Electroporation device, method for introducing bioactive substances, method for producing bio-derived products and bio-derived products

Abstract

This electric punching device 10 comprises: a channel 20 for circulating a liquid; a first electrode 30A which forms a first electric field area 60A in the channel 20 by supplying a pulse voltage; a second electrode 30B which forms a second electric field area 60B on a downstream side in a liquid circulation direction from the first electric field area 60A in the channel 20 by applying the pulse voltage; a cooling unit 40 which forms, between the first electric field area 60A and the second electric field area 60B in the channel 20, a cooling area 70 in which the liquid circulating in the channel 20 is cooled; and a voltage output unit 50 which outputs the pulse voltage for forming the first electric field area 60A and the second electrical field area 60B.

Description

[Technical Field]

[0001] The disclosed technologies relate to electroporation devices, methods for introducing bioactive substances, methods for producing bio-derived products, and bio-derived products. [Background technology]

[0002] Electroporation is a method of introducing substances into cells by creating pores in the cell membrane using electrical pulses. For example, by applying electrical pulses to a cell suspension, tiny pores are created in the cell membrane, and DNA (deoxyribonucleic acid) can be introduced into the cell, thereby enabling cell transformation. The following techniques are known for introducing bioactive substances such as DNA into cells using electroporation.

[0003] For example, Japanese Patent Publication No. 2007-7430 describes an electroporation apparatus comprising a fluid channel, electrodes arranged along the fluid channel and moving along the fluid channel, to which biological particles are subjected to an electric field suitable for electroporation, and a computer that controls the pulsed charge applied to the electrodes, wherein the apparatus is equipped with means for cooling the electrodes.

[0004] Japanese Patent Publication No. 2011-528550 describes an electroporation method that includes the steps of determining a first time constant (t1) representing the increase in conductivity in the electroporation medium when a pulse is applied, and determining a second time constant (t2) representing the discharge of the capacitor, setting the electroporation parameters such that t1 is greater than or equal to t2 and the duration of the pulse is shorter than either t1 or t2, and applying one or more electrical pulses to the sample to be electroporated under these electroporation parameters.

[0005] U.S. Patent Application Publication No. 2017 / 0283761 describes a device for electroporation of cells, comprising two electrodes. [Overview of the project] [Problems that the invention aims to solve]

[0006] A known flow process involves circulating a suspension containing the bio-derived material and the bioactive substance through a channel equipped with parallel plate electrodes, as a method for introducing bioactive substances such as DNA, RNA (ribonucleic acid), and proteins into biologically derived materials such as cells, cell derivatives, organelles, intracellular granules, and vesicles using electroporation.

[0007] The following are some of the problems with flow processes: The Joule heating generated when voltage is applied heats the suspension flowing through the channel, and this heat may damage biological materials. Furthermore, there is a concern that bumping may occur in the heated suspension, and the shock waves generated by this bumping may damage biological materials. In addition, if the electrode temperature becomes excessively high, the electrode itself will deteriorate, shortening the lifespan of the electroporation device.

[0008] The inventors of this invention have diligently investigated the temperature rise of the suspension flowing through the flow path during electroporation by a flow process. As a result, they have found that even under conditions where the temperatures of the electrodes and suspension, measured by averaging over time and space, are sufficiently suppressed, the electrodes and suspension can become locally and instantaneously hot. In other words, it is believed that the above problem can be resolved by suppressing the localized and instantaneous temperature rise of the electrodes and suspension.

[0009] The disclosed technology aims to suppress localized, instantaneous temperature increases of electrodes and suspensions during electroporation by a flow process. [Means for solving the problem]

[0010] The electroporation device according to the disclosed technology includes a flow path for flowing liquid, a first electrode that forms a first electric field region in the flow path when a pulse voltage is supplied, a second electrode that forms a second electric field region downstream of the first electric field region in the flow path in the direction of liquid flow when a pulse voltage is supplied, a cooling unit that forms a cooling region between the first electric field region and the second electric field region in the flow path for cooling the liquid flowing through the flow path, and a voltage output unit that outputs pulse voltages for forming the first electric field region and the second electric field region.

[0011] The electroporation device may further include a cooling mechanism for cooling the first electrode and the second electrode.

[0012] The electroporation device may include a plurality of pairs of conductive members provided along the direction of liquid flow on the opposing walls of the flow path, and a cooling mechanism for cooling each of the plurality of pairs of conductive members. In this case, the voltage output unit may supply a pulse voltage to each of two pairs of conductive members sandwiching at least one pair of conductive members from the plurality of pairs of conductive members, so that one of the two pairs of conductive members functions as a first electrode, the other of the two pairs of conductive members functions as a second electrode, and at least one pair of conductive members sandwiched between the two pairs of conductive members functions as a cooling unit. The voltage output unit may switch the conductive members from which the pulse voltage is supplied among the plurality of pairs of conductive members at a predetermined timing. The plurality of pairs of conductive members may have the same length in the direction of liquid flow.

