Cleaning method for flow channel device

Abstract

Provided is a cleaning method for a flow channel device for appropriately cleaning a flow channel. This cleaning method for a flow channel device is used in electroporation, in which a biologically active substance is introduced into a biologically derived material by using an electrode pair to apply an electric field to a suspension containing the biologically derived material and the biologically active substance, as the suspension is flowing. The cleaning method for a flow channel device includes: a step for stopping supply of the suspension in electroporation and using a gas to discharge the suspension from a suspension flow channel in the flow channel device; a step for cleaning the flow channel by supplying a cleaning liquid to the flow channel; and a step for stopping supply of the cleaning liquid and storing the flow channel device in a state in which the flow channel is filled with the cleaning liquid.

Description

Method for cleaning flow channel devices

[0001] The present invention relates to a method for cleaning a fluidic device used in electroporation, a process in which a suspension containing a bio-derived material and a bioactive substance is flowed through a suspension and an electric field is applied to the suspension to introduce a bioactive substance into a bio-derived material. More particularly, the present invention relates to a method for cleaning a fluidic device in which the fluidic channel is filled with a cleaning solution and stored.

[0002] In recent years, approvals for adeno-associated virus (AAV) gene therapy have increased, necessitating cost reductions in manufacturing and a stable supply of viral vectors. Triple transfection (TT) is the most widely used chemical transfection method for producing recombinant AAV (rAAV) gene therapy products. On the other hand, physical methods for transfecting cells with various substances, such as electroporation, are also known (Patent Documents 1 and 2). Hereinafter, electroporation may be abbreviated as EP. Electroporation involves creating transient pores in the cell membrane using high-voltage electrical pulses, through which plasmid DNA diffuses into the cell. However, standard static cuvette approaches and existing approaches have low throughput and are not suitable for large-scale production.

[0003] Patent documents 1 and 2 also disclose a continuous electroporation method in which a suspension containing biologically derived substances and bioactive substances is circulated in a channel in which an electrode pair is installed.

[0004] Japanese Patent Publication No. 2007-7430, U.S. Patent No. 11225638, Japanese Patent Publication No. 2023-125991

[0005] In batch-type EP (Electron Processing), for example, Patent Document 3 proposes cleaning the inside of the substance introduction unit with a cleaning solution after EP to suppress a decrease in introduction efficiency. Patent Documents 1 and 2 mentioned above do not describe a method for cleaning the flow path. The object of the present invention is to provide a method for cleaning a flow path device that properly cleans the flow path.

[0006] The above objective can be achieved with the following configuration. Invention [1] is a method for cleaning a flow channel device used in electroporation, in which a bioactive substance is introduced into a bio-derived substance by applying an electric field to the suspension using an electrode pair while a suspension containing a bio-derived substance and a bioactive substance is flowed through the device, and the method includes the steps of stopping the supply of the suspension in the electroporation and discharging the suspension from the flow channel of the flow channel device using gas, supplying a cleaning solution to the flow channel to clean the flow channel, and stopping the supply of the cleaning solution and storing the flow channel device with the flow channel filled with the cleaning solution.

[0007] Invention [2] is a method for cleaning a flow channel device according to Invention [1], wherein in the step of discharging the suspension from the flow channel, the gas is flowed in the opposite direction to the direction of liquid delivery in the flow channel of the suspension in electroporation. Invention [3] is a method for cleaning a flow channel device according to Invention [1] or [2], wherein the flow channel extends in the vertical direction, the direction of liquid delivery of the suspension between the electrode pair is in the direction upward in the vertical direction, and the direction in which the gas is flowed is in the direction downward in the vertical direction. Invention [4] is a method for cleaning a flow channel device according to any one of Inventions [1] to [3], wherein in the step of cleaning the flow channel, the cleaning solution is flowed in the same direction as the direction of liquid delivery in the flow channel of the suspension in electroporation. Invention [5] is a method for cleaning a flow channel device according to any one of Inventions [1] to [4], wherein the cleaning solution contains water. Invention [6] is a method for cleaning a flow channel device according to any one of Inventions [1] to [4], wherein the cleaning solution contains ion-exchanged water. Invention [7] is a method for cleaning a flow channel device used in electroporation, in which a bioactive substance is introduced into a bio-derived substance by applying an electric field to the suspension using an electrode pair while a suspension containing a bio-derived substance and a bioactive substance is flowed through the device, and the method includes the steps of stopping the supply of the suspension in electroporation and discharging the suspension from the flow channel of the flow channel device using gas, and supplying a cleaning solution to the flow channel to clean the flow channel, wherein in the step of discharging the suspension from the flow channel, the gas is flowed in the opposite direction to the flow direction of the suspension in the flow channel of electroporation.

[0008] According to the present invention, a method for cleaning a flow channel device that properly cleans the flow channel can be provided.

[0009] This is a schematic diagram showing a first example of an electroporation system having an electroporation apparatus to which the cleaning method for a flow channel device according to an embodiment of the present invention is applied. This is a schematic cross-sectional view showing an example of a flow channel device in an electroporation apparatus to which the cleaning method for a flow channel device according to an embodiment of the present invention is applied. This is a schematic cross-sectional view for explaining the operation of a flow channel device in an electroporation apparatus to which the cleaning method for a flow channel device according to an embodiment of the present invention is applied. This is a schematic diagram for explaining a first example of an electroporation method in a first example of an electroporation system according to an embodiment of the present invention. This is a schematic diagram for explaining a cleaning method for a flow channel device for a first example of an electroporation system according to an embodiment of the present invention. This is a schematic diagram showing a second example of an electroporation system having an electroporation apparatus to which the cleaning method for a flow channel device according to an embodiment of the present invention is applied.

[0010] The cleaning method for the flow channel device of the present invention will be described in detail below based on the preferred embodiments shown in the attached drawings. The figures described below are illustrative for illustrating the present invention and have been simplified for illustrative purposes; the present invention is not limited to the figures shown below. In the following, the "~" indicating a numerical range includes the numerical values ​​indicated on both sides. For example, ε is the numerical value ε α ~ numerical value ε β The range of ε is the numerical value ε α and the numerical value ε β This range includes ε α ≦ε≦ε βFurthermore, unless otherwise specified, the following error ranges are included for specific angles, parallelism, perpendicularity, and verticality, etc., which are generally acceptable in the relevant technical field. In addition, unless otherwise specified, the following error ranges are included for length, width, and thickness, etc., which are generally acceptable in the relevant technical field. Furthermore, unless otherwise specified, the following error ranges are included for temperature, flow rate, electrical conductivity, conductivity, and volume fraction, etc., which are generally acceptable in the relevant technical field.

[0011] (First Example of an Electroporation System) Figure 1 is a schematic diagram showing a first example of an electroporation system (EP system) having an electroporation apparatus (EP apparatus) to which the cleaning method for a flow channel device of an embodiment of the present invention is applied. Figure 2 is a schematic cross-sectional view showing an example of a flow channel device in an electroporation apparatus to which the cleaning method for a flow channel device of an embodiment of the present invention is applied. Figure 3 is a schematic cross-sectional view for explaining the operation of the flow channel device in an electroporation apparatus to which the cleaning method for a flow channel device of an embodiment of the present invention is applied. The EP system 10 shown in Figure 1 has an EP apparatus 11, and the EP apparatus 11 has a flow channel device 12. The EP apparatus 11 is an apparatus that performs electroporation to introduce a bioactive substance into a bio-derived substance by continuously supplying a suspension Q (see Figure 2) containing a bio-derived substance and a bioactive substance, and a sheath liquid s (see Figure 2) to the main flow channel 40 between an electrode pair 44 (see Figure 2) having electrodes arranged opposite each other, with the suspension Q sandwiched by the sheath liquid s, and applying an electric field to the suspension Q with the electrode pair 44. The EP device 11 is a continuous EP device that uses suspension Q and sheath liquid s, and performs EP by continuously supplying the suspension Q between the electrode pair 44 with the sheath liquid s sandwiching the suspension Q. Performing EP by continuously supplying suspension Q and sheath liquid s between the electrode pair 44 with the suspension Q sandwiched between the sheath liquid s is also called continuous electroporation or continuous EP. Since the suspension Q is supplied with the suspension Q sandwiched between the sheath liquid s, there are two flows of sheath liquid s, i.e., two sheath liquid flows. This results in a three-layered flow consisting of sheath liquid flow, suspension flow and sheath liquid flow. Performing EP by continuously supplying only suspension Q between the electrode pair 44 is also called continuous EP.

[0012] The EP device 11 includes a flow path device 12, a first pump 13a that supplies suspension Q to the flow path device 12, and second pumps 13b and third pumps 13c that supply sheath fluid s to the flow path device 12. The flow path device 12 is covered by, for example, a cover 66. The cover 66 is for protecting the flow path device 12 and preventing electric shock, and is made of a transparent material such as a transparent acrylic plate. The structure of the cover 66 can be, for example, a form that encloses only the exposed parts of the energized part of the flow path device, or a form that encloses the entire flow path device. In the case of the form that encloses the entire flow path device, outlets for various pipes such as the first pipe 14, the second pipe 15, the third pipe 16, and the discharge pipe 28 are required. The cover 66 may be fixed to the flow path device 12 with a tool, or it may be an open / close type with hinges, but in the case of an open / close type, an interlock mechanism using a sensor that detects when the cover 66 is opened is required. Furthermore, "transparent" means that the light transmittance in the visible light wavelength range of 380 to 780 nm is 40% or more, preferably 80% or more, and more preferably 90% or more. In the following description, unless otherwise specified, "transparent" means transparent to visible light, i.e., light in the wavelength range of 380 to 780 nm. Light transmittance is measured using the method specified in JIS (Japanese Industrial Standards) K 7375:2008, "Plastics - Method for determining total light transmittance and total light reflectance."

[0013] The flow path device 12 and the first pump 13a are connected by a first pipe 14. The flow path device 12 and the second pump 13b are connected by a second pipe 15. The flow path device 12 and the third pump 13c are connected by a third pipe 16. In addition, a discharge pipe 28 for discharging the suspension Q and sheath liquid s to the outside is connected to the flow path device 12. Furthermore, the EP device 11 has a power supply unit 18 for performing EP and a control unit 19 for controlling each part that constitutes the EP device 11. The control unit 19 also controls each part that constitutes the EP device 11 based on detection signals from, for example, the detection units 29, 29a, 29b and various sensors described later.

[0014] A tank 22 is connected to the first pump 13a by piping 20. Tank 22 stores suspension Q. Suspension Q is a mixture of a culture medium containing biologically derived substances and culture medium, and a bioactive substance. Suspension Q is used in EP without replacing the culture medium in the culture medium with EP buffer. In addition, between the first pump 13a and the tank 22, for example, a tank 23a for storing washing solution, a tank 23b for storing liquid culture medium, and a gas supply unit 23c for supplying gas such as air are branched off from piping 20 from the first pump 13a side. A valve 24a is provided between tank 23a and piping 20 to control the passage of washing solution from tank 23a to piping 20. A valve 24b is provided between tank 23b and piping 20 to control the passage of liquid culture medium from tank 23b to piping 20. A valve 24c is provided between the gas supply unit 23c and the piping 20 to control the passage of gas, such as air, from the gas supply unit 23c to the piping 20. Note that the liquid culture medium is also simply referred to as the culture medium.

