Electroporation method, method for producing useful substance, flow path device, and electroporation apparatus

Abstract

The present invention addresses the problem of providing an electroporation method that makes it possible to perform highly efficient electroporation, a method for producing a useful substance using the method, a flow path device for carrying out the method, and an electroporation apparatus including the flow path device. The problem is solved by forming a sheath flow that flows with a suspension and is in contact with electrodes, and by providing a restriction region where the thickness of a suspension flow formed of the suspension in the separation direction of a pair of the electrodes is restricted to 1-10 mm in the electroporation.

Description

Electroporation method, useful substance production method, flow channel device, and electroporation apparatus

[0001] The present invention relates to an electroporation method for introducing a bioactive substance into a biologically derived substance by applying an electric field to a suspension containing the biologically derived substance and the bioactive substance, a method for producing a useful substance using this method, a flow path device for performing this method, and an electroporation apparatus using this flow path device.

[0002] Electroporation, also known as electroporation, is a method for introducing substances into cells by creating holes in the cell membrane using an electric pulse. For example, by applying an electric field to a cell suspension using an electrode pair, minute holes are created in the cell membrane, and DNA (deoxyribonucleic acid) can be introduced into the cells, thereby transforming the cells.

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

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

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

[0006] In electroporation, the higher the concentration of biologically derived substances such as cells in the suspension, the higher the utilization efficiency of the bioactive substance. However, in flow electroporation, if the concentration of biologically derived substances in the suspension is too high, the introduction efficiency may decrease.

[0007] An object of the present invention is to provide an electroporation method capable of performing highly efficient electroporation, a method for producing useful substances using this electroporation method, a flow path device for carrying out this electroporation method, and an electroporation apparatus using this flow path device.

[0008] In order to achieve the above-mentioned objects, the present invention has the following configurations. [1] An electroporation method for introducing the bioactive substance into a suspension containing a biologically derived substance and a biologically active substance by applying an electric field to the suspension using an electrode pair, comprising: forming a sheath flow of a sheath liquid other than the suspension that flows together with the suspension and contacts the electrodes of the electrode pair; and further providing a regulated region through which only the suspension flow consisting of the suspension flows, the suspension flow flowing through the regulated region having a thickness of 1 to 10 mm in the direction of separation between the electrodes of the electrode pair, and merging the suspension flow with the sheath flow downstream of the regulated region. [2] The electroporation method according to [1], wherein the total thickness of the sheath flow in the direction of separation between the electrodes when merging with the suspension flow is equal to or less than the thickness of the suspension flow in the direction of separation between the electrodes in the regulated region. [3] The electroporation method according to [1], wherein the sheath liquid is recovered downstream of the electrode pair, thereby reducing the dilution rate of the suspension in the mixture of the suspension and sheath liquid after sheath liquid recovery to 2 times or less. [4] The electroporation method according to any one of [1] to [3], wherein the volume fraction of the biologically derived substance in the suspension is 10% or more. [5] The electroporation method according to [4], wherein the volume fraction of the biologically derived substance in the suspension is 40% or more. [6] The electroporation method according to any one of [1] to [5], wherein the suspension is a cell suspension and the sheath fluid is a medium used for culturing the cells in the cell suspension. [7] The electroporation method according to any one of [1] to [6], wherein the conductivity of the suspension or the concentration of the biologically derived substance in the suspension is measured prior to application of the electric field, and the electric field or pulse width applied by the electrode pair is adjusted according to the measurement result. [8] The electroporation method according to any one of [1] to [7], wherein the flow rate of the suspension is 1 mL / min or more. [9] The electroporation method according to any one of [1] to [8], wherein the flow rate of the sheath fluid is 0.1 mL / min or more.

[10] An electroporation method for introducing the bioactive substance into a biologically derived substance by applying an electric field to a suspension containing the biologically derived substance and the bioactive substance using an electrode pair, the method comprising: forming a sheath flow of a sheath liquid other than the suspension that flows together with the suspension and comes into contact with the electrodes that constitute the electrode pair; and further setting the thickness of the suspension flow consisting of the suspension in the direction of separation of the electrodes to 1 to 10 mm in at least a portion between the electrode pair.

[11] A method for producing a useful substance, comprising the electroporation method according to any one of [1] to

[10] .

[12] A flow path device used for electroporation, which introduces a bioactive substance into a suspension containing a biological substance and a biologically derived substance by applying an electric field to the suspension using an electrode pair, the flow path comprising: a main flow path; an electrode pair for applying an electric field to a liquid flowing through the main flow path; a supply port for supplying the suspension to the main flow path; a suspension flow path having a regulated region through which only the suspension flows; and a sheath liquid flow path through which only a liquid other than the suspension flows, the sheath liquid flow path being disposed downstream of the supply port and upstream of the electrode pair so that the sheath liquid flows into the regulated region; the thickness of the regulated region in the direction of separation between the electrodes constituting the electrode pair is 1 to 10 mm.