[0013] A method for introducing a bioactive substance according to the disclosed technology is a method for introducing a bioactive substance into a bio-derived substance while the suspension containing the bioactive substance flows through a channel, and includes the steps of: the suspension passing through a first electric field region; the suspension passing through a cooling region for cooling the suspension after passing through the first electric field region; and the suspension passing through a second electric field region after passing through the cooling region.

[0014] The process of passing the suspension through the electric field region and the process of passing the suspension through the cooling region may be repeated alternately at least twice each.

[0015] The cumulative amount of energy applied to the suspension while the suspension passes through a plurality of electric field regions including the first electric field region and the second electric field region may correspond to an energy amount predetermined as the energy required to introduce a bioactive substance into a bio-derived substance.

[0016] It is preferable that the local and instantaneous maximum temperature of the suspension in the flow path is 100°C or lower.

[0017] The plurality of electric field regions including the first electric field region and the second electric field region may each be formed by supplying a pulsed voltage to a cooled electrode.

[0018] The time required for the suspension to pass through one electric field region is preferably an integer multiple of the period of the pulsed voltage.

[0019] The bio-derived substance is not limited to cells and may be human-derived cells. Particularly preferably, it is HEK293 cells. The bioactive substance may be DNA.

[0020] The method for producing a bio-derived substance according to the disclosed technology includes a step of introducing a bioactive substance by the above-described introduction method.

[0021] The bio-derived substance according to the disclosed technology is produced by the above-described production method.

Advantages of the Invention

[0022] According to the disclosed technology, in electroporation by a flow process, it is possible to suppress a local and instantaneous temperature rise of the electrode and the suspension.

Brief Description of the Drawings

[0023] [Figure 1] It is a perspective view schematically showing the configuration of an electroporation device according to an embodiment of the disclosed technology. [Figure 2]This is a cross-sectional view along line 2-2 in Figure 1. [Figure 3] This figure shows an example of a pulse voltage output from the voltage output unit according to an embodiment of the disclosed technology. [Figure 4] This is a cross-sectional view showing an example of the configuration of a cooling unit according to an embodiment of the disclosed technology. [Figure 5] This is a perspective view showing the configuration of an electroporation device related to a comparative example. [Figure 6] This image shows the behavior of an electroporation device according to a comparative example, when a pulse voltage is applied to the electrode while a suspension is flowing through the channel. [Figure 7] This image shows the behavior of an electroporation device according to a comparative example, when a pulse voltage is applied to the electrode while a suspension is flowing through the channel. [Figure 8] This is a cross-sectional view showing the configuration of an electroporation device according to another embodiment of the disclosed technology. [Figure 9] This figure shows a simulation model of an electroporation device according to an embodiment of the disclosed technology. [Figure 10] This figure shows a simulation model of an electroporation device related to a comparative example. [Figure 11A] This is a contour map showing the temperature distribution of the fluid in a model relating to an embodiment of the disclosed technology. [Figure 11B] This is a contour map showing the temperature distribution of the fluid in a model relating to an embodiment of the disclosed technology. [Figure 12A] This is a contour plot showing the temperature distribution of the fluid in the model related to the comparative example. [Figure 12B] This is a contour plot showing the temperature distribution of the fluid in the model related to the comparative example. [Figure 13] This is a graph showing the time course of the fluid temperature in a model relating to an embodiment of the disclosed technology. [Figure 14] This is a graph showing the time course of the fluid temperature in a model relating to an embodiment of the disclosed technology. [Figure 15] This is a cross-sectional view showing an example of the configuration of an electroporation device according to another embodiment of the disclosed technology. [Figure 16] This is a cross-sectional view showing an example of the configuration of an electroporation device according to another embodiment of the disclosed technology. [Figure 17] This is a cross-sectional view illustrating an example of a use case of an electroporation device according to another embodiment of the disclosed technology. [Figure 18] This is a cross-sectional view illustrating an example of a use case of an electroporation device according to another embodiment of the disclosed technology. [Modes for carrying out the invention]

[0024] Hereinafter, an example of an embodiment of the disclosed technology will be described with reference to the drawings. In each drawing, identical or equivalent components and parts will be given the same reference numerals, and redundant descriptions will be omitted.

[0025] In this specification, "biogenic substances" refer to structures derived from living organisms, such as cells, cell derivatives, organelles, intracellular granules, and vesicles. Examples of biogenic substances include yeast, Escherichia coli, and animal cells, preferably animal cells, which may be human-derived cells. These may be human T cells, HEK293, A549, SF9, EB66, Daudi, Hela, Vero, MDCK, or HEK293 cells. "Bioactive substances" refer to any substance that exerts some effect on biogenic substances, and may include DNA, RNA, and proteins. Examples of bioactive substances include plasmids, linear DNA, mRNA, etc.

