines that the flow is stable and outputs a determination signal to the control unit 16. Based on the received determination signal, the control unit 16 controls each unit, such as applying an electric field to the suspension Q by the power supply unit 14.

[0067] (First Example of Electroporation Method) In a first example of the electroporation method (EP method), a suspension Q containing a biologically derived substance and a bioactive substance and a sheath fluid s are continuously fed into the main flow channel 30 between the electrode pair 34, i.e., the above-mentioned space 37, with the suspension Q sandwiched between the sheath fluid s, and an electric field is applied to the suspension Q by the electrode pair 34, thereby introducing the bioactive substance into the biologically derived substance. For example, the EP device 10 shown in FIGS. 1 and 2 is used in the first example of the EP method. In the first example of the EP method, the culture solution in the culture device 24 is aspirated by the pump 12a of the EP device 10, and the bioactive substance in the tank 23 is aspirated by the pump 12b. Without replacing the culture medium with an EP buffer, the culture solution in the culture device 24 and the bioactive substance in the tank 23 are mixed in the mixer 25 to produce the suspension Q. The suspension Q is supplied to the main flow channel 30 by the pump 12a through the first tube 13 and the inlet 31. Furthermore, the pump 20a supplies the sheath fluid s in the tank 22 to the main flow path 30 through the second tube 21a, the first inlet 31a, the through-hole 56a in the first introduction part 56, and the through-hole 52a in the first substrate 52. The pump 20b supplies the sheath fluid s in the tank 22 to the main flow path 30 through the second tube 21b, the second inlet 31b, the through-hole 58a in the second introduction part 58, and the through-hole 54a in the second substrate 54. As a result, a three-layer laminated flow of sheath fluid flow / suspension flow / sheath fluid flow is formed between the electrode pair 34, and the suspension Q and sheath fluid s are continuously sent vertically from below to above the electrode pair 34. At this time, the power supply unit 14 applies, for example, a pulse voltage to the electrode pair 34 to perform EP.

[0068] In the first example of the EP method, the liquid transfer direction Dd of the suspension Q and the sheath liquid s is a direction from bottom to top in the vertical direction z. When EP is performed by applying an electric field to the suspension Q using the power supply unit 14 and bubbles are generated between the opposing first and second electrodes 35 and 36, the generated bubbles have a smaller specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q. Therefore, bubbles generated between the electrode pair 34 are removed from between the electrode pair 34 by moving in the vertical direction z without remaining between the electrode pair 34 due to the transfer of the suspension Q and the sheath liquid s, thereby preventing the bubbles from interfering with the application of the electric field to the suspension Q. This allows the first example of the EP method to properly apply an electric field to the suspension Q, thereby optimizing the introduction efficiency and the utilization efficiency of the bioactive substance. Furthermore, even if the composition of the suspension Q is such that bubbles are likely to be generated when an electric field is applied to the suspension Q, if bubbles are generated between the electrode pair 34 as described above, the generated bubbles have a smaller specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q, and are removed from between the electrode pair 34 by moving in the vertical direction z without remaining between the electrode pair 34 due to the delivery of the suspension Q and sheath fluid s, thereby preventing the bubbles from interfering with the application of the electric field to the suspension Q. As a result, in the first example of the EP method, the electric field can be properly applied to the suspension Q, and as a result, the introduction efficiency and the utilization efficiency of the bioactive substance can be properly maintained.

[0069] The EP method may include, for example, a culturing step in which a culture solution containing a biologically derived material and a medium is obtained by cell culture prior to EP, and a mixing step in which the culture solution is mixed with a bioactive substance to form a suspension. Typically, prior to EP, biologically derived material (e.g., cells) is removed from the culture solution and suspended in an EP buffer to obtain a suspension. Replacing this medium with an EP buffer is simply referred to as medium exchange or buffer exchange. In a first example of the EP method, the culture medium obtained in the culturing step is mixed with a bioactive substance without being exchanged for an EP buffer, i.e., without adding an EP buffer, and the suspension is continuously pumped between the electrode pair 34. In this first example of the EP method, since the culture medium is not exchanged for an EP buffer, the resulting suspension contains more medium and has higher electrical conductivity than a suspension typically subjected to EP. Therefore, a large number of bubbles are generated when an electric field is applied. Even for such a suspension, the suspension is subjected to electroporation while moving vertically from bottom to top between the pair of electrodes, so that any bubbles generated between the pair of electrodes are removed from between the pair of electrodes by moving vertically, thereby preventing the bubbles from interfering with the application of an electric field to the suspension. The culturing step is carried out in the culturing device 24 as described above. The mixing step is carried out in the mixer 25 by using the pumps 12a and 12b to suck the culture solution from the culturing device 24 and the bioactive substance from the tank 23 as described above.

[0070] A first example of the EP method will be described in more detail below. FIG. 8 is a flowchart showing the first example of the electroporation method according to an embodiment of the present invention. As described above, a standby position for the head of the suspension Q is predetermined in the first tube 13, and standby positions for the head of the sheath fluid s are predetermined in each of the second tubes 21a and 21b. First, the sensor 38 (see FIGS. 1 and 2) located at the standby position for the head of the suspension Q in the first tube 13 begins to detect the standby position for the head of the suspension Q. Then, the sensors 39a and 39b (see FIGS. 1 and 2) located at the standby positions for the head of the sheath fluid s in the second tubes 21a and 21b begin to detect the standby positions for the head of the sheath fluid s in the second tubes 21a and 21b. The suspension Q is delivered to the first tube 13 by the pumps 12a and 12b. Furthermore, the sheath fluid s in the tank 22 is delivered to the second tube 21a by the pump 20a. The sheath fluid s in the tank 22 is sent to the second tube 21b by the pump 20b (step S10). At this time, as described above, the detection of the standby positions of the heads of the suspension Q and sheath fluid s has already been started by the sensor 38 (see FIGS. 1 and 2) and the sensors 39a and 39b (see FIGS. 1 and 2).