[13] The flow path device according to

[12] , wherein the angle formed between the main flow path and the sheath liquid flow path is 60° or less.

[14] The flow path device according to

[12] or

[13] , wherein the sheath liquid flow path has a region where the flow path is widened in the width direction downstream.

[15] The flow path device according to any of

[12] to

[14] , wherein the width of the main flow path is 2 mm or more.

[16] The flow channel device according to any one of

[12] to

[15] , which has an outlet downstream of the electrode for discharging the sheath flow from the main channel.

[17] An electroporation apparatus comprising the flow channel device according to any one of

[12] to

[16] and a pair of contacts for applying electricity to the electrodes of the flow channel device.

[18] A flow path device used for electroporation, which introduces a bioactive substance into a suspension containing a biological substance and a biologically derived substance by applying an electric field to the suspension using an electrode pair, the flow path comprising: a main flow path; an electrode pair which applies an electric field to a liquid flowing through the main flow path; a supply port which supplies the suspension to the main flow path; and a sheath flow forming means which forms a sheath flow in contact with the electrodes constituting the electrode pair downstream of the supply port in the main flow path, the sheath flow forming means also serving as a regulating means which regulates the thickness of the suspension flow flowing through the main flow path in the direction of separation between the electrodes constituting the electrode pair, and the spacing of the regulating means in the direction of separation between the electrodes is 1 to 10 mm.

[19] The flow path device according to

[18] , wherein the sheath flow forming means has a sheath liquid flow path which is provided upstream of the electrodes in the main flow path and is parallel to the flow direction of the liquid in the main flow path, in order to supply sheath liquid which forms the sheath flow, and the sheath liquid flow path acts as the regulating means.

[20] The flow channel device according to

[18] or

[19] , wherein the total thickness of the sheath fluid flow channel in the electrode spacing direction is equal to or less than the spacing between the electrodes in the sheath fluid flow channel.

[21] The flow channel device according to

[18] , wherein the sheath flow forming means has a convex portion protruding in the electrode spacing direction within the main flow channel, and a sheath fluid supply port opening downstream of the convex portion for supplying sheath fluid downstream in a direction oblique to the flow direction of the liquid in the main flow channel, the convex portion acting as a regulating means.

[22] The flow channel device according to

[21] , wherein the total thickness of the convex portion in the electrode spacing direction is equal to or less than the spacing between the convex portion in the electrode spacing direction.

[23] The flow channel device according to any of

[18] to

[22] , wherein an outlet for discharging the sheath flow from the main flow channel is provided downstream of the electrodes.

[24] An electroporation apparatus comprising the flow channel device according to any of

[18] to

[23] , and a pair of contacts for applying electricity to the electrodes of the flow channel device.

[0009] According to the present invention, highly efficient electroporation can be performed.

[0010] FIG. 1 is a conceptual diagram illustrating the electroporation method of the present invention. FIG. 2 is a schematic cross-sectional view of an example of a flow channel device of the present invention. FIG. 3 is a schematic top view of the flow channel device shown in FIG. 2. FIG. 4 is a schematic cross-sectional view of an example of a flow channel substrate of the flow channel device shown in FIG. 2. FIG. 5 is a conceptual diagram illustrating the flow channel substrate shown in FIG. 4. FIG. 6 is a conceptual diagram illustrating the operation of the flow channel device shown in FIG. 2. FIG. 7 is a schematic cross-sectional view of another example of a flow channel device of the present invention. FIG. 8 is a conceptual diagram illustrating the operation of the flow channel device shown in FIG. 7. FIG. 9 is a schematic cross-sectional view of another example of a flow channel device of the present invention. FIG. 10 is a conceptual diagram illustrating an example of feedback control in the electroporation method of the present invention. FIG. 11 is a conceptual diagram illustrating another example of feedback control in the electroporation method of the present invention.

[0011] The electroporation method, the method for producing a useful substance, the flow path device, and the electroporation apparatus of the present invention will be described in detail below with reference to preferred embodiments shown in the accompanying drawings.

[0012] The drawings described below are illustrative for explaining the present invention, and the present invention is not limited to the drawings shown below. In the following, the range of values ​​indicated by "to" includes the values ​​written on both sides.

[0013] 1 conceptually illustrates the electroporation method of the present invention. In the present invention, a suspension flow consisting of a suspension m containing a biologically derived substance and a bioactive substance is passed through a flow channel 104 provided between a first substrate 100 and a second substrate 102, while an electric field is applied to the suspension m by an electrode pair consisting of an electrode 106 and an electrode 108. An example of the electric field is a pulsed electric field. Application of this pulsed electric field forms minute pores in the membrane of the biologically derived substance, through which the bioactive substance is introduced into the biologically derived substance by electrophoresis.