[0026] [First Embodiment] Figure 1 is a schematic perspective view showing the configuration of an electroporation device 10 according to a first embodiment of the disclosed technology. Figure 2 is a cross-sectional view along line 2-2 in Figure 1. The electroporation device 10 is a device used for introducing bioactive substances into biological materials by electroporation using a flow process. For example, by using the electroporation device 10 to introduce DNA, an example of a bioactive substance, into cells, an example of a biological material, it is possible to create new cells with new genetic characteristics. The electroporation device 10 is composed of a flow channel 20, a first electrode 30A, a second electrode 30B, a cooling unit 40, and a voltage output unit 50.

[0027] The flow path 20 is a flow path for circulating a liquid (hereinafter referred to as suspension) containing biologically derived materials and bioactive substances. The flow path 20 includes an inlet 21 and an outlet 22. The inlet 21 is provided at one end of the flow path 20, and the outlet 22 is provided at the other end of the flow path 20. The flow path 20 is made of resin and an insulator such as glass. When the electroporation device 10 is used, the suspension flows into the flow path 20 from the inlet 21, flows through the flow path 20, and flows out of the flow path 20 from the outlet 22. In this embodiment, the area of ​​the cross-section of the flow path 20 perpendicular to the flow direction of the suspension is kept constant. Also, the flow velocity of the suspension flowing through the flow path 20 is kept constant. The liquid flow of the suspension flowing through the flow path 20 is formed by a liquid delivery means such as a pump (not shown).

[0028] The first electrode 30A has the form of a so-called parallel plate electrode and is composed of a pair of conductive members provided on the opposing walls of the flow channel 20. The first electrode 30A forms a first electric field region 60A in the flow channel 20 when a pulse voltage output from the voltage output unit 50 is supplied to it.

[0029] The second electrode 30B, like the first electrode 30A, has the form of a parallel plate electrode and is composed of a pair of conductive members provided on the opposing walls of the flow channel 20. The second electrode 30B is provided downstream of the first electrode 30A in the flow direction of the suspension flowing through the flow channel 20. The second electrode 30B is supplied with a pulse voltage output from the voltage output unit 50, thereby forming a second electric field region 60B downstream of the first electric field region 60A in the flow channel 20 in the flow direction of the suspension flowing through the flow channel 20. Preferably, the dimensions (length in the flow direction of the suspension) of the first electrode 30A and the second electrode 30B are the same. As the conductive members constituting the first electrode 30A and the second electrode 30B, for example, metals such as Cu and Al or conductive resins can be used.

[0030] The voltage output unit 50 supplies pulse voltages to the first electrode 30A and the second electrode 30B, respectively. Figure 3 shows an example of the pulse voltage output from the voltage output unit 50. The voltage output unit 50 outputs pulse voltages whose polarity alternates. The period T of the pulse voltage is the on period t. on and off period off This is the sum of the above. The time required for the suspension to pass through one of the first electric field region 60A and the second electric field region 60B (the region directly below the electrode) can be an integer multiple of the pulse voltage period T. This ensures that the voltage application time to the biological material contained in the suspension flowing through the channel 20 is uniform, regardless of the timing of the pulse voltage being turned on. For example, if the time required for the suspension to pass through one electric field region is 1 times the pulse voltage period T, then the voltage application time to the biological material during the period in which the biological material passes through one electric field region (the region directly below the electrode) is uniform regardless of the timing of the pulse voltage being turned on. on This is equal to [the given value]. Furthermore, although the suspension flowing through channel 20 is considered to have a flow velocity distribution, representative values ​​such as the maximum and average values ​​can be used when calculating the time required for the suspension to pass through the electric field region.

[0031] In the electroporation device 10 according to the present embodiment, the amount of energy E required to introduce a bioactive substance into a biological-derived material N The predetermined amount of energy as is divided and applied in the first electric field region 60A and the second electric field region 60B. That is, the cumulative amount of energy E applied to the suspension while the suspension passes through the first electric field region 60A and the second electric field region 60B G is equivalent to the required amount of energy E N (that is, so that E G ≒E N ), the on-period t of the voltage pulse on and the voltage level are set. By making the dimensions (lengths in the flow direction of the suspension) of the first electrode 30A and the second electrode 30B the same as each other, the required amount of energy E N can be evenly divided and applied in each electric field region.

[0032] The cooling unit 40 is provided between the first electrode 30A and the second electrode 30B of the flow path 20. The cooling unit 40 forms a cooling region 70 that cools the suspension flowing through the flow path 20 between the first electric field region 60A and the second electric field region 60B in the flow path 20. FIG. 4 is a cross-sectional view showing an example of the configuration of the cooling unit 40. The cooling unit 40 includes a heat conduction member 41 provided on the wall surfaces of the flow path 20 facing each other, and a cooling mechanism 42 connected to the heat conduction member 41. The heat conduction member 41 is preferably made of a metal having a relatively high thermal conductivity, such as Cu and Al. The cooling mechanism 42 may be, for example, a water cooling type in which a liquid as a cooling medium flows inside a jacket as illustrated in FIG. 4. From the viewpoint of cooling efficiency, it is preferable that the coolant is in direct contact with the heat conduction member 41. Also, by providing a flow path through which the cooling medium flows inside the heat conduction member 41, it is possible to integrally configure the heat conduction member 41 and the cooling mechanism 42. Further, the cooling mechanism 42 is not limited to the water cooling type, and may be, for example, an air cooling type or one using a Peltier element.