[0071] Next, it is determined whether the leading edge of the suspension Q and the leading edge of the sheath fluid s have reached their standby positions (step S12). In step S12, when the control unit 16 receives detection signals from the sensor 38 (see FIGS. 1 and 2) disposed at the standby position for the leading edge of the suspension Q and the sensors 39a, 39b (see FIGS. 1 and 2) disposed at the standby positions for the leading edge of the sheath fluid s, the control unit 16 determines that the leading edge of the suspension Q and the leading edge of the sheath fluid s have reached their standby positions. In step S12, when it is determined that the leading edges of the suspension Q and the sheath fluid s have all reached their standby positions, the control unit 16 temporarily stops driving the pumps 12a, 12b, 20a, and 20b, thereby stopping the delivery of the suspension Q and the sheath fluid s. Thereafter, with the leading edges of the suspension Q and the sheath fluid s all in their standby positions, the control unit 16 starts driving the pumps 12a, 12b, 20a, and 20b, thereby starting the delivery of the suspension Q and the sheath fluid s (step S14).

[0072] On the other hand, if the control unit 16 does not receive the above-mentioned detection signal, the control unit 16 determines that the front of the suspension Q and the front of the sheath fluid s have not reached the standby position. In this case, the control unit 16 continues to feed the front of the suspension Q and the sheath fluid s (step S26). It is again determined whether the front of the suspension Q and the front of the sheath fluid s have reached the standby position (step S12). Steps S12 and S26 are repeated until the control unit 16 determines that the fronts of all of the suspension Q and the sheath fluid s have reached the standby position.

[0073] After the feeding of the suspension Q and the sheath fluid s is resumed in step S14, the stability of the flow of the suspension Q and the sheath fluid s between the electrode pair 34 is determined (step S16). Step S16 is the determination process. In step S16 (determination process), as described above, for example, the measurement unit 19 of the determination unit 18 measures the electrical resistance of the flow of the suspension Q and the sheath fluid s between the electrode pair 34, and the stability of the flow is determined based on the electrical resistance between the electrode pair 34. For example, as described above, a range of electrical resistance between the electrode pair 34 indicating stable flow is set in advance, and if the measured electrical resistance between the electrode pair 34 is within the predetermined range, the determination unit 18 determines that the flow is stable, and outputs a determination signal to the control unit 16. In step S16 (determination process), the stability of the flow can also be determined by detecting the stability of the flow rate. In this case, as described above, the determination unit 18, for example, compares the set flow rate with the measured flow rate, and determines that the flow is stable when the set flow rate is maintained for a preset time after reaching the set flow rate, as shown in FIG. 7, and outputs a determination signal to the control unit 16. The determination step is performed before the application step described below. In step S16, the stability of the flow of the suspension Q and sheath liquid s between the electrode pair 34 can also be determined by the above-described time control. In step S16, the stability of the flow may be determined by any of the above-described methods.

[0074] If it is determined in step S16 that the flow is stable, the control unit 16 causes the power supply unit 14 to apply an electric field to the suspension Q (step S18). EP is performed in step S18. Step S18 is the application step. Since EP is continuous, the electric field is continuously applied for a predetermined period of time. If electrical resistance is used to determine the stability of the flow in step S16, as described above, the circuit breaker is used in step S16 to bring the determination unit 18 and the electrode pair 34 into a conductive state, and the power supply unit 14 and the electrode pair 34 are electrically insulated from each other to electrically insulate the power supply unit 14 and the determination unit 18, and then the measurement unit 19 measures the electrical resistance between the electrode pair 34. If electrical resistance is used to determine the stability of the flow in step S16, in step S18, as described above, the power supply unit 14 and the electrode pair 34 are brought into a conductive state by the circuit breaker, and the determination unit 18 and the electrode pair 34 are brought into an electrically insulated state, thereby electrically insulating the power supply unit 14 and the determination unit 18, and then an electric field is applied to the suspension Q by the power supply unit 14.

[0075] On the other hand, if it is not determined in step S16 that the flows are stable, the feeding of the suspension Q and the sheath fluid s continues (step S28). It is again determined whether the flows of the suspension Q and the sheath fluid s are stable (step S16). Steps S16 and S28 are repeated until it is determined that the flows of the suspension Q and the sheath fluid s are stable.

[0076] Next, after a predetermined time has elapsed since the application of the electric field in step S18 (application step), the discharge destination is changed (step S20). In step S20, the time elapsed since the application of the electric field in step S18 is measured, for example, by the control unit 16. Here, from the start of the supply of the suspension Q and the sheath fluid s (step S10) until the application of the electric field (step S18), the suspension Q and the sheath fluid s discharged from the discharge port 32 are sent to the waste tank 26 (see FIG. 1). After the application of the electric field (step S18), after a preset time has elapsed, for example, at time t ２ Then, the valve 29 (see FIG. 1) switches the liquid destination, and the liquid destination of the suspension Q and sheath liquid s discharged from the discharge port 32 is changed to the container 27 (see FIG. 1).