[0015] The gas supply unit 23c supplies gas, such as air, to the first pipe 14. The configuration of the gas supply unit 23c is not particularly limited as long as it can supply gas to the first pipe 14. For example, it may be a cylinder filled with a gas such as air, or a compressor that compresses and sends out a gas such as air. A valve 24d is provided between the tank 22 and the branch of the gas supply unit 23c to control the passage of the suspension Q from the tank 22 to the pipe 20. A liquid detection sensor 25 is provided between the first pump 13a and the branch of the tank 23a. When liquid is detected by the liquid detection sensor 25, a detection signal is output to the control unit 19. Any known valve can be used as appropriate for valves 24a, 24b, 24c, and 24d; for example, a pinch valve can be used. Any valve 24a, 24b, 24c, and 24d may be controlled to open or close by the control unit 19.

[0016] The second pump 13b and the third pump 13c are connected to a tank 27a and a gas supply unit 27b by piping 26. Tank 27a stores sheath fluid s for supplying sheath fluid s to the flow path device 12. The configuration of tank 27a is not particularly limited as long as it can store sheath fluid s. Gas supply unit 27b supplies gas, such as air, to the second pipe 15 and the third pipe 16. The configuration of gas supply unit 27b is not particularly limited as long as it can supply gas to the second pipe 15 and the third pipe 16, and is the same as that of gas supply unit 23c described above. Gas supply unit 27b may be, for example, a compressor that compresses and sends out a gas such as air, or a cylinder filled with a gas such as air. Tank 27a is provided with a valve 27c that controls the passage of suspension Q to piping 26. The gas supply unit 27b is provided with a valve 27d that controls the passage of air or other gas into the piping 26. Both valves 27c and 27d can be any known valve as appropriate; for example, a pinch valve can be used. Both valves 27c and 27d may be controlled to open or close by a control unit 19, for example.

[0017] In the first piping 14, between the flow path device 12 and the first pump 13a, a detection unit 29, a flow sensor 30, a valve 31a, a valve 31b, and a pressure sensor 32 are provided in this order from the flow path device 12 side. In the first piping 14, a branch passage 33a is provided between valve 31a and valve 31b, and a recovery container 33b is connected to this branch passage 33a. In the first piping 14, a fourth piping 17 is connected to a branch section 33c between valve 31b and the pressure sensor 32. The fourth piping 17 is connected to a discharge pipe 28, connecting the first piping 14 and the discharge pipe 28. The fourth piping 17 is used when supplying a gas such as air to the main flow path 40 of the flow path device 12. A valve 31d is provided in the fourth piping 17. Valves 31a, 31b, 31c, and 31d control the passage of fluids such as gas and liquid in the first pipe 14, the fourth pipe 17, and the branch passage 33a. Any known valve can be used for valves 31a, 31b, 31c, and 31d; for example, pinch valves can be used. Valves 31a, 31b, 31c, and 31d may also be controlled by the control unit 19, for example.

[0018] In the second piping 15, a detection unit 29a, a valve 34a, a flow sensor 30a, and a pressure sensor 32 are provided in this order from the flow device 12 side between the flow device 12 and the second pump 13b. In the second piping 15, a branch passage 33a is provided between the valve 34a and the flow sensor 30a, and a recovery container 33b is connected to this branch passage 33a. A valve 34b is provided in the branch passage 33a. In the third piping 16, a detection unit 29b, a valve 34a, a flow sensor 30a, and a pressure sensor 32 are provided in this order from the flow device 12 side between the flow device 12 and the third pump 13c. In the third piping 16, a branch passage 33a is provided between the valve 34a and the flow sensor 30a, and a recovery container 33b is connected to this branch passage 33a. A valve 34b is provided in the branch passage 33a. Valves 34a and 34b control the passage of fluids such as gas and liquid in the second pipe 15, the third pipe 16, and the branch passage 33a. Any known valve can be used for valves 34a and 34b; for example, pinch valves can be used. Both valves 34a and 34b may be controlled by the control unit 19, for example.

[0019] The detection unit 29 described above is used in combination, for example, a conductivity sensor and an ultrasonic sensor. This configuration makes it possible to determine the type of fluid supplied to the first pipe 14. Specifically, the ultrasonic sensor is used to distinguish between gases and liquids. However, while the ultrasonic sensor can distinguish between gases and liquids, it is difficult to accurately determine the type of liquid. On the other hand, the conductivity sensor can distinguish between liquids such as culture media, culture media, suspensions, and washing solutions by utilizing the difference in conductivity. In this way, by combining the conductivity sensor and the ultrasonic sensor, it is possible to distinguish between gases such as air, culture media, culture media, suspensions, and washing solutions passing through the first pipe 14. This makes it possible to distinguish between the discharge of suspension Q using gas, the passage of washing solution through the first pipe 14, and the passage of gas through the first pipe 14 in the washing method. Furthermore, since it is possible to distinguish between gases, etc., passing through the first pipe 14, it is possible to detect control problems such as faulty valve installation or valves being able to open and close. In addition, the detection unit 29 uses the ultrasonic sensor to detect the position of the leading edge of suspension Q in the first pipe 14. In the detection unit 29, it is preferable to position the ultrasonic sensor that detects the leading position of the suspension Q closer to the flow channel device 12 than the conductivity sensor. The conductivity sensor can measure the concentration of cells contained in the suspension Q by utilizing the difference in conductivity depending on the cell concentration. For this reason, for example, if the cell concentration measured by the conductivity sensor is less than a preset cell concentration, the supply of suspension Q from the first pump 13a may be stopped.

[0020] Furthermore, the detection units 29a and 29b described above detect, for example, the position of the leading edge of the sheath liquid s in the second pipe 15 and the third pipe 16. The configuration of the detection units 29a and 29b is not particularly limited as long as they can detect the position of the leading edge of the sheath liquid s in the second pipe 15 and the third pipe 16; for example, ultrasonic sensors can be used. With ultrasonic sensors, since ultrasound propagates more easily through liquids and solids than through gases, false detections are less likely when biologically derived materials are used. Also, as described above, since ultrasound propagates more easily through liquids and solids than through gases, a decrease in detection accuracy is suppressed even when the concentration of biologically derived materials in the suspension Q is high. For these reasons, it is preferable that the detection unit 29 used for detecting the suspension Q has an ultrasonic sensor. It is also preferable that the detection units 29a and 29b used for detecting the sheath liquid s have ultrasonic sensors.

[0021] The discharge pipe 28 has, for example, two branched paths 28b and 28c, which are branched at the branching section 28a. In the discharge pipe 28, a valve 35 and a detection unit 36 ​​are arranged in this order from the flow path device 12 side, between the branching section 28a. The detection unit 36 ​​has, for example, a liquid detection sensor 36a and a temperature sensor 36b. Here, if the temperature of the suspension Q and sheath liquid s immediately after EP is abnormally high due to some abnormality such as insufficient flow rate, abnormal conductivity of the culture medium, or abnormal conductivity of the suspension, it can lead to electrode deterioration and cell damage due to discharge inside the flow path device. By providing the temperature sensor 36b in the detection unit 36 ​​and measuring the temperature of the suspension Q and sheath liquid s immediately after EP, an abnormality can be detected, and if an abnormality is detected, the supply of suspension Q and sheath liquid s can be stopped, or the supply of current to the electrode pair can be stopped. A valve 31e is provided in the fourth pipe 17, facing the valve 35 across the connection section 28d between the discharge pipe 28 and the fourth pipe 17. Valve 31e controls the passage of fluids such as gas and liquid in the discharge pipe 28 and the fourth pipe 17. Valve 35 controls the passage of fluids such as gas and liquid in the discharge pipe 28. Both valve 31e and valve 35 can be any known valve as appropriate; for example, a pinch valve can be used. Both valve 31e and valve 35 may be controlled to open or close by the control unit 19, for example.

[0022] A waste liquid tank 37 is connected to the end of branch line 28b. A valve 39a is provided between branch section 28a and waste liquid tank 37. A container 38 is connected to the end of branch line 28c. A valve 39b is provided between branch section 28a and container 38. The waste liquid tank 37 stores liquids such as suspension Q and sheath liquid s discharged from the flow channel device 12 that are not suitable for post-cultivation. The waste liquid tank 37 also stores cleaning liquid discharged from the flow channel device 12. The configuration of the waste liquid tank 37 is not particularly limited as long as it can store liquids such as suspension Q and sheath liquid s, as well as cleaning liquid, discharged from the flow channel device 12. The container 38 stores liquids from suspension Q and sheath liquid s after EP treatment that are discharged from the flow channel device 12 and are used for post-cultivation. The container 38 is not particularly limited in its configuration, as long as it can store the suspension Q and sheath fluid s, etc., discharged to the outside from the flow path device 12. Also, although Figure 1 shows one container 38, the number of containers 38 is not particularly limited and there may be multiple containers 38. In this case, the discharge pipe 28 is configured to have branching paths corresponding to the number of containers 38.

[0023] Valves 39a and 39b switch the destination of the liquid discharged from the flow path device 12 to the waste liquid tank 37 or container 38. Valves 39a or 39b send the liquid discharged from the flow path device 12 to the waste liquid tank 37 or container 38. For example, the suspension Q and sheath liquid s discharged from the outlet 41d from the start of supplying the suspension Q and sheath liquid s until the application of the electric field are supplied and stored in the waste liquid tank 37. For example, the electroporated suspension Q is supplied and stored in the container 38 after a predetermined time has elapsed following the application of an electric field to the suspension Q. The configuration of valves 39a and 39b is not particularly limited as long as they can switch the destination of the liquid to the waste liquid tank 37 or container 38, and known valves can be used as appropriate, for example, pinch valves can be used. Furthermore, valves 39a and 39b may be controllable by the control unit 19, in which case the control unit 19 can switch the destination of the liquid to the waste liquid tank 37 or the container 38.

[0024] Hereinafter, the flow path device 12 will be mainly described with reference to FIGS. 2 and 3. As shown in FIG. 2, the flow path device 12 includes a main flow path 40, a supply port 41a for supplying the suspension Q to the main flow path 40, a discharge port 41d for discharging the treated suspension after continuous EP, and a first supply port 41b and a second supply port 41c for supplying the sheath fluid s to the main flow path 40. A first pipe 14 is connected to the supply port 41a. A second pipe 15 is connected to the first supply port 41b. A third pipe 16 is connected to the second supply port 41c. A discharge pipe 28 is connected to the discharge port 41d.