[0033] While the suspension passes through the cooling region 70, the suspension is continuously cooled. Therefore, the longer the length of the cooling section 40 (cooling region 70) in the direction of suspension flow, the lower the temperature of the suspension can be. The electroporation device 10 sets the lengths of the first electrode 30A (first electric field region 60A), the second electrode 30B (second electric field region 60B), and the cooling section 40 (cooling region 70) in the direction of suspension flow so that the local and instantaneous maximum temperature of the suspension in the flow path 20 is 100°C or less. In other words, the length of the cooling section 40 is set so that the temperature of the suspension can be lowered to a temperature of 100°C or less even if heated in the second electric field region 60B before the suspension reaches the second electric field region.

[0034] According to the electroporation device 10 of this embodiment, an introduction method is realized in which a bioactive substance is introduced into a bio-derived material while the suspension containing the bio-derived material and the bioactive substance flows through the channel 20. That is, the introduction method according to the embodiment of the disclosed technology includes the steps of: the suspension passing through a first electric field region 60A; the suspension passing through a cooling region 70 for cooling the suspension after passing through the first electric field region; and the suspension passing through a second electric field region 60B after passing through the cooling region 70.

[0035] Figure 5 is a perspective view showing the configuration of an electroporation device 10X according to a comparative example that does not have a cooling section. The electroporation device 10X according to the comparative example comprises a flow channel 20 and a single electrode 30. Figures 6 and 7 are images showing the electroporation device 10X according to the comparative example when a suspension is flowed through the flow channel 20 and a pulse voltage is applied to the electrode 30. ru.

[0036] As shown in the images on the right of Figure 6 and the right of Figure 7, with the electroporation device 10X of the comparative example, the suspension flowing through the channel 20 is heated by Joule heat generated when voltage is applied, causing the suspension to boil over (see the right of Figure 7) and a discharge phenomenon at the electrode outlet end (see the right of Figure 6). With the electroporation device 10X of the comparative example, biological materials contained in the suspension may be damaged by heat and shock waves associated with boiling over and discharge. Furthermore, the electrode 30 may deteriorate, potentially shortening the lifespan of the electroporation device 10X.

[0037] On the other hand, according to the electroporation device 10 according to an embodiment of the disclosed technology, since it has a first electrode 30A and a second electrode 30B that are separated from each other, the amount of energy E required to introduce a bioactive substance into a bio-derived material is N This can be divided and applied in a first electric field region 60A and a second electric field region 60B. This allows the amount of energy in one electric field region to be suppressed, thereby suppressing local and instantaneous temperature increases of the electrodes and suspension.

[0038] Here, as a method to suppress the local, instantaneous temperature rise of the electrodes and suspension, the pulse voltage period T and off period t are used. off It is conceivable to lengthen the interval. However, in this case, the amount of energy E required to introduce a bioactive substance into a bio-derived material is N To ensure this, it is necessary to reduce the flow rate of the suspension, which reduces processing efficiency. According to the electroporation device 10 according to an embodiment of the disclosed technology, the required amount of energy E can be reduced without reducing the flow rate of the suspension. N It is possible to provide this. In other words, according to the electroporation device 10 according to an embodiment of the disclosed technology, in electroporation by a flow process, it is possible to suppress local, instantaneous temperature rises of the electrodes and suspension without reducing processing efficiency (throughput).

[0039] Furthermore, according to the electroporation device 10 according to an embodiment of the disclosed technology, since there is a cooling section 40 between the first electrode 30A and the second electrode 30B, the suspension whose temperature has risen by passing through the first electric field region 60A is cooled before passing through the second electric field region 60B. This makes it possible to suppress local and instantaneous maximum temperatures that are determined according to the cumulative temperature change of the suspension.

[0040] The electroporation device 10 according to the embodiment of the disclosed technology can also be modified as shown in Figure 8. That is, the electroporation device 10 may include a cooling mechanism 45 for cooling the first electrode 30A and the second electrode 30B. The cooling mechanism 45 may be a water-cooled type in which a liquid as a cooling medium flows inside the jacket, similar to the cooling mechanism 42 that constitutes the cooling unit 40. From the viewpoint of cooling efficiency, it is preferable that the cooling liquid is in direct contact with the first electrode 30A and the second electrode 30B. The cooling mechanism 45 is not limited to a water-cooled type, but may be, for example, an air-cooled type or one using a Peltier element. Although Figure 8 illustrates a configuration in which the first electrode 30A, the second electrode 30B and the heat conduction member 41 are cooled by separate cooling mechanisms, the cooling mechanism may be configured to cool the first electrode 30A, the second electrode 30B and the heat conduction member 41 integrally.