[0077] After it is determined in step S16 that the flow has stabilized, it is determined whether a predetermined time has elapsed (step S22). The time elapsed since it was determined in step S16 that the flow has stabilized is measured, for example, by the control unit 16, and the control unit 16 determines whether the predetermined time has elapsed. If it is determined in step S22 that the time elapsed since it was determined in step S16 that the flow has stabilized has elapsed the predetermined time, the control unit 16 controls the power supply unit 14 to stop applying the electric field to the suspension Q and further stops the pumps 12a, 12b and the pumps 20a, 20b to stop the delivery of the suspension Q and the sheath fluid s (step S24). On the other hand, if it is determined in step S22 that the time elapsed since it was determined in step S16 that the flow has stabilized has not elapsed the predetermined time, the delivery of the suspension Q and the sheath fluid s and the application of the electric field are continued until it is determined that the time elapsed since it was determined in step S16 that the flow has stabilized has elapsed the predetermined time. If it is determined in step S22 that the preset time has elapsed, the control unit 16 controls the power supply unit 14 to stop applying the electric field to the suspension Q, and further stops the pumps 12a, 12b and the pumps 20a, 20b to stop the delivery of the suspension Q and the sheath fluid s (step S24).

[0078] (Second Example of Electroporation Apparatus) FIG. 9 is a schematic cross-sectional view showing a second example of a flow path device of an electroporation apparatus (EP apparatus) according to an embodiment of the present invention. In FIG. 9, components identical to those of the EP apparatus 10 shown in FIGS. 1 and 2 are designated by the same reference numerals, and detailed description thereof will be omitted. The EP apparatus 10a shown in FIG. 9 differs from the EP apparatus 10 shown in FIGS. 1 and 2 in that it includes an auxiliary electrode 60, which is electrically connected to the measurement unit 19 of the determination unit 18. The EP apparatus 10a is otherwise similar in configuration to the EP apparatus 10 shown in FIGS. 1 and 2. In the EP apparatus 10a, the auxiliary electrode 60 is provided downstream of the electrode pair 34 in the main flow path 30. The auxiliary electrode 60 is used to measure the electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s. The determination unit 18 uses the auxiliary electrode 60 to measure the electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s. The electrical resistance in the thickness direction measured using the auxiliary electrode 60 includes impedance.

[0079] The auxiliary electrode 60 has a first electrode 61 and a second electrode 62 arranged opposite to each other. The first electrode 61 has an electrode surface 61a, which is flat. The second electrode 62 has an electrode surface 62a, which is flat. The electrode surface 61a of the first electrode 61 and the electrode surface 62a of the second electrode 62 are arranged opposite to each other and parallel to each other. The auxiliary electrode 60 is a counter electrode having the first electrode 61 and the second electrode 62. The electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s using the auxiliary electrode 60 is the electrical resistance between the first electrode 61 and the second electrode 62 constituting the auxiliary electrode 60 for the flow of the suspension Q and the sheath fluid s. The electrode surface 61a of the first electrode 61 and the electrode surface 36a of the second electrode 36 are each arranged facing the inner surface 30c of the main flow channel 30. The electrode surface 61 a of the first electrode 61 and the electrode surface 61 a of the second electrode 62 each constitute the inner surface 30 c of the main flow channel 30, and for example, when EP is performed and the suspension Q and the sheath fluid s are supplied to the main flow channel 30, the electrode surface 61 a comes into contact with the sheath fluid s. The first electrode 61 is electrically connected to the measurement unit 19 by a wiring 19 a. The second electrode 62 is electrically connected to the measurement unit 19 by a wiring 19 b.

[0080] The EP device 10a uses the measurement unit 19 and auxiliary electrode 60 of the determination unit 18 to measure, for example, impedance as the electrical resistance in the thickness direction of the flow of the suspension Q and sheath fluid s. Even in the case of the auxiliary electrode 60, the electrical resistance or impedance in the thickness direction when the flow of the suspension Q and sheath fluid s is stable is measured in advance, as shown in FIG. 4 , and a range of the electrical resistance or impedance in the thickness direction indicating that the flow is stable is preset. The preset range of the electrical resistance or impedance in the thickness direction is stored in the determination unit 18. The determination unit 18 determines that the flow is stable when the measured value of the electrical resistance or impedance in the thickness direction measured using the auxiliary electrode 60 is within the preset range. Information indicating the determination result of the determination unit 18 is output to the control unit 16. The control unit 16 controls various components, such as applying an electric field to the suspension Q using the power supply unit 14, based on the received determination result. By providing the auxiliary electrode 60 downstream of the electrode pair 34 in the main flow path 30, if the flow of the suspension Q and the sheath liquid s is stable at the auxiliary electrode 60 downstream of the electrode pair 34, the flow of the suspension Q and the sheath liquid s will also be stable between the electrode pairs 34 upstream of the auxiliary electrode 60.

[0081] Although the EP device 10a is configured such that the auxiliary electrode 60 is provided downstream of the electrode pair 34 in the main flow path 30, this is not limiting. For example, the auxiliary electrode 60 may be provided upstream of the electrode pair 34 in the main flow path 30. More specifically, the auxiliary electrode 60 may be provided in a region of the main flow path 30 corresponding to the junction 59 and the electrode pair 34. When the auxiliary electrode 60 is provided upstream of the main flow path 30 of the electrode pair 34, if the flow of the suspension Q and sheath fluid s is stable at the auxiliary electrode 60, the flow of the suspension Q and sheath fluid s is also stable between the electrode pair 34 downstream of the auxiliary electrode 60. The EP device 10a can achieve the same effects as the EP device 10 described above. The EP device 10a can also properly apply an electric field to the suspension Q, resulting in high introduction efficiency and bioactive substance utilization efficiency, reduced suspension Q waste, and high-productivity EP.