[0025] The main flow path 40 is a linear flow path. Also, in the direction orthogonal to the extending direction D of the main flow path 40, the cross-sectional shape is, for example, a quadrilateral with all interior angles being 90°. The above-described cross-sectional shape of the main flow path 40 is not limited to a quadrilateral with all interior angles being 90°. Ｌ The flow path device 12 shown in FIG. 2 is arranged such that, for example, the extending direction D of the linear main flow path 40 is orthogonal to the horizontal plane Hp. The main flow path 40 extends in the vertical direction z. The extending direction D of the main flow path 40 is the vertical direction z orthogonal to the horizontal plane Hp. The width direction of the main flow path 40 is the direction x orthogonal to the vertical direction z and is also the direction orthogonal to the extending direction D of the main flow path 40. When it is said that the extending direction D of the linear main flow path 40 is orthogonal to the above-described horizontal plane Hp, it means that the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, but an allowance of ±10° with respect to 90° is permitted. Therefore, when the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, the extending direction D of the main flow path 40 is parallel to the vertical direction z, but an inclination of ±10° with respect to the vertical direction z is permitted for the extending direction D of the main flow path 40. The extending direction D of the main flow path 40

[0026] The flow path device 12 shown in FIG. 2 is arranged such that, for example, the extending direction D of the linear main flow path 40 is orthogonal to the horizontal plane Hp. The main flow path 40 extends in the vertical direction z. The extending direction D of the main flow path 40 is the vertical direction z orthogonal to the horizontal plane Hp. The width direction of the main flow path 40 is the direction x orthogonal to the vertical direction z and is also the direction orthogonal to the extending direction D of the main flow path 40. When it is said that the extending direction D of the linear main flow path 40 is orthogonal to the above-described horizontal plane Hp, it means that the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, but an allowance of ±10° with respect to 90° is permitted. Therefore, when the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, the extending direction D of the main flow path 40 is parallel to the vertical direction z, but an inclination of ±10° with respect to the vertical direction z is permitted for the extending direction D of the main flow path 40. The extending direction D of the main flow path 40 Ｌ is arranged to be orthogonal. The main flow path 40 extends in the vertical direction z. The extending direction D of the main flow path 40 Ｌ is the vertical direction z orthogonal to the horizontal plane Hp. The width direction of the main flow path 40 is the direction x orthogonal to the vertical direction z and is also the direction orthogonal to the extending direction D of the main flow path 40. When it is said that the extending direction D of the linear main flow path 40 is orthogonal to the above-described horizontal plane Hp, it means that the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, but an allowance of ±10° with respect to 90° is permitted. Therefore, when the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, the extending direction D of the main flow path 40 is parallel to the vertical direction z, but an inclination of ±10° with respect to the vertical direction z is permitted for the extending direction D of the main flow path 40. The extending direction D of the main flow path 40 Ｌ and is also the direction orthogonal to the extending direction D. When it is said that the extending direction D of the linear main flow path 40 is orthogonal to the above-described horizontal plane Hp, it means that the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, but an allowance of ±10° with respect to 90° is permitted. Therefore, when the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp is 90°, the extending direction D of the main flow path 40 is parallel to the vertical direction z, but an inclination of ±10° with respect to the vertical direction z is permitted for the extending direction D of the main flow path 40. The extending direction D of the main flow path 40 Ｌ is orthogonal, it means that the angle of the extending direction D of the main flow path with respect to the horizontal plane Hp Ｌ is 90°, but an allowance of ±10° with respect to 90° is permitted. For this reason, when the angle of the extending direction D of the main flow path 40 with respect to the horizontal plane Hp Ｌ is 90°, the extending direction D of the main flow path 40 Ｌ is parallel to the vertical direction z, but the extending direction D of the main flow path 40 Ｌ is permitted an inclination of ±10° with respect to the vertical direction z. The extending direction D of the main flow path 40 Ｌis the direction Df in which the suspension Q flows within the main flow path 40. In the EP device 11, the liquid feeding direction Dd of the suspension Q between the electrode pairs is a direction from the lower side to the upper side in the vertical direction. The suspension Q flows within the main flow path 40 in a direction from the lower side to the upper side in the vertical direction z. The suspension Q is supplied from the supply port 41a, flows along the direction Df shown in FIG. 2 in the main flow path 40, and is discharged from the discharge port 41d. In the main flow path 40, the supply port 41a side is the upstream side, and the discharge port 41d side is the downstream side. The direction Df is also the direction from the upstream to the downstream of the main flow path 40. The sheath liquid s is supplied from the first supply port 41b and the second supply port 41c, flows along the direction Df shown in FIG. 2 in the main flow path 40, and is discharged from the discharge port 41d.

[0027] As shown in FIG. 2, a supply port 41a for supplying the suspension Q to the main flow path 40 is provided, for example, on the surface 12a of the flow path device 12 at one end 40a of the main flow path 40. At the other end 40b of the main flow path 40, a discharge port 41d for discharging liquids such as the suspension Q and the sheath liquid s to the outside is provided, for example, on the back surface 12b of the flow path device 12. In the main flow path 40, the supply port 41a side is the upstream side, and the discharge port 41d side is the downstream side. Further, as shown in FIG. 2, a first supply port 41b for supplying the sheath liquid s to the main flow path 40 is provided, for example, on the surface 12a of the flow path device 12 at one end 40a of the main flow path 40. A second supply port 41c for supplying the sheath liquid s to the main flow path 40 is provided, for example, on the back surface 12b of the flow path device 12. For example, the positions of the first supply port 41b and the second supply port 41c in the extending direction D Ｌ are the same.

[0028] The first pipe 14 described above is used to supply the suspension Q when the suspension Q is delivered to the main channel 40. In addition to the suspension Q, the first pipe 14 is also used to transport gases such as air and cleaning liquid. The second pipe 15 and the third pipe 16 are used to supply the sheath liquid s when the sheath liquid s is delivered to the main channel 40 so as to surround the suspension Q. The configuration of the first pipe 14 is not particularly limited, and depending on the viscosity of the suspension Q, the amount of suspension Q delivered, the type and amount of cleaning liquid delivered, and the type and supply pressure of the gas, known in the art can be used as appropriate, and for example, it is made of a flexible material such as a silicone tube. The configurations of the second pipe 15 and the third pipe 16 are not particularly limited, and depending on the viscosity of the sheath liquid s, the amount of sheath liquid s delivered, known in the art can be used as appropriate, and for example, they are made of a flexible material such as a silicone tube. The configuration of the fourth pipe 17 is not particularly limited, and depending on the type of gas passing through it and the gas supply pressure, etc., known pipes in the art can be used as appropriate, for example, a flexible material such as a silicone tube. The configuration of the discharge pipe 28 is not particularly limited, and depending on the viscosity of the discharged suspension Q and sheath liquid s, the amount of suspension Q and sheath liquid s delivered, etc., known pipes in the art can be used as appropriate, for example, a flexible material such as a silicone tube.

[0029] Although a configuration with a second pump 13b and a third pump 13c has been described, the system is not limited to this, and a configuration with one pump for the first supply port 41b and the second supply port 41c is also possible. That is, the sheath fluid s may be supplied to the flow path device 12 with one pump. As described above, the supply port 41a is provided on the surface 12a of the flow path device 12 and the discharge port 41d is provided on the back surface 12b of the flow path device 12, but the system is not limited to this. For example, one of the supply port 41a and the discharge port 41d may be provided on the surface 12a of the flow path device 12 and the other on the back surface 12b. The first pump 13a, the second pump 13b, and the third pump 13c described above deliver a culture medium containing water, culture medium, cells, etc. The configurations of the first pump 13a, the second pump 13b, and the third pump 13c are not particularly limited, and known ones can be used as appropriate, but examples include tube pumps and syringe pumps.

[0030] In the main flow channel 40, an electrode pair 44 is provided downstream of the confluence section 59 where the suspension flow and the sheath liquid flow merge. The electrode pair 44 comprises electrodes arranged opposite each other and applies an electric field to the suspension Q flowing through the main flow channel 40. The electrode pair 44 that applies an electric field to the suspension Q has a first electrode 45 and a second electrode 46 arranged opposite each other. The first electrode 45 has an electrode surface 45a, which is a flat surface. The second electrode 46 has an electrode surface 46a, which is a flat surface. The electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 are arranged opposite each other and parallel to each other. The electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 are each arranged facing the inner surface 40c of the main flow channel 40. The electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 each constitute the inner surface 40c of the main flow channel 40. For example, when EP is performed, if the suspension Q and the sheath liquid s are supplied to the main flow channel 40, they come into contact with the sheath liquid s. The space 47 between the opposing electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 is the space between the electrode pair 44.

[0031] The first electrode 45 and the second electrode 46 of the electrode pair 44 are electrically connected to the power supply unit 18. The power supply unit 18 applies a voltage to the electrode pair 44. That is, the power supply unit 18 applies a voltage to the opposing first electrode 45 and second electrode 46. The configuration of the power supply unit 18 is not particularly limited, as long as it can apply a pulse voltage to the electrode pair 44 and adjust the pulse width and pulse period. For example, a pulse power supply can be used as the power supply unit 18.

[0032] For example, the detection unit 29 described above is provided near the supply port 41a of the first pipe 14. Also, for example, the detection unit 29a described above is provided near the first supply port 41b of the second pipe 15, and the detection unit 29b is provided near the second supply port 41c of the third pipe 16. The vicinity of the supply port 41a is within 10 cm from the supply port 41a, preferably within 5 cm. The vicinity of the first supply port 41b is within 10 cm from the first supply port 41b, preferably within 5 cm. The vicinity of the second supply port 41c is within 10 cm from the second supply port 41c, preferably within 5 cm.

[0033] Here, the leading position of the suspension Q in the first pipe 14 refers to the liquid level at the position of the suspension Q closest to the flow device 12 in the first pipe 14. The leading positions of the sheath liquid s in the second pipe 15 and the third pipe 16 refer to the liquid level at the position of the sheath liquid s closest to the flow device 12 in the second pipe 15, and the liquid level at the position of the sheath liquid s closest to the flow device 12 in the third pipe 16. The detection unit 29 detects the leading position of the suspension Q in the first pipe 14. The detection units 29a and 29b detect the leading positions of the sheath liquid s in the second pipe 15 and the third pipe 16. Furthermore, it is preferable that the detection unit 29 transmits a detection signal to the control unit 19 when it detects the leading position of the suspension Q in the first pipe 14. It is also preferable that the detection units 29a and 29b transmit a detection signal to the control unit 19 when they detect the leading position of the sheath liquid s in the second pipe 15 and the third pipe 16. The control unit 19 is connected to the above-mentioned detection units 29, 29a, and 29b, and receives detection signals from the detection units 29, 29a, and 29b. Based on the detection signals, it controls the operation of the first pump 13a, the second pump 13b, and the third pump 13c, as well as the opening and closing of each valve.

[0034] In the EP device 11, the standby position at the beginning of the suspension Q in the first piping 14 is predetermined. For this reason, it is preferable to position the detection unit 29 at the predetermined standby position at the beginning of the suspension Q in the first piping 14. The standby position in the first piping 14 is, for example, near the supply port 41a of the first piping 14.