[0041] The local and instantaneous highest temperature of the suspension flowing through the channel 20 is determined by the on-period t of the pulse voltage supplied to the first electrode 30A and the second electrode 30B. on It rises during the off period t of the pulse voltage. off The pulse voltage decreases during the off-period t as the first electrode 30A and the second electrode 30B are cooled. off This makes it possible to increase the rate of decrease in the local and instantaneous maximum temperature of the suspension. As a result, the local and instantaneous maximum temperature of the suspension can be suppressed. In addition, since the temperatures of the first electrode 30A and the second electrode 30B themselves are also suppressed, it is possible to suppress the progression of degradation of these electrodes.

[0042] [Computational Fluid Dynamics Analysis] Computational fluid dynamics (CFD) analysis was performed on an electroporation device according to an embodiment of the disclosed technology. Figure 9 shows a simulation model M1 of the electroporation device according to an embodiment of the disclosed technology, with the upper panel showing a lateral view and the middle and lower panels showing a top view. Model M1 according to the embodiment of the disclosed technology has a cooling section 40 between the first electrode 30A and the second electrode 30B, and the first electrode 30A and the second electrode 30B are cooled by a method equivalent to water cooling. Figure 10 shows a simulation model M2 of an electroporation device according to a comparative example, with the upper panel showing a lateral view and the middle and lower panels showing a top view. The model according to the comparative example does not have a cooling section between the first electrode 30A and the second electrode 30B, and the first electrode 30A and the second electrode 30B are cooled by a method equivalent to water cooling.

[0043] (1) Dimensions of the flow path model In Model M1, which is an embodiment of the disclosed technology shown in Figure 9, the dimensions of each part are as follows. L1 (length of the first electrode 30A): 5mm L2 (distance between the first electrode 30A and the cooling section 40): 1 mm L3 (length of cooling section 40): 50mm L4 (distance between cooling section 40 and second electrode 30B): 1 mm L5 (length of the second electrode 30B): 5mm L6 (First electrode 30A, second electrode 30 B (and thickness of cooling section 40): 1 mm L7 (thickness of the first electrode 30A, the second electrode 30B, and the cooling section 40): 1 mm L8 (height of channel 20): 2mm L9 (width of channel 20): 2mm In the comparative example shown in Figure 10, Model M2, the dimensions of each part are as follows. L11 (length of the first electrode 30A): 5mm L12 (distance between the first electrode 30A and the second electrode 30B): 1 mm L13 (length of the second electrode 30B): 5mm L14 (thickness of the first electrode 30A, the second electrode 30B, and the cooling section 40): 1 mm L15 (thickness of the first electrode 30A, the second electrode 30B, and the cooling section 40): 1 mm L16 (width of channel 20): 2mm

[0044] (2) Analysis software (common to both the examples and comparative examples) Ansys 2020R1

[0045] (3) Mesh conditions (common to both the examples and comparative examples) Hexa Mesh Standard Cell Size 0.2mm Mesh refinement is performed only on the channel walls (standard conditions for Ansys inflation function).

[0046] (4) Analysis model (common to both the examples and comparative examples) Viscosity model: k-epsilon (standard conditions) Potential: Application of potential equation, Joule heating incorporated.

[0047] (5) Physical properties (common to both examples and comparative examples) The physical properties of the fluid flowing through the channel 20, the first electrode 30A, the second electrode 30B, and the cooling unit 40 were set to the values ​​shown in Table 1 below. The physical properties of the fluid are equivalent to those of physiological saline, and the physical properties of the first electrode 30A, the second electrode 30B, and the cooling unit 40 are equivalent to those of copper. [Table 1]

[0048] (6) Boundary conditions (a) Momentum Inlet speed: 66.7mm / s (b) fever Hatched areas in the middle of Figure 9 and the middle of Figure 10 Convection Convective heat transfer coefficient: 7000 [W / m 2 ·k] Free flow temperature: 283.15 [K] (equivalent to 10°C water cooling) The boundary where different materials meet coupled All other walls Heat flux: 0[W / m 2 (Equivalent to thermal insulation) Temperature of the fluid flowing in from the inlet: 300[K] (c) Potential Hatching in the lower part of Figure 9 and the lower part of Figure 10 Pulse voltage application (see Figure 3) Voltage: Apply +195V and -195V alternately. Period T:75ms On period t on :5ms Off period off :70ms All other walls Current density: 0[A / m 2 ]

[0049] (7) Calculation conditions Unsteady-state analysis Time step: 5ms Regarding convergence: After confirming the convergence of the model through steady-state analysis under equivalent conditions, transient analysis was performed.