[0082] (Second Example of Electroporation Method) In a second example of the electroporation method (EP method), for example, an EP device 10a shown in Fig. 9 is used. The second example of the EP method differs from the first example of the EP method in that the determination step (step S16) for determining the stability of the flow of the suspension and sheath fluid between the electrode pair 34 does not measure the electrical resistance between the electrode pair 34, but rather the electrical resistance in the thickness direction of the flow of the suspension Q and sheath fluid s measured using the auxiliary electrode 60. The other steps are the same as those in the first example of the EP method, and therefore detailed description thereof will be omitted.

[0083] In step S16, for example, the auxiliary electrode 60 is used to measure the electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s by the measuring unit 19 of the determining unit 18, and the stability of the flow is determined based on the electrical resistance in the thickness direction measured using the auxiliary electrode 60. For example, as described above, a range of electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s indicating stability of the flow is set in advance, and if the measured electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s falls within the predetermined range, the determining unit 18 determines that the flow is stable. Note that, for example, if the auxiliary electrode 60 is provided upstream of the electrode pair 34 in the main flow path 30, in step S16 (determination step), the electrical resistance in the thickness direction of the flow of the suspension Q and the sheath fluid s is measured by the auxiliary electrode 60 upstream of the electrode pair 34 in the flow path, and the determining unit 18 determines the stability of the flow of the suspension Q and the sheath fluid s based on the electrical resistance in the thickness direction. The determining unit 18 outputs information indicative of the determination result to the control unit 16. The second example of the EP method can achieve the same effect as the first example of the EP method.

[0084] The above-described EP device and EP method can both be used to produce useful substances such as target proteins or viruses. That is, the method for producing useful substances includes the above-described EP method. Each part of the EP device will be described below.

[0085] [Distance D Between Electrode Pairs] The distance D between the electrode pair 34 (see Figures 2 and 9) is the distance between the electrode surface 35a of the first electrode 35 and the electrode surface 36a of the second electrode 36 in the opposing direction, and is preferably 1 to 10 mm. The lower limit of the distance D between the electrode pair is more preferably 2 mm, and even more preferably 3 mm. The upper limit of the distance D between the electrode pair is more preferably 6 mm, and even more preferably 5 mm. If the distance D between the electrode pair is too large, an excessively large electric field may be generated, resulting in heat generation, electric field concentration at the electrode edges, boiling or discharge, and a decrease in survival rate or introduction efficiency. From the viewpoint of preventing heat generation, the distance D between the electrode pair is preferably 1 to 6 mm. Furthermore, a distance D between the electrode pair of 1 to 3 mm is more preferable because it suppresses the generation of heat and discharge and also suppresses a decrease in survival rate or introduction efficiency of biological substances such as cells due to heat generation or discharge. The distance D between the electrode pair corresponds to the thickness of the flow path substrate 55. The distance D between the electrode pair is also referred to as the inter-electrode gap or gap. The distance D between the electrode pair can be measured by measuring the length of the relevant portion using a vernier caliper or a micrometer.

[0086] <Electrode Pair> The first electrode 35 and the second electrode 36 constituting the electrode pair 34 are made of, for example, a metal material or a carbon material. Specifically, a configuration similar to that of the electrodes described in WO 2023 / 157673 can be adopted.

[0087] (Thickness) The thickness of the first electrode 35 and the second electrode 36 is not particularly limited, but is preferably 0.5 to 10 mm, and more preferably 1 to 5 mm. The thickness of the first electrode 35 and the second electrode 36 can be measured at the corresponding portion using a vernier caliper or a micrometer.

[0088] <Auxiliary Electrode> The first electrode 61 and the second electrode 62 constituting the auxiliary electrode 60 have the same configuration as the first electrode 35 and the second electrode 36 constituting the electrode pair 34, and are made of, for example, the metal material described above. The thicknesses of the first electrode 61 and the second electrode 62 are not particularly limited, but, like the first electrode 35 and the second electrode 36, are preferably 0.5 to 10 mm, and more preferably 1 to 5 mm. The thicknesses of the first electrode 61 and the second electrode 62 described above can be measured at the corresponding portions using a vernier caliper or a micrometer. The distance D between the electrode pair is also not particularly limited, but, like the first electrode 35 and the second electrode 36, is preferably 1 to 10 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. The auxiliary electrode 60 is arranged in the extension direction D of the main flow channel 30. Ｌ The length at this point is preferably shorter than that of the electrode pair 34, and more preferably 2 to 10 mm.

[0089] <Configuration of Flow Channel Device> There are no particular limitations on the materials that make up the first introduction section 56, the first substrate 52, the flow channel substrate 55, the second substrate 54, and the second introduction section 58 of the flow channel device 11 in Fig. 2. Various materials can be used as the materials that make up these components, as long as they have sufficient resistance to the suspension, or the suspension and the sheath liquid, and can ensure the necessary rigidity.

[0090] <Suspension> The suspension to be subjected to electroporation (EP) contains a biologically derived substance and a bioactive substance. Furthermore, the suspension does not have the culture medium replaced with an EP buffer, nor does it have an EP buffer added. The electrical conductivity of the suspension is preferably 2 mS / cm or higher, more preferably 3 mS / cm or higher, and even more preferably 4 mS / cm or higher. Typically, before EP, the culture medium of the suspension is replaced with an EP buffer, and the medium in the suspension is replaced with the EP buffer. This reduces the electrical conductivity of the suspension, and reduces the generation of bubbles due to the application of an electric field. Suspensions with the above electrical conductivities are not normally suitable for EP, but can be used in the method of the present invention. The electrical conductivity (mS / cm) is the value measured using an electrical conductivity meter on a suspension at 25°C. Electrical conductivity (mS / cm) is synonymous with electrical conductivity, and electrical conductivity has the same units as electrical conductivity. Methods for adjusting the electrical conductivity include, for example, adjusting the volume fraction of the biologically derived substance contained in the suspension and changing the type of culture medium.