[0035] The control unit 19 of the EP device 11 is connected to the first pump 13a, second pump 13b, and third pump 13c of the EP device 11, as well as to the detection units 29, 29a, and 29b, in addition to the power supply unit 18 (not shown in the diagram). The control unit 19 controls the operation of the first pump 13a, second pump 13b, and third pump 13c, and controls the timing of the start and stop of the supply of suspension Q and sheath liquid s. The control unit 19 also adjusts the magnitude, pulse width, and pulse period of the voltage applied by the power supply unit 18 to the electrode pair 44. Furthermore, the control unit 19 adjusts the timing of the operation of the first pump 13a, second pump 13b, and third pump 13c, i.e., the timing of the supply of suspension Q and sheath liquid s, and the timing of the voltage application by the power supply unit 18 to the electrode pair 44. The control unit 19 can adjust the timing of the operation of the first pump 13a, the second pump 13b, and the third pump 13c using various control means such as contact signals, serial communication, and LAN (Local Area Network) communication, thereby controlling the timing of the delivery of the suspension Q and the sheath liquid s.

[0036] The control unit 19 may be configured as a computer that functions when a program is executed, or as a dedicated device configured with a dedicated circuit. The control unit 19 may be configured to be located separately from the flow path device 12. It may also be configured to be remotely controlled by the control unit 19. The configuration of remote control by the control unit 19 is not particularly limited, and known methods such as the aforementioned LAN communication can be used. The aforementioned computer and dedicated device have, for example, a processor. The processor may be configured as one or more hardware components, and the type of hardware is not limited. For example, a processor may consist of programmable logic devices such as a CPU (Central Processing Unit), MPU (Micro Processing Unit), FPGA (Field Programmable Gate Array), dedicated circuits for performing specific processing such as an ASIC (Application Specific Integrated Circuit), and hardware such as a GPU (Graphic Processing Unit) or NPU (Neural Processing Unit).

[0037] In the EP device 11, the flow channel device 12 is configured, for example, as shown in Figure 2, to include a first substrate 52, a second substrate 54, a first introduction section 56, a second introduction section 58, and a flow channel substrate 55 provided between the first substrate 52 and the second substrate 54. The first introduction section 56, the first substrate 52, the flow channel substrate 55, the second substrate 54, and the second introduction section 58 are stacked in that order. A first electrode 45 is incorporated into the first substrate 52, and a second electrode 46 is incorporated into the second substrate 54, forming an electrode pair 44. As described above, the electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 each constitute the inner surface 40c of the main flow channel 40. As described above, the flow channel device 12 has a supply port 41a for supplying the suspension Q, a main flow channel 40, an outlet 41d for discharging the treated suspension after continuous EP, and a first supply port 41b and a second supply port 41c for receiving the sheath liquid s. The main flow channel 40 is formed in a substantially straight line between the first substrate 52 and the second substrate 54.

[0038] A through-hole 56b extending in the stacking direction Ds of the first introduction section 56, first substrate 52, flow path substrate 55, second substrate 54, and second introduction section 58 is provided in the first introduction section 56. Furthermore, a through-hole 52b is provided in the first substrate 52, which communicates with the through-hole 56b of the first introduction section 56 and extends in the stacking direction Ds. The through-hole 52b communicates with the uppermost part of the main flow path 40. The opening connecting the through-hole 52b and the main flow path 40 is the suspension inlet 55d. Suspension Q flows into the main flow path 40 through the suspension inlet 55d. The opening on the opposite side of the through-hole 56b of the first introduction section 56 from the first substrate 52 is the supply port 41a. The supply port 41a communicates with the uppermost part of the main flow path 40 through the through-hole 56b of the first introduction section 56 and the through-hole 52b of the first substrate 52. Furthermore, the opening connecting the discharge port 41d and the main flow path 40 is the outlet port 40d. The stacking direction Ds is parallel to the direction y which is perpendicular to the vertical direction z. Also, the opposing direction of the electrodes in the electrode pair 44 is parallel to the direction y which is perpendicular to the vertical direction z.

[0039] Furthermore, the first substrate 52 is provided with a through hole 52a that communicates with the main flow path 40. The second substrate 54 is provided with a through hole 54a that communicates with the main flow path 40. The through holes 52a and 54a are provided at an angle to the main flow path 40. The first introduction section 56 is provided with a through hole 56a that communicates with the through hole 52a of the first substrate 52. Furthermore, the second introduction section 58 is provided with a through hole 58a that communicates with the through hole 54a of the second substrate 54. The through hole 56a communicates with the first supply port 41b and the through hole 52a of the first substrate 52, extends from the first supply port 41b in the stacking direction Ds, and at the boundary with the first substrate 52 is in the extending direction D of the main flow path 40. Ｌ It is bent in this way. For convenience, a through hole 56a of this shape is called a roughly L-shaped through hole 56a. The through hole 58a communicates with the second supply port 41c and the through hole 54a of the second substrate 54, extends from the second supply port 41c in the stacking direction Ds, and at the boundary with the second substrate 54 is in the extension direction D of the main flow path 40. ＬIt is bent in this way. For convenience, a through hole 58a of this shape is called a roughly L-shaped through hole 58a. The through hole 56a of the first introduction section 56 and the through hole 52a of the first substrate 52, and the through hole 58a of the second introduction section 58 and the through hole 54a of the second substrate 54 are arranged symmetrically with respect to the main flow path 40 which extends in the vertical direction z. The sheath liquid flow path 64 extends from the first supply port 41b to the through holes 56a and 52a. The sheath liquid flow path 65 extends from the second supply port 41c to the through holes 58a and 54a. The sheath liquid flow paths 64 and 65 are channels through which only sheath liquid s flows, and which allow the sheath liquid s to flow into the main flow path 40. The through hole 52a is in communication with the sheath liquid confluence port 52c provided in the main flow path 40, which will be described later. The through-hole 54a is in communication with the sheath liquid confluence port 54c provided in the main flow path 40, which will be described later.

[0040] The first supply port 41b communicates with the main flow path 40 through a substantially L-shaped through hole 56a provided in the first introduction section 56 and through a through hole 52a provided in the first substrate 52. Here, the sheath liquid flow path 64 is provided so as to be inclined downstream with respect to the liquid flow direction in the main flow path 40, and opens downstream of the suspension supply port and upstream of the electrode pair 44 so that the sheath liquid s flows in. The region upstream of the main flow path 40 through which only the suspension Q flows is the suspension flow path.

[0041] Furthermore, the through-hole 56a (part of the sheath liquid flow path) may be widened in the width direction toward downstream from a point where the substantially L-shaped flow path is bent, for example, so that the flow velocity in the width direction of the sheath liquid flow is uniform. The same applies to the through-hole 58a (part of the sheath liquid flow path) which will be described later. That is, it is preferable that the sheath liquid flow path 65 has a region in the through-hole 58a where the flow path widens in the width direction toward downstream. As a result, even if the width of the main flow path 40 is wide, the flow velocity distribution downstream becomes uniform, and EP can be applied uniformly to 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 downstream, so that the flow velocity distribution is also uniform in the suspension flow path. In addition, the through-hole 56a may be provided spanning the first introduction section 56 and the first substrate 52.

[0042] On the other hand, the second supply port 41c communicates with the main flow path 40 through a substantially L-shaped through hole 58a provided in the second introduction section 58 and through a through hole 54a provided in the second substrate 54. Here, as described above, the through holes 58a and 54a are sheath liquid flow paths 65, similar to the through holes 56a and 52a. Therefore, the above description of the through holes 56a and 52a also applies to the through holes 58a and 54a. The through hole 58a may span across the second introduction section 58 and the second substrate 54. Furthermore, the opening of the through hole 52a to the main flow path 40 and the opening of the through hole 54a to the main flow path 40 become sheath liquid confluence ports 52c and 54c, which allow the sheath liquid s to flow into the main flow path 40 of the flow path device 12. Furthermore, it is preferable that the widths of the suspension supply port of suspension Q and the sheath liquid confluence ports 52c and 54c of sheath liquid s are approximately the same. In addition, in the flow path device 12, the region where the sheath liquid confluence port 52c and the sheath liquid confluence port 54c face each other becomes a confluence section 59 where the suspension flow and the sheath liquid flow merge.

[0043] A sheath liquid confluence port 52c is provided on the inner surface 40c of the main flow channel 40 to form a sheath liquid flow that contacts the electrodes of the electrode pair 44. The sheath liquid confluence port 52c is located downstream of the suspension inlet 55d and upstream of the electrode pair 44. Through holes 52a and 56a form a sheath liquid flow channel 64 in the main flow channel 40 that merges the sheath liquid flow with the suspension Q at the confluence 59. A second pipe 15 is connected to the sheath liquid flow channel 64. The second pipe 15 is connected to the sheath liquid confluence port 52c via the first supply port 41b, through holes 56a and 52a. A sheath liquid confluence port 54c is provided on the inner surface 40c of the main flow channel 40 to form a sheath liquid flow that contacts the electrodes of the electrode pair 44. The sheath liquid confluence port 54c is located downstream of the suspension inlet 55d and upstream of the electrode pair 44. Through holes 54a and 58a form a sheath liquid flow path 65 in which the sheath liquid flow is merged with the suspension Q at the confluence 59 in the main flow path 40. A third pipe 16 is connected to the sheath liquid flow path 65. The third pipe 16 is connected to the sheath liquid confluence port 54c via the second supply port 41c, through hole 58a, and through hole 54a. It is preferable that the sheath liquid flow paths 64 and 65 have a region in the width direction toward downstream. As a result, even if the width of the main flow path 40 is wide, the flow velocity distribution downstream becomes uniform, and EP can be uniformly applied to the suspension Q. At this time, it is preferable that the suspension flow path also has a region in the width direction toward downstream, so that the flow velocity distribution is uniform in the suspension flow path as well.

[0044] In the flow channel device 12, the suspension Q is supplied from the first pipe 14 to the supply port 41a, flows into the main flow channel 40 from the uppermost part, and reaches the outlet port 41d from the main flow channel 40. The sheath liquid s is supplied from the second pipe 15 to the first supply port 41b, and from the third pipe 16 to the second supply port 41c. The sheath liquid s that flows into the first supply port 41b is supplied to the main flow channel 40 via the through hole 56a provided in the first introduction section 56 and the through hole 52a provided in the first substrate 52, that is, via the sheath liquid flow channel 64, and reaches the outlet port 41d from the main flow channel 40. The sheath liquid s that flows into the second supply port 41c is supplied to the main flow channel 40 via the through hole 58a provided in the second introduction section 58 and the through hole 54a provided in the second substrate 54, that is, via the sheath liquid flow channel 65, and reaches the outlet port 41d from the main flow channel 40.

[0045] In the embodiment shown in Figure 2, the sheath fluid flow path is composed of through holes 56a (58a) and 52a (54a). However, the sheath fluid flow path only needs to have through holes 52a (54a), and the flow path configuration is designed appropriately according to the device configuration. In the sheath fluid flow path 64 composed of through holes 56a and 52a, the sheath fluid flow path 64 only needs to have through hole 52a, and the configuration of the sheath fluid flow path is determined appropriately according to the configuration of the flow path device.