[0050] (8) Analysis results Figures 11A and 11B are contour plots showing the temperature distribution of the fluid in Model M1 according to an embodiment of the disclosed technology. Figure 11A shows the temperature distribution in a cross-section at the center of the width direction of the flow path 20, and Figure 11B shows the temperature distribution in a cross-section near the wall surface of the flow path 20. Figures 12A and 12B are contour plots showing the temperature distribution of the fluid in Model M2 according to a comparative example. Figure 12A shows the temperature distribution in a cross-section at the center of the width direction of the flow path 20, and Figure 12B shows the temperature distribution in a cross-section near the wall surface of the flow path 20. As shown in Figures 11A and 11B, according to Model M1 according to an embodiment of the disclosed technology, the fluid heated in the first electric field region is cooled in the cooling region and then flows into the second electric field region. As shown in Figures 12A and 12B, according to Model M2 according to a comparative example, the fluid heated in the first electric field region is further heated in the second electric field region without being cooled.

[0051] Figures 13 and 14 are graphs showing the time course of the fluid temperature in Model M1 according to an embodiment of the disclosed technology, respectively. Figure 13 shows the time course of the fluid temperature in the period immediately after the start of pulse voltage application, and Figure 14 shows the time course of the fluid temperature in the period before and after 4 seconds have elapsed since the start of pulse voltage application. In Figures 13 and 14, the time course of the maximum fluid temperature is shown by a solid line, and the time course of the temperature at the center of the width direction of the flow path 20 at the end of the second electrode 30B (the location marked with a triangle in Figure 9) is shown by a dotted line.

[0052] Table 2 below shows whether or not there are any points where the fluid temperature exceeds 100°C in Model M1, which is an embodiment of the disclosed technology, and Model M2, which is a comparative example. As shown in Table 2, it was confirmed that in Model M1, which is an embodiment of the disclosed technology, there are no points where the fluid temperature exceeds 100°C, that is, the maximum temperature of the fluid is 100°C or lower. [Table 2]

[0053] [Second Embodiment] Figure 15 is a cross-sectional view showing an example of the configuration of an electroporation device 10A according to a second embodiment of the disclosed technology. The electroporation device 10A according to this embodiment includes a first electrode 30A and a second electrode 30B, as well as a third electrode 30C and a fourth electrode 30D. Furthermore, the electroporation device 10A according to this embodiment includes a first cooling unit 40A, a second cooling unit 40B, and a third cooling unit 40C.

[0054] The third electrode 30C, when a pulse voltage is supplied, forms a third electric field region 60C downstream of the second electric field region 60B in the flow path 20 in the direction of suspension flow. The fourth electrode 30D forms a fourth electric field region 60D downstream of the third electric field region 60C in the flow path 20 in the direction of suspension flow. The first cooling section 40A forms a first cooling region 70A between the first electric field region 60A and the second electric field region 60B in the flow path 20. The second cooling section 40B forms a second cooling region 70B between the second electric field region 60B and the third electric field region 60C in the flow path 20. The third cooling section 40C forms a third cooling region 70C between the third electric field region 60C and the fourth electric field region 60D in the flow path 20.

[0055] The voltage output unit 50 supplies pulse voltages to the first electrode 30A, the second electrode 30B, the third electric field region 60C, and the fourth electrode 30D, respectively. The amount of energy E required to introduce a bioactive substance into a biological material. N However, it is given by dividing it into the first to fourth electric field regions 60A to 60D. By making the dimensions (length in the direction of suspension flow) of the first to fourth electrodes 30A to 30D the same, the required amount of energy E in each electric field region is obtained. N It can be divided equally and given to each person.

[0056] According to the electroporation device 10A of this embodiment, similar to the first embodiment, an introduction method is realized in which a bioactive substance is introduced into a bio-derived material while the suspension containing the bio-derived material and the bioactive substance flows through a channel. The introduction method according to this embodiment involves repeatedly performing, alternately, two or more steps each: the step of the suspension passing through an electric field region and the step of the suspension passing through a cooling region (in the example shown in Figure 15, the step of the suspension passing through the electric field region is performed four times, and the step of the suspension passing through the cooling region is performed three times).

[0057] The electroporation device 10A according to this embodiment can form more electric field regions than the electroporation device 10 according to the first embodiment (see Figures 1 and 2), so the amount of energy in one electric field region can be further suppressed, and the local, instantaneous temperature rise of the electrodes and suspension can be further suppressed. The number of electrodes may be three or five or more. Cooling units are provided between each electrode.

[0058] [Third Embodiment] Figure 16 is a cross-sectional view showing an example of the configuration of an electroporation device 10B according to a third embodiment of the disclosed technology. The electroporation device 10B according to this embodiment has nine pairs of conductive members 31a to 31i provided on the mutually opposing wall surfaces of the flow path 20. The conductive members 31a to 31i are arranged along the flow direction of the suspension flowing through the flow path 20. It is preferable that the dimensions (length in the flow direction of the suspension) of the conductive members 31a to 31i are the same. It is also preferable that the conductive members 31a to 31i are made of metals such as Cu and Al, which have relatively high electrical conductivity and thermal conductivity. The number of conductive members is not limited and can be increased or decreased as appropriate.