[0091] Typically, the culture medium (liquid components other than biologically derived substances) for the suspension of an EP target contains less than 0.1 g / L of poloxamer because the culture medium is exchanged with an EP buffer. The sodium chloride concentration is also less than 0.01 g / L. On the other hand, in the present invention, the culture medium is not exchanged with an EP buffer, so the poloxamer content of the medium is 0.1 g / L or more. The poloxamer content may be 1 g / L or more, 3 g / L or more, or 5 g / L or more. In the present invention, even if the poloxamer content is high, interference with the application of an electric field to the suspension Q due to air bubbles can be suppressed. The culture medium for the suspension of an EP target contains sodium chloride, and the sodium chloride content is 0.001 g / L or more, more preferably 0.01 g / L or more, 0.1 g / L or more, or 1 g / L or more. The sodium chloride content can be determined by centrifuging the suspension at 300 G for 15 minutes, collecting the supernatant, and measuring it by high-performance liquid chromatography. The suspension flow rate is preferably 1 mL / min or more, more preferably 2 mL / min or more, and even more preferably 3 mL / min or more. The upper limit of the suspension flow rate is preferably 1000 mL / min. A suspension flow rate of 1 mL / min or more is preferable in that it allows for a large amount of EP to be processed. The flow rate can be measured by installing various flow sensors (ultrasonic, Coriolis, thermal MEMS) in the liquid transfer flow path. Alternatively, the flow rate may be calculated from the weight change of the collection container and the liquid transfer time.

[0092] (Biologically derived products) Biologically derived products are not particularly limited, and specific examples include cells, organelles, intracellular granules and vesicles, and bacteria. Among these, cells are preferred, animal cells are more preferred, mammalian cells are even more preferred, and human or Chinese hamster-derived cells are most preferred, due to the superior effects of the present invention. Specific examples of cells include human T cells, HEK (Human Embryonic Kidney) 293, A549, SF9, EB66, Daudi, HeLa, Vero, MDCK, BHK (Baby Hamster Kidney), CHO (Chinese Hamster Ovary), NS0, SP2 / 0, hybridoma, etc. In terms of pharmaceutical production, gene transfer using HEK293 or CHO is most commonly used.

[0093] (Bioactive Substances) Bioactive substances are substances such as nucleic acids (e.g., DNA, RNA) and proteins that, when introduced into a biologically derived substance, exert some effect on the biologically derived substance. Examples of nucleic acids include plasmids, linear DNA, and mRNA (messenger RNA), with plasmids being particularly preferred. The concentration of the bioactive substance in the suspension is preferably 1 to 1000 μg / mL, more preferably 2.5 to 500 μg / mL, and even more preferably 10 to 200 μg / mL. The concentration of the bioactive substance in the suspension relative to the medium in the suspension is preferably 10 to 500 μg / mL. The concentration of the bioactive substance per biologically derived substance in the suspension is preferably 20 pg / unit or less, more preferably 5 pg / unit or less, even more preferably 1 pg / unit or less, and particularly preferably 0.5 pg / unit or less. The lower the concentration of the bioactive substance per biologically derived substance in the suspension, the less the amount of bioactive substance used per biologically derived substance, thereby reducing costs.

[0094] Although there is no limitation on the concentration of the biologically derived substance in suspension Q, it is preferable that the volume fraction of the biologically derived substance in suspension Q is 20% or more. In EP, by increasing the concentration of the biologically derived substance in suspension Q, the amount of biologically derived substance processed can be increased compared to when the same amount of a low-concentration suspension is introduced. For example, doubling the concentration allows for twice the processing, thereby improving the EP throughput and utilization efficiency of the biologically derived substance. In addition, in EP, as described above, bioactive substances are incorporated into the biologically derived substance by electrophoresis. Therefore, even if the concentration of the biologically derived substance is increased, there is no need to increase the amount of bioactive substance accordingly, and the utilization efficiency of the bioactive substance can also be improved.

[0095] By having a volume fraction of biologically derived substances in suspension Q of 20% or more, the above-mentioned effects can be suitably obtained, enabling efficient processing. Furthermore, if the concentration of suspension Q is too high, the viscosity of the cell suspension increases rapidly, potentially clogging the flow path. However, by setting the volume fraction of biologically derived substances in suspension Q to 70% or less, clogging of the flow path caused by an excessively high concentration of suspension Q can be suitably prevented. The volume fraction of biologically derived substances in suspension Q is more preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. The upper limit of the volume fraction of biologically derived substances in suspension Q is more preferably 65%, and even more preferably 60%. The volume fraction of biologically derived substances in suspension Q can be determined by measuring the average diameter and concentration (number per unit volume) of the biologically derived substances by image analysis (for example, Vi-CELL XR (Beckman Coulter)), and calculating the volume fraction (= 4 ÷ 3 × (average diameter ÷ 2) 3 ×π×concentration×100(%)) can be calculated. The volume fraction is calculated assuming that the biologically derived substances are spherical. More specifically, for example, if the average diameter of the biologically derived substances in suspension Q is 20 μm and the concentration is 40×10 ６ When the volume fraction of the biologically derived material in the suspension Q is 17%, the volume fraction of the biologically derived material in the suspension Q can be calculated to be 17%.