[0046] Furthermore, the sheath liquid flow path 65, which is composed of through holes 54a and 58a, only requires through hole 54a to function as the sheath liquid flow path 65, and the configuration of the sheath liquid flow path is appropriately determined according to the configuration of the flow path device. Therefore, upstream of the electrode pair 44, as conceptually shown in Figure 3, a sheath liquid flow consisting of sheath liquid s is formed on both sides in the opposite direction to the suspension flow of suspension Q, forming a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, and this three-layered flow flows into the space between the electrode pair. In the EP device 11, the suspension Q and sheath liquid s are continuously supplied between the electrode pair 44, i.e., into the space 47 described above, in a state where they form a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, and by applying, for example, a pulsed electric field to the suspension Q in the state of the three-layered flow using the first electrode 45 and the second electrode 46, continuous EP processing using the sheath liquid flow can be performed as described above. Note that in Figure 3, the first pipe 14, second pipe 15, third pipe 16, fourth pipe 17, and discharge pipe 28, power supply unit 18, control unit 19, and valves 31e and 35 shown in Figure 2 are omitted from the illustration.

[0047] As described above, the sheath fluid s supplied from the first supply port 41b slopes downstream and flows into the main flow path 40 via the sheath fluid confluence port 52c. On the other hand, the sheath fluid s supplied from the second supply port 41c slopes downstream and flows into the main flow path 40 via the sheath fluid confluence port 54c.

[0048] In the flow channel device 12, in the three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow in the continuous EP described above, the thickness Dm of the suspension flow (see Figure 3) is controlled to 1 to 10 mm. The thickness Dm of the suspension flow 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 regulated area is preferably 2 mm, and more preferably 3 mm.

[0049] Furthermore, there are no restrictions on the thickness ds of the sheath liquid flow due to the sheath liquid s (see Figure 3), but the thickness of the sheath liquid flow when it merges 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.

[0050] In such a flow channel device 12, there are no restrictions on the angle formed by the suspension flow and the sheath liquid flow when the suspension flow and the sheath liquid flow are merged, that is, the angle formed by the main flow channel 40 and the through holes 52a and 54a, but it is preferable that it be small. Specifically, the extending direction D of the main flow channel 40 Ｌ Furthermore, the angle formed by the extending direction of the through-hole 52a and the extending direction of the 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. Also, when the suspension flow and the sheath liquid flow are joined, it is preferable that the difference in flow velocity between the two is small. Specifically, when the suspension flow and the sheath liquid flow are joined, the flow velocity of the sheath liquid flow 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%, that is, the suspension flow and the sheath liquid flow are at the same velocity.

[0051] In the flow channel device 12 shown in 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 channel substrate 55, between the flow channel substrate 55 and the second substrate 54, and between the second substrate 54 and the second introduction section 58.

[0052] In the EP device 11, the first pump 13a, the second pump 13b, and the third pump 13c deliver the suspension Q and sheath liquid s between the electrode pair 44, i.e., in the aforementioned space 47, such that the delivery direction Dd of the suspension Q and sheath liquid s is from below to above in the vertical direction z. When EP is performed by applying an electric field to the suspension Q using the power supply unit 18, and bubbles are generated between the opposing first electrode 45 and second electrode 46, the generated bubbles have a lower specific gravity than the suspension Q and sheath liquid s, and therefore move more easily in the vertical direction z than the suspension Q and sheath liquid s. For this reason, bubbles generated between the electrode pair 44 are removed from between the electrode pair 44 by moving in the vertical direction z without accumulating between the electrode pair 44 due to the delivery of the suspension Q and sheath liquid s, thereby suppressing the adverse effect of bubbles on the application of the electric field to the suspension Q. As a result, the EP device 11 can properly apply an electric field to the suspension Q, thereby enabling optimal introduction efficiency and utilization efficiency of bioactive substances.

[0053] In the EP device 11, as shown in Figure 3, a sheath liquid flow consisting of a sheath liquid s different from the suspension is formed between the suspension flow and the first electrode 45 and the second electrode 46, along with the suspension flow, to perform continuous EP.

[0054] (First example of electroporation method) The first example of the electroporation method (EP method) is a method of introducing a bioactive substance into a bio-derived substance by continuously supplying a suspension Q containing a bio-derived substance and a bioactive substance, along with a sheath liquid s, to the main channel 40 between the electrode pair 44, i.e., in the space 47 described above, with the suspension Q sandwiched between the sheath liquid s, and applying an electric field to the suspension Q with the electrode pair 44. For example, the EP system 10 having the EP device 11 shown in Figure 1 is used in the first example of the EP method. Figure 4 is a schematic diagram for illustrating the first example of the electroporation method in the first example of the electroporation system of the embodiment of the present invention. In Figure 4, the same reference numerals are used for components identical to those in the EP system 10 shown in Figure 1, and their detailed explanation is omitted.

[0055] In the first example of the EP method, as shown in Figure 4, the suspension Q in the tank 22 is supplied to the main flow path 40 by the first pump 13a through the piping 20, the first piping 14, and the supply port 41a. At this time, in piping 20, valve 24d is open, but valves 24a, 24b, and 24c are closed. Also, in the first piping 14, valves 31a and 31b are open, but valve 31c is closed. In the fourth piping 17, valves 31d and 31e are closed. Valve 35 of the discharge pipe 28 and valve 39b of the branch passage 28c are open, but valve 39a of the branch passage 28b is closed. Note that the path R from the tank 22 to the piping 20, the first pump 13a, the first piping 14, the flow path device 12, the discharge pipe 28, and the container 38 shown in Figure 4. １ This will be designated as Route 1. Route R １ The valve is open. When the suspension Q is supplied to the main flow path 40 of the flow device 12 by the first pump 13a, the valve 31d of the fourth pipe 17 may be opened and the valve 31e may be closed. In this case, the fourth pipe 17 functions as a damper for the first pump 13a, reducing the pulsation of the first pump 13a.

[0056] Furthermore, the second pump 13b supplies the sheath fluid s in the tank 27a to the main flow path 40 through the piping 26, the second piping 15, the first supply port 41b, the through hole 56a provided in the first introduction section 56, and the through hole 52a provided in the first substrate 52. At this time, valve 27c is open. Also, valve 34a of the second piping 15 is open, and valve 34b is closed. The path R from the tank 27a to the piping 26, the second pump 13b, and the flow path device 12 shown in Figure 4. ２ This will be the second route. Route R ２ The valve is open.

[0057] Furthermore, the third pump 13c supplies the sheath fluid s in the tank 27a to the main flow path 40 through the piping 26, the third piping 16, the second supply port 41c, the through hole 58a provided in the second introduction section 58, and the through hole 54a provided in the second substrate 54. At this time, valve 27c is open. Also, valve 34a of the third piping 16 is open, and valve 34b is closed. The path R from the tank 27a to the piping 26, the third pump 13c, and the flow path device 12 shown in Figure 4. ３ This will be the third route. Route R ３ The valve is open.

[0058] During EP, a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow is formed between the electrode pair 44, and the suspension Q and sheath liquid s are continuously delivered between the electrode pair 44 in a direction from bottom to top in the vertical direction z. At this time, the power supply unit 18 applies, for example, a pulse voltage to the electrode pair 44 to perform EP. After EP, the suspension Q and sheath liquid s are discharged from the outlet 41d into the discharge pipe 28, pass through the branching passage 28c, and are stored in the container 38.

[0059] In the first example of the EP method, the direction Dd of supplying the suspension Q and sheath liquid s is 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 18, and bubbles are generated between the opposing first electrode 45 and second electrode 46, the generated bubbles have a lower specific gravity than the suspension Q and therefore move more easily in the vertical direction z than the suspension Q. For this reason, bubbles generated between the electrode pair 44 are removed from between the electrode pair 44 by moving in the vertical direction z as the suspension Q and sheath liquid s are supplied, without remaining between the electrode pair 44, thus suppressing the adverse effect of bubbles on the application of the electric field to the suspension Q. As a result, in the first example of the EP method, the application of an electric field to the suspension Q can be performed appropriately, and as a result, the introduction efficiency and the utilization efficiency of bioactive substances can be made appropriate. Furthermore, even if the composition of suspension Q is such that bubbles are easily generated when an electric field is applied to suspension Q, if bubbles are generated between the electrode pair 44 as described above, the generated bubbles have a lower specific gravity than suspension Q, and therefore move more easily in the vertical direction z than suspension Q. As a result of the supply of suspension Q and sheath liquid s, the bubbles do not remain between the electrode pair 44 but are removed by moving in the vertical direction z, thereby suppressing the adverse effects of bubbles on the application of an electric field to suspension Q. In this way, in the first example of the EP method, the application of an electric field to suspension Q can be carried out appropriately, and as a result, the introduction efficiency and the utilization efficiency of bioactive substances can be made appropriate.

[0060] Normally, before performing electroporation (EP), biological substances (e.g., cells) are removed from the culture medium and suspended in EP buffer to obtain a suspension. Replacing this medium with EP buffer is simply called medium exchange or buffer exchange. In the first example of the EP method, without replacing the medium in the culture medium with EP buffer, i.e., without adding EP buffer, a suspension Q containing biological substances and medium, mixed with a bioactive substance, is continuously delivered between the electrode pair 44. In the first example of the EP method, since the medium in the culture medium is not replaced with EP buffer, the suspension contains more medium and has higher electrical conductivity than a suspension that is normally EP'd. Therefore, a large amount of bubbles are generated when an electric field is applied. Even with such a suspension, since the suspension is EP'd while moving vertically from bottom to top between the electrode pair, the bubbles generated between the electrode pair are removed by moving vertically, and the adverse effects of bubbles on the application of the electric field to the suspension can be suppressed.

[0061] <Method for Cleaning the Flow Channel Device> In a continuous electroporation (EP) system, the same flow channel device 12 may be used repeatedly. Therefore, after performing EP, the flow channel device 12 is cleaned. The method for cleaning the flow channel device 12 is described below. Figures 5 and 6 are schematic diagrams illustrating the method for cleaning the flow channel device for a first example of an electroporation system according to an embodiment of the present invention. In Figures 5 and 6, the same components as those in the EP system 10 shown in Figure 1 are denoted by the same reference numerals, and their detailed descriptions are omitted.

[0062] First, the supply of suspension Q and sheath fluid s is stopped, and suspension Q is discharged from the suspension Q flow path of the flow path device 12 using gas. That is, suspension Q is discharged from the main flow path 40 using gas. Specifically, the first pump 13a, the second pump 13b, and the third pump 13c are stopped. Next, as shown in Figure 5, valve 35 is closed and valve 31e is opened. Also, valve 24d of tank 22 and valve 31b of first piping 14 are closed, and valves 24c, 31c, and 31d are opened. Furthermore, valve 34a of second piping 15 and valve 34a of third piping 16 are closed. Next, a gas such as air is supplied from the gas supply unit 23c, passes through the piping 20, the first pump 13a, and a portion of the first piping 14, and then through the fourth piping 17 and a portion of the discharge pipe 28 to the discharge port 41d, where the gas passes from the other end 40b of the main flow path 40 to the other end 40a, and then through the supply port 41a. As a result, the suspension Q is discharged from the main flow path 40. If sheath liquid s remains in the main flow path 40, the sheath liquid s is also discharged from the main flow path 40. The discharged suspension Q and sheath liquid s pass through the first piping 14 and through the branch passage 33a to the recovery container 33b. In EP, the direction of liquid delivery of the suspension Q in the main flow path 40 is from bottom to top in the vertical direction z, but in the process of discharging the suspension Q from the flow path, the gas is flowed from top to bottom in the vertical direction z relative to the main flow path 40.