[0059] The electroporation device 10B has a cooling mechanism 45 that cools each of the conductive members 31a to 31i individually. The cooling mechanism 45 may be a water-cooled type in which a liquid as a cooling medium flows inside a jacket, as illustrated in Figure 16. From the viewpoint of cooling efficiency, it is preferable that the cooling liquid is in direct contact with the conductive members 31a to 31i. It is also possible to integrate the conductive members 31a to 31i and the cooling mechanism 45 by providing a flow path for the cooling medium to flow inside the conductive members 31a to 31i. Furthermore, the cooling mechanism 45 is not limited to a water-cooled type, but may be an air-cooled type or use a Peltier element. Although Figure 16 illustrates a configuration in which the conductive members 31a to 31i are cooled by separate cooling mechanisms, the cooling mechanism may be configured to cool the conductive members 31a to 31i integrally.

[0060] The voltage output unit 50 is capable of supplying pulse voltages independently to the conductive members 31a to 31i. The voltage output unit 50 supplies pulse voltages to each of two pairs of conductive members, with at least one pair of conductive members sandwiched between them. As a result, of the two pairs of conductive members to which the voltage pulses are supplied, the conductive member on the upstream side in the direction of suspension flow functions as a first electrode that forms a first electric field region in the flow path 20. Also, of the two pairs of conductive members to which the voltage pulses are supplied, the conductive member on the downstream side in the direction of suspension flow functions as a second electrode that forms a second electric field region downstream of the first electric field region in the flow path in the direction of suspension flow. Furthermore, at least one pair of conductive members, which are not supplied with voltage pulses and are sandwiched between the two pairs of conductive members to which the voltage pulses are supplied, functions as a cooling section that forms a cooling region between the first electric field region and the second electric field region. In other words, the conductive members 31a to 31i also function as heat conductive members that transfer heat from the suspension to the cooling mechanism 45.

[0061] For example, as shown in Figure 17, the voltage output unit 50 supplies pulse voltages to conductive members 31a and 31e, respectively. As a result, conductive member 31a functions as a first electrode forming a first electric field region 60A in the flow path, and conductive member 31e functions as a second electrode forming a second electric field region 60B downstream of the first electric field region 60A in the flow path in the direction of suspension flow. Conductive members 31b, 31c, and 31d, which are sandwiched between conductive members 31a and 31e and to which no voltage pulses are supplied, function as a first cooling section forming a first cooling region 70A between the first electric field region 60A and the second electric field region 60B in the flow path 20. Furthermore, conductive members 31f, 31g, 31h, and 31i, to which no voltage pulses are supplied, function as a second cooling section forming a second cooling region 70B downstream of the second electric field region 60B in the flow path 20 in the direction of suspension flow.

[0062] The voltage output unit 50 switches the conductive member to which it supplies the pulse voltage from among a plurality of pairs of conductive members at a predetermined timing. The voltage output unit 50 switches the conductive member to which it supplies the pulse voltage to conductive members 31b and 31f, for example, as shown in Figure 18. As a result, conductive member 31b functions as a first electrode that forms a first electric field region 60A in the flow path, and conductive member 31f functions as a second electrode that forms a second electric field region 60B downstream of the first electric field region 60A in the flow path in the direction of suspension flow. Conductive members 31c, 31d, and 31e, which are sandwiched between conductive members 31b and 31f and to which no voltage pulse is supplied, function as a first cooling section that forms a first cooling region 70A between the first electric field region 60A and the second electric field region 60B in the flow path 20. Furthermore, the conductive members 31g, 31h, and 31i, to which no voltage pulses are supplied, function as a second cooling section that forms a second cooling section 70B downstream of the second electric field section 60B in the flow path in the direction of suspension flow. In addition, the conductive member 31a, to which no voltage pulses are supplied, functions as a third cooling section that forms a third cooling section 70C upstream of the first electric field section 60A in the flow path in the direction of suspension flow.

[0063] The voltage output unit 50 may, for example, switch the conductive member supplying the pulse voltage when the cumulative time since the start of operation of the electroporation device 10B exceeds a predetermined threshold. Alternatively, the voltage output unit 50 may switch the conductive member supplying the pulse voltage when the cumulative processing amount of the suspension in the electroporation device 10B exceeds a predetermined threshold. Furthermore, the voltage output unit 50 may switch the conductive member supplying the pulse voltage when the number of pulses of the pulse voltage it has output exceeds a predetermined threshold. Additionally, the voltage output unit 50 may switch the conductive member supplying the pulse voltage when it detects an abnormality. The abnormality may be, for example, an abnormality in the current value, resistance value, or metal concentration value acquired in in-line measurement. The voltage output unit 50 may also switch the conductive member supplying the pulse voltage by sequentially shifting two pairs of conductive members supplying the pulse voltage along the flow direction of the suspension.