[0096] Liquid media typically used in the culture of animal cells can be used. Various companies sell media based on phosphate-buffered saline (PBS) optimized for culturing cells such as HEK293. The media may contain, but are not limited to, amino acids, salts, sugars (e.g., glucose), vitamins, hormones, growth factors, lipids, and trace elements. The pH (hydrogen ion exponent) of the medium is 6 to 8, preferably 6.8 to 7.6, and more preferably 7.2 to 7.6. An antifoaming agent may also be added to the medium. Silicone-based antifoaming agents are preferred, with dimethicone being particularly preferred. Antifoaming agents containing polydimethylsiloxane are preferred, and polydimethylsiloxane containing finely powdered silica (simethicone) is more preferred. The rate of simethicone addition relative to the volume of culture solution is not particularly limited, but is preferably 5 mg / hr / L or less, more preferably 2 mg / hr / L or less. A block copolymer of polyoxypropylene and polyoxyethylene may also be added to the medium. As the copolymer of polyoxypropylene and polyoxyethylene, poloxamer is preferred, and poloxamer-188 is more preferred.

[0097] In the present invention, it is sufficient that the thickness Dm of the suspension flow is 1 to 10 mm in a portion between the electrode pair, and that the thickness Dm of the suspension flow is 1 to 10 mm between the electrode pair and at the upstream end of the electrodes. Regarding the thickness of at least a portion of the suspension flow between the electrode pair, a suspension flow having a cell concentration of 80% or more is identified, and the thickness of the identified suspension flow is measured.

[0098] <Sheath Liquid> Various liquids other than suspensions can be used as the sheath liquid flow. A preferred example of a sheath liquid is a culture medium used for suspensions. This culture medium is preferably a culture medium used for growing biological substances such as cells. As described above, there are two sheath liquid flows. The flow rates of the two sheath liquid flows are preferably 0.1 mL / min or more. The upper limit of the flow rate of the sheath liquid is preferably equal to or less than the flow rate of the suspension. Setting the flow rate of the sheath liquid to 0.1 mL / min or more is preferable in that it can effectively prevent the suspension from contacting the electrodes. Setting the flow rate of the sheath liquid to equal to or less than the flow rate of the suspension is also preferable in that it can reduce the amount of sheath liquid and prevent dilution of the suspension.

[0099] The pump preferably delivers the suspension at a flow rate of 1 mL / min or more, more preferably at a flow rate of 2 mL / min or more, and even more preferably at a flow rate of 3 mL / min or more. The upper limit of the flow rate of the suspension delivered by the pump is preferably 10 mL / min. The pump preferably delivers the sheath fluid forming each of the two sheath fluid streams at a flow rate of 0.1 mL / min or more. The upper limit of the flow rate of the sheath fluid delivered by the pump to each of the two sheath fluid streams is preferably equal to or less than the flow rate of the suspension.

[0100] Here, in EP, when an electric field is applied, holes open in the membrane (or shell) of the biological material, allowing the bioactive substance to enter the biological material through electrophoresis. The holes formed in the membrane of the biological material gradually close over time, but if the liquid components (osmotic pressure, ion concentration, etc.) outside the biological material suddenly change before the holes close, for example, due to operations such as diluting the suspension, the movement of ions and medium through the holes can cause a sudden change in the component ratio within the biological material, as well as damage such as deformation and expansion. As a result, for example, in the case of substance introduction into cells, the viability of the biological material can decrease, potentially reducing the introduction efficiency and utilization efficiency of the bioactive substance during EP. If the thickness ds of the sheath liquid flow is greater than the thickness Dm of the suspension flow and the dilution rate of the suspension Q is high, for example, in the case of substance introduction into cells, the viability of the biological material can decrease (become damaged), resulting in low EP efficiency. As a result of investigations by the inventors, it was confirmed that, for example, when a cell suspension is diluted with a culture medium sheath fluid flow, a significant decrease in cell viability is often observed when the dilution rate is more than 2. For this reason, as described above, by making the total thickness of the sheath fluid flow at the time of merging with the suspension flow less than the thickness of the suspension flow in the regulated region, i.e., by making the dilution rate of suspension Q less than 2, it is possible to suppress the problems caused by a large amount of sheath fluid s being mixed into the biological substance, such as a decrease in the viability of the biological substance in the case of substance introduction into cells, and thereby enable efficient processing.

[0101] In order to more suitably obtain this effect, the total thickness of the sheath liquid flow at the time of merging with the suspension flow is preferably 1 time or less, more preferably 0.8 time or less, and even more preferably 0.5 time or less, of the thickness of the suspension flow in the regulated region, as described above. In other words, the dilution ratio of the suspension Q downstream of the electrode pair is preferably 2 time or less, more preferably 1.8 time or less, and even more preferably 1.5 time or less, as described above.

[0102] <Electroporation Voltage> The electroporation voltage is set so that the electric field applied to the suspension between the electrode pair has a desired electric field strength, taking into account the thickness of the suspension flow, the electrical conductivity (electrical conductivity) of the suspension flow, the thickness of the sheath liquid flow, and the electrical conductivity (electrical conductivity) of the sheath liquid flow. Note that the electrical resistance of the electrodes is preferably sufficiently lower than the resistance of the suspension and sheath liquid, as this may result in a decrease in the introduction efficiency and utilization efficiency of the bioactive substance during EP. The optimal value of the electric field applied to the suspension varies depending on the type and size of the biological material, but is typically approximately 100 to 2000 V / cm. The voltage is preferably a pulse voltage. Furthermore, a bipolar pulse (alternating positive and negative) may be used to uniformly distribute electrode reactions (e.g., gas generation by electrolysis, electrode deterioration, etc.).

[0103] <Pulse Width> The optimum value of the pulse width varies depending on the type of biological substance, etc., but is usually 0.1 to 100 ms (milliseconds), preferably about 1 to 10 ms.