[0063] After the suspension Q is discharged from the main channel 40, the suspension Q passes through the path R described below. ４To prevent backflow, valve 31e is closed, and valve 27d, valve 34a of the second pipe 15, and valve 34a of the third pipe 16 are opened. Valve 27c, valve 34b of the second pipe 15, and valve 34b of the third pipe 16 are closed. Valves 35 and 39a are opened, and valve 39b is closed. In this state, a gas such as air is supplied from the gas supply unit 27b and supplied to the main flow path 40 through pipe 26, the second pipe 15, the third pipe 16, the first supply port 41b, and the second supply port 41c, discharged from the main flow path 40 to the discharge pipe 28, and the sheath liquid s is discharged from the branch passage 28b to the waste liquid tank 37. In this way, the sheath liquid s between the flow path devices 12 or the sheath liquid s between valve 34a and the flow path device 12 may be discharged downstream of valve 27c. It is preferable to discharge the suspension Q from the main channel 40, and then discharge the sheath liquid s from the sheath liquid channels 64 and 65 and the main channel 40, because this suppresses the entry of suspension Q into the sheath liquid channels 64 and 65.

[0064] As described above, in the step of discharging the suspension Q from the flow path, the gas is flowed in the opposite direction to the liquid delivery direction of the suspension Q in the main flow path 40 during EP. This allows the suspension Q and sheath liquid s to be efficiently discharged from the main flow path 40, and the residue of suspension Q and sheath liquid s is suppressed. If suspension Q remains in the flow path device 12, dead cells, damaged cells, proteins, nucleic acids (hereinafter referred to as cell debris) contained in the suspension Q will accumulate on or near the electrode surface of the electrode pair, forming precipitates of cell debris on the electrode surface and hindering the uniform application of an electric field between the electrodes. In addition, if culture medium components such as salts of suspension Q and sheath liquid s remain, electrochemical reactions will occur on the electrode surface, causing deterioration of the electrode surface. For this reason, it is undesirable to leave suspension Q and sheath liquid s in the main flow path 40, as this will reduce the introduction efficiency during subsequent EPs and deteriorate the electrodes. Furthermore, the path R shown in Figure 5, from the gas supply unit 23c, through the piping 20, the first pump 13a, part of the first piping 14, the fourth piping 17, part of the discharge pipe 28, the flow path device 12, the first piping 14, the branching path 33a, and the recovery container 33b, is described. ４ This will be designated as the fourth route. Route R ４ The valve is open.

[0065] Next, a cleaning solution is supplied to the flow path to clean it. The cleaning solution includes, for example, water or deionized water. Specifically, as shown in Figure 6, the cleaning solution in tank 23a is supplied to the main flow path 40 through piping 20, the first piping 14 and the supply port 41a, discharged from the main flow path 40 to the discharge pipe 28, and discharged from the branch passage 28b to the waste liquid tank 37. At this time, valve 24a is open, but valves 24b, 24c and 24d are closed. Also, valves 31a and 31b are open, but valves 31c, 31d and 31e are closed. Valve 34a of the second piping 15 and valve 34a of the third piping 16 are closed. In the discharge pipe 28, valve 39b is closed, but valves 35 and 39a are open. In the process of cleaning the flow path, the cleaning solution is flowed in the same direction as the liquid delivery direction of the suspension Q in the main flow path 40 in EP. In other words, the cleaning fluid is flowed in the main channel 40 from the bottom to the top in the vertical direction z. As a result, the entire interior of the main channel 40 is reliably filled with the cleaning fluid, so that even if the suspension Q and sheath fluid s remain when the suspension Q and sheath fluid s are discharged using gas, the remaining suspension Q and sheath fluid s are reliably washed away by the cleaning fluid. Note that the path R from the tank 23a shown in Figure 6 to the piping 20, the first pump 13a, the first piping 14, the flow path device 12, the discharge pipe 28, the branching path 28b, and the waste liquid tank 37 ５ This is designated as the fifth route. It is preferable to discharge the suspension Q from the main channel 40 using the gas described above, and to clean the channel, at least twice, from the viewpoint of more reliably washing away the suspension Q and sheath liquid s from the main channel 40.

[0066] Next, the supply of the cleaning solution is stopped, and the flow channel device 12 is stored with the flow channel filled with the cleaning solution. That is, the flow channel device 12 is sealed and stored using the cleaning solution. Specifically, after stopping the supply of the cleaning solution from the tank 23a to the main flow channel 40 while the flow channel is being cleaned, the valve 35 is further closed to fill the piping 20, the first pump 13a, the first piping 14, the main flow channel 40, and a portion of the discharge pipe 28 with the cleaning solution. In this way, the flow channel device 12 is stored with the main flow channel 40 filled with the cleaning solution. This prevents the culture medium contained in the suspension Q and sheath liquid s from drying out or agglomerating, even if suspension Q and sheath liquid s remain. This prevents the formation of cell debris precipitates on the electrode surface, suppresses electrochemical degradation of the electrode surface, and as a result, improves the durability of the flow channel device 12. The storage period for the flow channel device 12 sealed using the cleaning solution described above is permissible up to 7 days, but preferably within 1 day. It is preferable to perform the following EP within one day after sealing the flow channel device 12 with a cleaning solution.

[0067] As described above, by properly cleaning the flow path of the flow path device 12, the electrode pair 44 arranged in the main flow path 40 can be properly cleaned. Furthermore, by storing the flow path device 12 with the main flow path 40 filled with cleaning solution, electrode deterioration can be suppressed as described above, and as a result, the durability of the flow path device 12 is improved. The cleaning of the flow path device 12 described above is preferably performed after one continuous EP is performed, regardless of the duration of the EP. When performing the above cleaning method for the flow path device, it is preferable that the valve 34a of the second pipe 15 and the valve 34a of the third pipe 16 are both closed so that cleaning solution and gas do not enter the second pipe 15 and the third pipe 16.

[0068] <Pre-EP Process> As described above, the same flow channel device 12 may be reused repeatedly. When EP is performed again after cleaning the flow channel device 12, the flow channel device 12 is stored with the main flow channel 40 etc. filled with cleaning fluid. Therefore, first, the cleaning fluid filling the main flow channel 40 etc. of the flow channel device 12 is drained. Specifically in this case, the path R shown in Figure 5 is used.４ , in other words, the cleaning solution is discharged to the recovery container 33b via the fourth route. More specifically, valve 31e is opened. Also, valve 24d of tank 22 and valve 31b of first piping 14 are closed, and valves 24c, 31c, and 31d are opened. Next, gas is supplied from the gas supply unit 23c, passes through piping 20, the first pump 13a, a part of the first piping 14, and through a part of the discharge pipe 28 from the fourth piping 17 to the discharge port 41d, where the gas passes from the other end 40b of the main flow path 40 to the one end 40a, and then passes through the supply port 41a. As a result, the cleaning solution is discharged from the main flow path 40. The cleaning solution discharged from the main flow path 40 passes through the first piping 14 and through the branch passage 33a to the recovery container 33b. In the process of discharging the cleaning solution from the flow path, the gas is flowed in the opposite direction to the liquid delivery direction of the suspension Q in the main flow path 40 of EP. This allows the washing solution to be efficiently discharged from the flow path, suppressing residual washing solution. If washing solution remains in the flow path device 12, it will mix with the suspension Q during subsequent EPs, reducing cell viability and decreasing the cell concentration and plasmid concentration in the suspension Q, thereby reducing the introduction efficiency. Furthermore, the amount of fluid required to stabilize the flow of suspension Q and sheath fluid s increases, requiring a large amount of fluid to be delivered to suspension Q and sheath fluid s before EP.

[0069] Next, only the liquid culture medium is passed through the main channel 40 of the flow channel device 12 (culture medium seasoning). Specifically, valve 24b is opened, and valves 24a, 24c, and 24c are closed. Also, valves 31a and 31b are opened, and valves 31c, 31d, and 31e are closed. In the discharge pipe 28, valve 39b is closed, and valves 35 and 39a are opened. As a result, the liquid culture medium from tank 23b is supplied to the main channel 40 through piping 20, the first piping 14, and the supply port 41a, discharged from the main channel 40 to the discharge pipe 28, and discharged to the waste liquid tank 37 from the branching passage 28b. If cell debris has accumulated on or near the electrode surface after the cleaning solution has been discharged from the flow channel using gas, the cell debris is removed by passing only the liquid culture medium through the main channel 40 of the flow channel device 12 as described above. This suppresses the formation of cellular debris deposits on the electrode surface and also inhibits electrochemical degradation of the electrode surface.

[0070] Next, the liquid culture medium in the flow channel device 12 is discharged from the flow channel device 12 using gas. Specifically, gas is used to discharge the medium through the path R shown in Figure 5. ４Specifically, the liquid culture medium is discharged to the recovery container 33b via the fourth route. More specifically, valve 35 is closed and valve 31e is opened. Also, valve 24d of tank 22 and valve 31b of first piping 14 are closed, and valves 24c, 31c, and 31d are opened. Next, a gas such as air is supplied from the gas supply unit 23c, passes through piping 20, the first pump 13a, a part of the first piping 14, and through a part of the discharge pipe 28 from the fourth piping 17 to the discharge port 41d, where the gas passes from the other end 40b of the main flow path 40 to the one end 40a, and then passes through the supply port 41a. As a result, the liquid culture medium is discharged from the main flow path 40. The liquid culture medium discharged from the main flow path 40 passes through the first piping 14 and through the branch passage 33a to the recovery container 33b. In this way, after discharging the washing solution from the flow channel, only the liquid culture medium is passed through the main flow channel 40 of the flow channel device 12, and then the liquid culture medium is discharged from the flow channel device 12. This suppresses changes in cell concentration during EP, damage to cells, and changes in the concentration of plasmids, etc., caused by the washing solution containing water supplied during washing and storage.

[0071] Next, the first pump 13a, the second pump 13b, and the third pump 13c are de-aerated. For the first pump 13a, valve 24d is opened, and valves 24a, 24b, and 24c are closed. Also, valves 31b and 31c are opened, and valves 31a and 31d are closed. In this state, the suspension Q is passed from tank 22 through the first pump 13a. The suspension Q that has passed through the first pump 13a is discharged to the recovery container 33b through the branching passage 33a. For the second pump 13b, valves 27c and 34b are opened, and valve 34a is closed. In this state, the sheath liquid is passed from tank 27a through the second pump 13b. The sheath liquid s that has passed through the second pump 13b is discharged to the recovery container 33b through the branching passage 33a. For the third pump 13c, valves 27c and 34b are opened, and valve 34a is closed. In this state, the sheath liquid is passed from tank 27a to the third pump 13c. The sheath liquid s that has passed through the third pump 13c is discharged to the recovery container 33b through the branch passage 33a. After defoaming of the first pump 13a, the second pump 13b, and the third pump 13c is completed, the continuous EP described above is performed. After performing the continuous EP, the cleaning described above is performed. This allows the flow path to be properly cleaned and suppresses the decrease in introduction efficiency when EP is performed next time, thus allowing stable introduction efficiency to be maintained when EP is performed. Note that defoaming of the first pump 13a, the second pump 13b, and the third pump 13c is also performed before the first EP is performed.