[0064] Figures 16 and 17 illustrate an example where three adjacent conductive members are used to form a cooling region between the first electric field region 60A and the second electric field region 60B. However, the number of conductive members forming the cooling region between the first electric field region 60A and the second electric field region 60B can be increased or decreased as appropriate.

[0065] The electroporation device 10B according to this embodiment, like the electroporation device 10 according to the first embodiment, makes it possible to suppress localized, instantaneous temperature rises of the electrodes and suspension. Furthermore, the electroporation device 10B according to this embodiment makes it possible to switch the electrodes that form the electric field region by switching the conductive member that supplies the pulse voltage. As a result, even if the conductive member that has been used as an electrode deteriorates and its function as an electrode decreases, another conductive member that has not deteriorated can be used as an electrode, thus reducing the frequency of replacement of the electroporation device. Replacing the electroporation device involves a loss of suspension. Also, the liquid flow of the suspension becomes unstable before and after the replacement of the electroporation device. Therefore, it is preferable to have a low frequency of replacement of the electroporation device. In addition, by making the dimensions (length in the direction of suspension flow) of the conductive members 31a to 31i the same, it is possible to suppress changes in the environment in the flow path 20 before and after switching the conductive member to which the pulse voltage is supplied.

[0066] Furthermore, the disclosure of Japanese Patent Application No. 2021-072675, filed on April 22, 2021, is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A channel for circulating liquid, A pulse voltage is supplied to the first electrode which forms a first electric field region in the flow path, A pulse voltage is supplied to a second electrode which forms a second electric field region downstream of the first electric field region in the flow path in the direction of liquid flow, A cooling section is provided to form a cooling region between the first electric field region and the second electric field region in the flow path, for cooling the liquid flowing through the flow path. A voltage output unit that outputs pulse voltages for forming the first electric field region and the second electric field region, A plurality of pairs of conductive members are provided along the direction of liquid flow on the mutually opposing wall surfaces of the flow path, A cooling mechanism for cooling each of the aforementioned multiple pairs of conductive members, Includes, The voltage output unit supplies a pulse voltage to each of two pairs of conductive members that sandwich at least one pair of conductive members from the plurality of pairs of conductive members, so that one of the two pairs of conductive members functions as the first electrode, the other of the two pairs of conductive members functions as the second electrode, and at least one pair of conductive members sandwiched between the two pairs of conductive members functions as the cooling unit. Electroporation device.

2. The voltage output unit switches the conductive member that supplies the pulse voltage from among the plurality of pairs of conductive members at a predetermined timing. The electroporation device according to claim 1.

3. The plurality of conductive members have the same length in the direction of liquid flow. The electroporation device according to claim 1 or claim 2.

4. A method for introducing a bioactive substance into a bio-derived substance while a suspension containing a bio-derived substance and a bioactive substance flows through a channel, The suspension comprises the step of passing through a first electric field region, The suspension passes through a cooling region after passing through the first electric field region, The suspension passes through a second electric field region after passing through the cooling region, Includes, Multiple pairs of conductive members are provided on the opposing walls of the flow path along the direction of flow of the suspension, and a cooling mechanism is provided to cool each of the multiple pairs of conductive members. By supplying a pulse voltage to each of two pairs of conductive members sandwiching at least one pair of conductive members from the plurality of pairs of conductive members, the first electric field region is formed by one of the two pairs of conductive members, the second electric field region is formed by the other of the two pairs of conductive members, and the cooling region is formed by at least one pair of conductive members sandwiched between the two pairs of conductive members. Installation method.

5. The process of the suspension passing through the electric field region and the process of the suspension passing through the cooling region are repeated alternately at least twice each. The introduction method according to claim 4.

6. The cumulative amount of energy supplied to the suspension as it passes through a plurality of electric field regions, including the first and second electric field regions, corresponds to a predetermined amount of energy required to introduce the bioactive substance into the bio-derived material. The introduction method according to claim 4 or claim 5.

7. The local and instantaneous maximum temperature of the suspension within the flow path is 100°C or less. The introduction method according to any one of claims 4 to 6.

8. The multiple electric field regions, including the first and second electric field regions, are each formed by supplying a pulsed voltage to a cooled electrode. The introduction method according to any one of claims 4 to 7.

9. Each of the multiple electric field regions, including the first electric field region and the second electric field region, is formed by supplying a pulse voltage to an electrode. The time required for the suspension to pass through one electric field region is an integer multiple of the period of the pulse voltage. The introduction method according to any one of claims 4 to 8.

10. The aforementioned biologically derived material is human-derived cells, The aforementioned bioactive substance is DNA. The introduction method according to any one of claims 4 to 9.

11. A method for producing a bio-derived product, comprising the step of introducing a bioactive substance by the introduction method described in any one of claims 4 to 10.

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