[0104] <Pulse Period (Pulse Interval)> The pulse period is preferably synchronized (an integer multiple of) the time it takes for the biologically derived substance to pass through the electrode (electrode length L (see Figures 2 and 9)). For example, a pulse voltage is applied 1 to 5 times, preferably once, on average while the biologically derived substance passes through the electrode (electrode length L). The time it takes for the biologically derived substance to pass through the electrode (electrode length L) is determined by the flow rate and the cross-sectional area of ​​the flow channel. When the cross-sectional shape of the flow channel is a rectangle with all interior angles at 90°, the cross-sectional area of ​​the flow channel is (electrode width W) × (distance D between the electrode pair). The electrode width W is shown in Figure 1, and the distance D between the electrode pair is shown in Figures 2 and 9.

[0105] This specification incorporates by reference the contents of International Application No. PCT / JP2024 / 012393, including (1) pre-culture of cells, (2) adjustment of cell concentration (concentration, etc.), (3) addition and mixing of nucleic acid, (4) electroporation (gene introduction), (5) culture for virus production (main culture), and (6) virus recovery and purification.

[0106] <Method for producing a useful substance> As described above, the method for producing a useful substance includes the EP method of the present invention. In the method for producing a useful substance, preferably, the biologically derived substance is a cell, and the bioactive substance is a nucleic acid. By using the EP method of the present invention, the nucleic acid is introduced into the cell, and the cell into which the nucleic acid has been introduced is cultured, thereby causing the cell to produce the useful substance.

[0107] The type of useful substance is not particularly limited, but is preferably a protein or a virus. Examples of useful substances include recombinant polypeptide chains, recombinant secreted polypeptide chains, antigen-binding proteins, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, bispecific antibodies, Fc fusion proteins, fragmented immune immunoglobulins, single-chain antibodies (scFv), and non-enveloped viruses. More specifically, non-enveloped viruses include adeno-associated viruses, adenoviruses, lentiviruses, baculoviruses, and retroviruses. Non-enveloped viruses are known in the art and are described in International Publication No. WO 2015 / 005430, which is incorporated herein by reference.

[0108] The useful substance is preferably a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, or an adeno-associated virus. The method for producing the useful substance may include recovery and purification steps.

[0109] The electroporation method, useful substance production method, and electroporation apparatus of the present invention can be suitably used for the production of gene therapy drugs, etc. The present invention is basically configured as described above. The electroporation method, useful substance production method, and electroporation apparatus of the present invention have been described in detail above, but the present invention is not limited to the above-described embodiments, and various improvements and modifications may be made without departing from the spirit and scope of the present invention.

[0110] 10, 10a EP device 11 flow path device 11a front surface 11b back surface 12a, 12b, 20a, 20b pump 13 first tube 14 power supply unit 15 piping 16 control unit 18 determination unit 21a, 21b second tube 22, 23 tank 24 culture device 25 mixer 26 waste tank 27 container 28 third tube 29 valve 30 main flow path 30a, 30b end 30c inner surface 31 inlet 31a first inlet 31b second inlet 32 ​​outlet 34 electrode pair 35, 61 first electrode 35a, 36a, 61a, 62a electrode surface 36, 62 second electrode 37 space 38, 39a, 39b sensor 40 electroporation system 52 First substrate 52a, 52b, 54a, 56a, 56b, 58a Through-hole 52c, 54c Sheath liquid supply port 54 Second substrate 55 Flow path substrate 55a, 55b Convex portion 55c Downstream end portion 55d Suspension supply port 56 First introduction portion 58 Second introduction portion 59 Confluence portion 60 Auxiliary electrode 64, 65 Sheath liquid flow path 70, 72 Line indicating change in flow rate of sheath liquid 71 Line indicating change in flow rate of suspension Bs Interface D Distance Dd Liquid transfer direction Df Direction D Ｌ Extension direction Dm, ds Thickness Ds Stacking direction Hp Horizontal plane L Electrode length Q Suspension W Electrode width s Sheath liquid x, y directions z Vertical direction

Claims

1. An electroporation method in which a suspension containing a biologically derived substance and a bioactive substance and a sheath liquid are continuously fed through a main flow path between a pair of electrodes so that the suspension is sandwiched between the sheath liquid, and an electric field is applied to the suspension by the pair of electrodes, thereby introducing the bioactive substance into the biologically derived substance, the electroporation method comprising an application step of applying an electric field to the suspension after the flow of the suspension and the sheath liquid between the pair of electrodes has stabilized.

2. The electroporation method of claim 1, wherein when the sheath fluid is fed to the main flow path so as to sandwich the suspension, a first tube is used to supply the suspension and a second tube is used to supply the sheath fluid, a standby position for the front of the suspension is predetermined in the first tube, and a standby position for the front of the sheath fluid is predetermined in the second tube, and feeding of the suspension and the sheath fluid begins after the fronts of the suspension and the sheath fluid have all reached the standby positions.

3. The electroporation method according to claim 1 or 2, further comprising, before the application step, a determination step of determining stability of the flow of the suspension and the sheath fluid between the electrode pair, wherein the determination step measures the electrical resistance of the flow of the suspension and the sheath fluid between the electrode pair and determines stability of the flow based on the electrical resistance, or the determination step detects stability of the flow rate of the suspension in the first tube and the flow rate of the sheath fluid in the second tube to determine stability of the flow.

4. The electroporation method of claim 3, wherein the determination step measures the electrical resistance of the flow of the suspension and the sheath liquid between the electrode pair on the upstream or downstream side of the main flow path of the electrode pair, and determines the stability of the flow based on the electrical resistance.