[0072] When performing EP, for example, the leading standby position of the suspension Q in the first piping 14 is predetermined. First, a detection unit 29 (see Figure 1) positioned at the leading standby position of the suspension Q starts detecting the leading standby position of the suspension Q in the first piping 14. The first pump 13a sends the suspension Q to the first piping 14. The second pump 13b sends the sheath liquid s in the tank 27a to the second piping 15. The third pump 13c sends the sheath liquid s in the tank 27a to the third piping 16. Detection of the leading standby position of the suspension Q is started by the detection unit 29 (see Figure 1).

[0073] Next, it is determined whether the leading edge of the suspension Q has reached the standby position. When the control unit 19 receives a detection signal from the detection unit 29 (see Figure 1) located at the standby position of the leading edge of the suspension Q, the control unit 19 determines that the leading edge of the suspension Q has reached the standby position. If it is determined that the leading edge of the suspension Q has reached the standby position, the operation of the first pump 13a, the second pump 13b, and the third pump 13c is temporarily stopped, and the supply of the suspension Q and sheath fluid s is stopped. Then, with the leading edge of the suspension Q in the standby position, the operation of the first pump 13a, the second pump 13b, and the third pump 13c is started, and the supply of the suspension Q and sheath fluid s is started.

[0074] On the other hand, if the control unit 19 has not received the above-mentioned detection signal, the control unit 19 determines that the leading edge of the suspension Q has not reached the standby position. In this case, the supply of the leading edge of the suspension Q and the sheath liquid s continues. The control unit 19 then determines again whether the leading edge of the suspension Q has reached the standby position. The reception of the detection signal is repeated until the control unit 19 determines that the leading edge of the suspension Q has reached the standby position.

[0075] The suspension Q and sheath fluid s are pumped, and the control unit 19 applies an electric field to the suspension Q between the electrode pairs 44 using the power supply unit 18, thereby performing the electroporation process (EP). Since it is a continuous EP, the electric field is applied continuously for a predetermined period of time. After a preset time has elapsed since the application of the electric field, the control unit 19 controls the power supply unit 18 to stop the application of the electric field to the suspension Q. Furthermore, the operation of the first pump 13a, the second pump 13b, and the third pump 13c is stopped, and the pumping of the suspension Q and sheath fluid s is stopped.

[0076] (Second example of an electroporation system) Figure 7 is a schematic diagram showing a first example of an electroporation system (EP system) having an electroporation apparatus (EP apparatus) to which the cleaning method for the flow channel device of an embodiment of the present invention is applied. In Figure 7, the same reference numerals are used for components identical to those in the EP system 10 shown in Figure 1, and their detailed descriptions are omitted. The EP system 10a shown in Figure 7 differs from the EP system 10 shown in Figure 1 in that it mixes the culture medium and the bioactive substance, and is otherwise the same as the EP system 10 shown in Figure 1.

[0077] EP system 10a, compared to EP system 10, includes a tank 70 for storing bioactive substances, a culture medium containing biologically derived materials and a culture culture medium, and a mixer 72 for mixing the bioactive substances. The mixer 72 mixes the culture medium containing biologically derived materials and a culture culture medium with the bioactive substances. The mixture of the culture medium and the bioactive substances is called suspension Q, and suspension Q includes a culture medium. The configuration of the mixer 72 is not particularly limited as long as it can mix the culture medium and the bioactive substances, and known mixers can be used as appropriate. The bioactive substances in tank 70 are mixed with the culture medium delivered to the first pipe 14 by the first pump 13a in the mixer 72 without exchanging the culture medium in the culture medium with the electroporation (EP) buffer to form suspension Q.

[0078] In the first piping 14, a temperature sensor 73 and a mixer 72 are provided between the detection unit 29 and the flow sensor 30, in that order from the detection unit 29 side. In the first piping 14, piping 71 is connected between the mixer 72 and the flow sensor 30. A tank 70 is provided at the end of piping 71 opposite to the first piping 14. A bioactive substance is stored in the tank 70, for example, in a dispersed state in liquid. The configuration of the tank 70 is not particularly limited as long as it can store the bioactive substance. Although not shown in the figures, the tank 70 is equipped with a pump for sending the bioactive substance, such as a plasmid, to the piping 71. For example, a syringe pump is used as the pump. In the piping 71, a valve 71a and a liquid detection sensor 71b are provided in that order from the first piping 14 side. The liquid detection sensor 71b can determine whether or not the bioactive substance, such as a plasmid, has been sent to the piping 71. Furthermore, a damper 74 is provided in the first piping 14 between the flow sensor 30 and the valve 31a. The damper 74 prevents pulsation in the first piping 14 caused by the first pump 13a. The configuration of the damper 74 is not particularly limited as long as it can prevent the pulsation in the first piping 14 as described above.

[0079] In the piping 20, a conductivity sensor 75 is provided between the liquid detection sensor 25 and the branch section to which the tank 23a is connected. The tank 22 stores a bioactive substance, for example, in a dispersed state in a liquid. The bioactive substance is supplied to a culture medium containing biologically derived material and culture medium through the piping 71 and mixed in the mixer 72 to create a suspension Q.

[0080] (Second example of electroporation method) The second example of the electroporation method (EP method) differs from the first example of the EP method in that, instead of using a suspension Q, a bioactive substance is mixed with a culture medium containing biologically derived material and culture medium to create a suspension Q. The other steps are the same as those of the first example of the EP method, so a detailed explanation is omitted. During EP, the culture medium in the culture medium is not replaced with an EP buffer, that is, no EP buffer is added, and a bioactive substance is mixed with a culture medium containing biologically derived material and culture medium in the mixer 72 to create a suspension Q. Also, before EP, the bioactive substance is delivered from the tank 70 through the piping 71 to the confluence of the first piping 14. The cleaning method for the flow channel device 12 is as described above, so a detailed explanation is omitted, but the flow channel can be properly cleaned and the electrode pair 44 arranged in the main flow channel 40 can be properly cleaned. As a result, as described above, the decrease in introduction efficiency when EP is performed next time can be suppressed, and furthermore, electrode degradation can be suppressed, and as a result, the durability of the flow channel device 12 is also improved.

[0081] Furthermore, in all of the above examples, the first pipe 14 through which the suspension Q flows is cleaned, but since the sheath liquid s uses culture media and does not contain cells, etc., cleaning of the second pipe 15 and third pipe 16 through which the sheath liquid s flows, as well as the sheath liquid flow path, is unnecessary. Also, although the above continuous EP is an example using suspension Q and sheath liquid s, it is not limited to this, and even when using only suspension Q, the flow path can be properly cleaned in the same way as the above-described method of cleaning the flow path, and the decrease in introduction efficiency when the continuous EP is performed next time can be suppressed, so that the introduction efficiency can be maintained stably over a long period of time when performing continuous EP. In addition, in the first and second examples of the above EP method, the EP method described in PCT / JP / 2025 / 016650 or International Publication No. 2024 / 248133 may be used.

[0082] The EP method described above can be used to produce useful substances such as target proteins or viruses. In other words, the method for producing useful substances includes the EP method described above. The parts of the EP apparatus will be described below.

[0083] [Main channel] As described above, for example, the main channel 40 extends in the direction D Ｌ The cross-sectional shape in the direction perpendicular to it is a quadrilateral with all interior angles at 90°.

[0084] [Distance D between electrode pairs] The distance D between electrode pairs 44 (see Figure 2) is the distance in the opposing direction between the electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46, and is preferably 1 to 10 mm. The lower limit of the distance D between electrode pairs is more preferably 2 mm, and even more preferably 3 mm. The upper limit of the distance D between electrode pairs is more preferably 6 mm, and even more preferably 5 mm. If the distance D between electrode pairs becomes too large, an excessive electric field will be generated, causing heat generation, electric field concentration at the electrode ends, boiling or discharge, which may reduce the survival rate or introduction efficiency. From the viewpoint of preventing heat generation, the distance D between electrode pairs is preferably 1 to 6 mm. Furthermore, a distance D between electrode pairs of 1 to 3 mm is even more preferable because it suppresses the generation of heat generation and discharge, and also suppresses the reduction in the survival rate or introduction efficiency of biological products such as cells due to heat generation or discharge. Note that the distance D between electrode pairs corresponds to the thickness of the flow channel substrate 55. The distance D between electrode pairs is also called the inter-electrode gap or gap. The distance D between the aforementioned electrode pairs can be measured by measuring the length of the corresponding portion using a caliper or micrometer.

[0085] <Electrode Pair> The first electrode 45 and the second electrode 46 constituting the electrode pair 44 are made of, for example, a metallic material or a carbon material. Specifically, a configuration similar to that of the electrodes described in International Publication No. 2023 / 157673 can be adopted.

[0086] (Thickness) The thickness of the first electrode 45 and the second electrode 46 are not particularly limited, but are preferably 0.5 to 10 mm, and more preferably 1 to 5 mm. The thickness of the first electrode 45 and the second electrode 46 can both be measured using calipers or a micrometer.

[0087] <Configuration of the Flowing Device> The materials constituting the first introduction section 56, the first substrate 52, the flowing substrate 55, the second substrate 54, and the second introduction section 58 of the flowing device 12 in Figure 2 are not particularly limited. Various materials can be used as these constituting materials, for example, as long as they have sufficient resistance to suspensions, or suspensions and sheath fluids, and can ensure the necessary rigidity. In the flowing device 12, the first introduction section 56, the first substrate 52, the flowing substrate 55, the second substrate 54, and the second introduction section 58 may be formed by various known methods. Regarding the configuration of the EP apparatus, the apparatus configuration described in PCT / JP / 2025 / 016650 or International Publication No. 2024 / 248133 may be used.

[0088] <Liquid Detection Sensor> The liquid detection sensor detects the liquid in the piping, and for example, an ultrasonic sensor is used. <Pressure Sensor> The pressure sensor measures the discharge pressure of the first pump 13a, the second pump 13b, and the third pump 13c. The pressure sensor is not particularly limited as long as it can measure the discharge pressure of the first pump 13a, the second pump 13b, and the third pump 13c. For example, a diaphragm type pressure sensor is used.

[0089] <Flow Sensor> The flow sensor is not particularly limited in its configuration, as long as it can measure the flow rate of the suspension Q in the first pipe 14, the flow rate of the sheath liquid s in the second pipe 15, and the flow rate of the sheath liquid s in the third pipe 16. For example, ultrasonic, Coriolis, or thermal MEMS (Micro Electro Mechanical Systems) type flow sensors can be used. <Temperature Sensor> The temperature sensor installed in the first pipe 14 measures the temperature of the suspension Q before EP. The temperature sensor installed in the discharge pipe 28 measures the temperature of the discharged suspension Q and sheath liquid s after EP. This makes it possible to detect any abnormalities such as insufficient flow rate, abnormal conductivity of the culture medium, or abnormal conductivity of the suspension, as described above.