5. An electroporation method according to claim 1 or 2, wherein the timing of feeding the suspension and the sheath liquid is simultaneous.

6. An electroporation method according to claim 1 or 2, wherein the timing of feeding the suspension and the sheath liquid is such that the sheath liquid is fed before the suspension.

7. An electroporation method in which a suspension containing a biological substance and a bioactive substance and a sheath fluid are continuously fed into a main flow path between a pair of electrodes so as to sandwich the suspension between the sheath fluid, and an electric field is applied to the suspension by the pair of electrodes, thereby introducing the bioactive substance into the biological substance; wherein when the sheath fluid is fed into the main flow path so as to sandwich the suspension, a first tube is used to supply the suspension and a second tube is used to supply the sheath fluid, a standby position for the head of the suspension is predetermined in the first tube, and a standby position for the head of the sheath fluid is predetermined in the second tube, the feeding of the suspension and the sheath fluid is started after it is detected that the heads of the suspension and the sheath fluid have all reached the standby position, and after the feeding of the suspension and the sheath fluid, an electric field is applied to the suspension after the flows of the suspension and the sheath fluid between the pair of electrodes have stabilized.

8. An electroporation method according to claim 1 or 7, wherein in the application step, the electric field is applied to the suspension after a predetermined time has elapsed after the start of the delivery of the suspension and the sheath liquid.

9. The electroporation method of claim 7, wherein the position of the front of the suspension in the first tube is detected with an ultrasonic sensor.

10. A method for producing a useful substance, comprising the electroporation method according to any one of claims 1 to 9.

11. An electroporation device that introduces a bioactive substance into a biologically derived substance by continuously feeding a suspension containing the biologically derived substance and a bioactive substance and applying an electric field, the electroporation device comprising: a main flow path; an electrode pair having electrodes arranged opposite each other and applying the electric field to the suspension flowing through the main flow path; a suspension supply port that supplies the suspension to the main flow path; a sheath fluid supply port that is provided in the main flow path downstream of the suspension supply port and supplies sheath fluid to the main flow path; a pump that feeds the suspension and the sheath fluid; and a power supply unit that applies an electric field to the suspension after the flows of the suspension and the sheath fluid between the electrode pair have stabilized.

12. An electroporation device as described in claim 11, comprising: a sheath fluid flow path that is connected to the sheath fluid supply port and that merges the sheath flow with the suspension in the main flow path; a first tube that is connected to the suspension supply port and that supplies the suspension to the main flow path; and a second tube that is connected to the sheath fluid supply port and that supplies the sheath fluid into the main flow path, wherein a standby position for the front of the suspension is predetermined in the first tube and a standby position for the front of the sheath fluid is predetermined in the second tube, and further comprising a control unit that starts the delivery of the suspension and the sheath fluid by the pump after all of the fronts of the suspension and the sheath fluid have reached the standby positions.

13. An electroporation device as described in claim 12, further comprising a determination unit that determines the stability of the flow of the suspension and the sheath fluid between the electrode pair, wherein the determination unit measures the electrical resistance of the flow of the suspension and the sheath fluid between the electrode pair and determines the stability of the flow based on the electrical resistance, or the determination unit detects the stability of the flow rate of the suspension in the first tube and the flow rate of the sheath fluid in the second tube and determines the stability of the flow.

14. An electroporation device as described in claim 13, which has an auxiliary electrode provided upstream or downstream of the electrode pair in the main flow path, and the determination unit uses the auxiliary electrode to measure the electrical resistance in the thickness direction of the flow of the suspension and the sheath liquid, and determines the stability of the flow based on the electrical resistance.

15. An electroporation device as described in claim 12, further comprising a sensor that detects the leading position of the suspension in the first tube and the leading position of the sheath fluid in the second tube, and the control unit controls the pump to deliver the suspension and the sheath fluid based on the leading position of the suspension in the first tube and the leading position of the sheath fluid in the second tube detected by the sensor.

16. An electroporation device for introducing a bioactive substance into a biologically derived substance by continuously feeding a suspension containing the biologically derived substance and a bioactive substance and applying an electric field to the suspension, the device comprising: a main flow path; an electrode pair having electrodes arranged opposite to each other and applying the electric field to the suspension flowing through the main flow path; a power supply unit for applying the electric field to the suspension; a suspension supply port for supplying the suspension to the main flow path; a sheath fluid supply port provided in the main flow path downstream of the suspension supply port and supplying the sheath fluid to the main flow path; a pump for feeding the suspension and the sheath fluid; a first tube connected to the suspension supply port for supplying the suspension to the main flow path; a second tube connected to the sheath fluid supply port for supplying the sheath fluid into the main flow path; a sensor for detecting the position of the front of the suspension in the first tube and the position of the front of the sheath fluid in the second tube; and a control unit for controlling the feeding of the suspension and the sheath fluid by the pump. an electroporation device, wherein a standby position for the head of the suspension is predetermined in the first tube, and a standby position for the head of the sheath fluid is predetermined in the second tube; the control unit starts the pump to feed the suspension and the sheath fluid after the sensor detects that the heads of the suspension and the sheath fluid have all reached the standby position; and after the suspension and the sheath fluid have been fed, the power supply unit applies the electric field to the suspension after the flows of the suspension and the sheath fluid have stabilized between the electrode pair.

17. An electroporation device as described in claim 12 or 15, wherein the control unit starts the pump to deliver the suspension and the sheath liquid, and after a preset time has elapsed, the power supply unit applies the electric field to the suspension.

18. An electroporation apparatus according to claim 15 or 16, wherein the sensor that detects the position of the front of the suspension in the first tube is an ultrasonic sensor.

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