[0090] <Conductivity Sensor> The conductivity sensor detects, for example, culture medium and a culture solution containing cells. Furthermore, since the conductivity of the washing solution and the culture solution containing cells differ, the conductivity sensor can distinguish between the washing solution and the culture solution containing cells. The configuration of the conductivity sensor is not particularly limited, and known conductivity sensors can be used as appropriate. In the EP system 10 of Figure 1, a liquid detection sensor 25 is provided in the piping 20 to supply the suspension Q or washing solution to the first pump 13a. On the other hand, in the EP system 10a of Figure 7, instead of suspension Q, a culture solution containing biologically derived material and culture medium is supplied to the first pump 13a. The conductivity sensor 75 can measure the concentration of cells in the culture solution by utilizing the difference in conductivity depending on the cell concentration. For this reason, it is preferable to provide the conductivity sensor 75. For example, if the measured cell concentration is less than a preset cell concentration, the supply to the first pump 13a may be stopped. Note that the liquid detection sensor 25 alone cannot distinguish between the culture solution and the washing solution. Therefore, by placing the conductivity sensor 75 on the tank 23a side of the liquid detection sensor 25, it is possible to distinguish between the culture solution and the washing solution by utilizing the difference in conductivity.

[0091] <Cleaning Solution> The cleaning solution is used to clean the main channel 40 as described above. The channel is properly cleaned by the cleaning solution. The cleaning solution is also used to store the channel device 12, and the channel device 12 is sealed with the cleaning solution. The cleaning solution contains water or deionized water as described above. For example, ultrapure water, RO (Reverse Osmosis) water produced using a reverse osmosis membrane, distilled water, and deionized water can be used as the cleaning solution. From the viewpoint of suppressing electrode degradation due to electrochemical reactions, the water used as the cleaning solution preferably has an conductivity of 0.01 mS / cm or less.

[0092] <Suspension> The suspension to be subjected to electroporation (EP) contains biologically derived substances and bioactive substances. Furthermore, the suspension is not one in which the culture medium has been replaced with EP buffer, nor has EP buffer been added. In other words, the suspension does not contain EP buffer, nor is it substantially free of salts (e.g., sodium chloride), nor is it substantially free of poloxamers.

[0093] (Biologically Derived Substances) Biologically derived substances are not particularly limited, but 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, for the reasons that the effects of the present invention are superior. 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, hybridomas, etc. In terms of pharmaceutical manufacturing, gene transfer using HEK293 or CHO is most commonly used.

[0094] (Bioactive Substances) Bioactive substances are substances that, when introduced into biologically derived materials such as nucleic acids (e.g., DNA, RNA) and proteins, exert some effect on those materials. 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 culture medium is preferably 10 to 500 μg / mL. The concentration of the bioactive substance per biologically derived material in the suspension is preferably 20 pg / particle or less, more preferably 5 pg / particle or less, even more preferably 1 pg / particle or less, and particularly preferably 0.5 pg / particle or less. The lower the concentration of the bioactive substance per biologically derived material in the suspension, the less bioactive substance can be used per biologically derived material, thus reducing costs.

[0095] There are no restrictions on the concentration of biologically derived substances in suspension Q, but it is preferable that the volume fraction of biologically derived substances in suspension Q is 20% or more.

[0096] 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 is determined by measuring the average diameter and concentration (number of particles per unit volume) of the biologically derived substances by image analysis (e.g., Vi-CELL XR (Beckman Coulter), etc.), and calculating the volume fraction (= 4 ÷ 3 × (average diameter ÷ 2)). 3 The volume fraction can be calculated as (π × concentration × 100 (%)). Note that the volume fraction is calculated assuming that the biological material is spherical. More specifically, for example, if the average diameter of the biological material in suspension Q is 20 μm and the concentration is 40 × 10 ６ If the concentration is per mL (milliliters), the volume fraction of biologically derived substances in suspension Q can be calculated as 17%.

[0097] As the culture medium, liquid culture media commonly used for culturing animal cells can be used. Various companies sell culture media optimized for cell culture, such as HEK293 based on PBS (phosphate-buffered saline). The culture medium contains, but is not particularly limited to, amino acids, salts, sugars (glucose, etc.), vitamins, hormones, growth factors, lipids, trace elements, etc. The pH (hydrogen ion concentration) of the culture medium is 6 to 8, preferably 6.8 to 7.6, and more preferably 7.2 to 7.6.

[0098] <Sheath Fluid> Any liquid other than a suspension can be used as the sheath fluid for the sheath fluid flow. Among these, a culture medium used for suspensions is a preferred example of the sheath fluid. This culture medium is preferably one that has been used for the proliferation of living organisms such as cells.

[0099] <Electroporation Voltage> The electroporation voltage is set so that the electric field applied to the suspension between the electrode pair reaches the desired electric field strength, taking into account the thickness of the suspension flow, the electrical conductivity of the suspension flow, the thickness of the sheath liquid flow, and the electrical conductivity of the sheath liquid flow. It is preferable that the electrical resistance of the electrodes is sufficiently lower than the resistance of the suspension and sheath liquid, as this may cause a decrease in the introduction efficiency and utilization efficiency of bioactive substances in electroporation. The optimal value of the electric field applied to the suspension varies depending on the type and size of the bio-derived material, but is usually around 100 to 2000 V / cm. The voltage is preferably a pulsed voltage. In addition, a bipolar pulse (alternating between positive and negative) may be used to homogenize the electrode reaction (gas generation due to electrolysis, electrode degradation, etc.).

[0100] <Pulse width> The optimal pulse width varies depending on the type of biological material, but is usually 0.1 to 100 ms (milliseconds), preferably 1 to 10 ms.

[0101] <Pulse Period (Pulse Interval)> The pulse period is preferably synchronized (an integer multiple) with the time it takes for the biological material to pass through the electrode (electrode length L (see Figure 2)). For example, an average of 1 to 5 pulses, preferably 1 pulse voltage, is applied while the biological material passes through the electrode (electrode length L). The time it takes for the biological material to pass through the electrode (electrode length L) is determined by the flow rate and the cross-sectional area of ​​the flow path. If the cross-sectional shape of the flow path is a rectangle with all interior angles at 90°, the cross-sectional area of ​​the flow path is (electrode width W) × (distance between electrode pairs D). Note that the electrode width W is not shown. The distance between electrode pairs D is shown in Figure 2.

[0102] This specification incorporates, by reference, the provisions of International Application No. PCT / JP2024 / 012393, specifically (1) cell pre-culture, (2) adjustment of cell concentration (concentration, etc.), (3) addition and mixing of nucleic acids, (4) electroporation (gene transfer), (5) culture for virus production (main culture), and (6) recovery and purification of the virus.

[0103] The method for cleaning fluidic devices of the present invention is suitably applicable to the manufacture of gene therapy drugs and the like. The present invention is basically configured as described above. Although the method for cleaning fluidic devices of the present invention has been described in detail above, the present invention is not limited to the embodiments described above, and various improvements or modifications may be made without departing from the spirit of the present invention. All publications, patents and patent applications cited herein are incorporated herein by direct reference.

[0104] 10, 10a EP system 11 EP device 12 flow path device 12a front surface 12b back surface 13a first pump 13b second pump 13c third pump 14 first piping 15 second piping 16 third piping 17 fourth piping 18 power supply unit 19 control unit 20, 26, 71 piping 22, 23a, 23b, 27a, 70 tank 23c gas supply unit 24a, 24b, 24c, 24d, 27c, 27d valve 25 liquid detection sensor 27b gas supply unit 28 discharge pipe 28a branching section 28b, 28c, 33a branching path 28d connection section 29, 29a, 29b, 36 detection unit 30, 30a flow sensor 31a, 31b, 31c, 31d, 31e Valves 32 Pressure sensor 33b Recovery container 33c Branch section 34a, 34b, 35, 39a, 39b, 71a Valves 36a Liquid detection sensor 36b Temperature sensor 37 Waste liquid tank 38 Container 40 Main flow path 40a End 40b End 40c Inner surface 40d Outlet 41a Supply port 41b First supply port 41c Second supply port 41d Discharge port 44 Electrode pair 45 First electrode 45a, 46a Electrode surface 46 Second electrode 47 Space 52 First substrate 52a, 52b, 54a, 56a, 56b, 58a Through hole 52c, 54c Sheath liquid confluence port 54 Second substrate 55 Flow channel substrate 55d Suspension inlet 56 First introduction section 58 Second introduction section 59 Confluence section 64, 65 Sheath liquid flow channel 66 Cover 71b Liquid detection sensor 72 Mixer 73 Temperature sensor 74 Damper 75 Conductivity sensor D Distance D Ｌ Extension direction Dd, liquid delivery direction Df, direction Dm, thickness Ds, stacking direction Hp, horizontal plane L, electrode length Q, suspension R １ , R ２ , R ３ , R ４ , R ５ Path ds Thickness s Sheath fluid x direction y direction z Vertical direction

Claims

1. A method for cleaning a flow channel device used in electroporation, wherein a suspension containing a bio-derived substance and a bioactive substance is flowed through the device, and an electric field is applied to the suspension by an electrode pair to introduce a bioactive substance into the bio-derived substance, the method comprising: stopping the supply of the suspension in the electroporation and discharging the suspension from the suspension flow channel of the flow channel device using gas; supplying a cleaning solution to the flow channel to clean the flow channel; and stopping the supply of the cleaning solution and storing the flow channel device with the flow channel filled with the cleaning solution.

2. A method for cleaning a flow channel device according to claim 1, wherein, in the step of discharging the suspension from the flow channel, the gas is flowed in the opposite direction to the direction of liquid delivery of the suspension in the flow channel during electroporation.

3. The method for cleaning a flow channel device according to claim 1, wherein the flow channel extends in a vertical direction, the direction of delivery of the suspension between the electrode pair is upward in the vertical direction, and the direction of flow of the gas is downward in the vertical direction.

4. A method for cleaning a flow channel device according to claim 1, wherein, in the step of cleaning the flow channel, the cleaning solution is flowed in the same direction as the flow direction of the suspension in the flow channel in the electroporation.

5. The method for cleaning a flow channel device according to claim 1, wherein the cleaning solution contains water.

6. The method for cleaning a flow channel device according to claim 1, wherein the cleaning solution contains ion-exchanged water.

7. A method for cleaning a flow channel device used in electroporation, wherein a bioactive substance is introduced into a bio-derived substance by applying an electric field to the suspension containing a bio-derived substance and a bioactive substance using an electrode pair while the suspension is flowing, the method comprising: stopping the supply of the suspension in the electroporation and discharging the suspension from the flow channel of the flow channel device using gas; and supplying a cleaning solution to the flow channel to clean the flow channel, wherein in the step of discharging the suspension from the flow channel, the gas is flowed in the opposite direction to the flow direction of the suspension in the flow channel in the electroporation.

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