[0014] In the present invention, as shown in FIG. 1 , a sheath flow consisting of a sheath fluid s, separate from the suspension, is formed between the suspension flow and the electrodes 106 and 108, along with the suspension flow, to perform flow electroporation. By using such a sheath flow, even when the concentration of biological substances in the suspension m is high, the flow of biological substances can be prevented from stagnating near the wall of the flow channel 104, enabling highly efficient flow electroporation. According to the inventors' studies, when the concentration of biological substances in the suspension is high but there is no sheath flow, the flow of biological substances stagnates very close to the wall of the flow channel. In this stagnation layer, the biological substances are in close contact with the flowing region, resulting in high electrical resistance. This stagnation layer consumes a large amount of the electric field applied by the electrode pair. As a result, the electric field applied to the flowing region where an electric field should be applied is reduced, preventing sufficient electric field application and reducing the efficiency of electroporation. Furthermore, in the absence of a sheath flow, the biological substances stagnate as a stagnation layer near the electrodes, resulting in repeated application of an electric field to the biological substances. As a result, the biological material burns near the electrode, and the burnt material adheres to the surface of the electrode. When such foreign matter adheres to the electrode surface, it blocks the electric field applied by the electrode, reducing the efficiency of electroporation.

[0015] Furthermore, the inventors have found, through their investigations, that in flow electroporation using such a sheath flow, highly efficient flow electroporation can be achieved by increasing the thickness dm of the suspension flow in the direction of separation between the electrodes constituting the electrode pair. Specifically, highly efficient flow electroporation can be achieved by setting the thickness dm of the suspension flow in the direction of separation between the electrodes constituting the electrode pair to 1 to 10 mm (1.0 to 10.0 mm, the same applies below). That is, in the illustrated example, highly efficient flow electroporation can be achieved by setting the thickness dm of the suspension flow in the direction of separation between electrode 106 and electrode 108 to 1 to 10 mm. Note that, in the present invention, it is sufficient that the thickness dm of the suspension flow is 1 to 10 mm in a portion between the electrode pair, and that the thickness dm of the suspension flow is 1 to 10 mm between the electrode pair and at the upstream ends of the electrodes. Furthermore, in the present invention, based on the area of ​​one electrode exposed to the main channel, the thickness dm of the suspension flow is preferably the above-mentioned thickness over a region of 30% or more, more preferably over a region of 50% or more, and even more preferably over a region of 70% or more. In this case, since the two electrodes are the same size, either electrode in the electrode pair can be used as the reference. The electroporation method of the present invention enables more efficient flow electroporation by performing flow electroporation with a thicker suspension flow thickness dm. Specifically, the present invention enables more efficient flow electroporation with higher throughput, introduction efficiency, and bioactive substance utilization efficiency. Throughput refers to the number of cells processed per unit time. Furthermore, by performing flow electroporation with a thicker suspension flow thickness dm, the electroporation method of the present invention enables efficient large-scale flow electroporation of highly concentrated suspensions while increasing the processing volume, thereby achieving high productivity.

[0016] In the electroporation method of the present invention, there is no limitation on the flow rate of the suspension (suspension flow), but it is preferably 1 mL / min or more, more preferably 2 mL / min or more, and even more preferably 3 mL / min or more. Note that "mL" stands for milliliter. A suspension flow rate of 1 mL / min or more is preferable because it allows for a larger electroporation throughput. Note that the suspension flow rate is preferably 1000 mL / min or less.

[0017] Furthermore, in the electroporation method of the present invention, there is no limitation on the flow rate of the sheath liquid (sheath liquid flow), but the flow rate of the sheath liquid is preferably 0.1 mL / min or more. Note that this sheath flow rate is the flow rate of the sheath flow on one electrode side. Therefore, the total flow rate of the sheath flow is preferably 0.2 mL / min or more. One electrode side refers to one side of the electrode pair. Setting the flow rate of the sheath liquid to 0.1 mL / min or more is preferable in that it can effectively prevent the suspension from contacting the electrodes. Note that the flow rate of the sheath flow is preferably equal to or less than the flow rate of the suspension. Setting the flow rate of the sheath flow to be equal to or less than the flow rate of the suspension is preferable in that it can reduce the amount of sheath liquid and prevent dilution of the suspension.

[0018] In the following description, the thickness of the suspension flow in the direction of separation between the electrodes that make up the electrode pair will also be simply referred to as the "suspension flow thickness." The same applies to the thickness of the sheath flow. In the following description, a suspension with a high concentration of biologically derived substances will also be simply referred to as a "high-concentration suspension" or a "high-concentration suspension."

[0019] As described above, flow electroporation using a sheath flow enables efficient treatment even when the concentration of the suspension m is high. In the following description, unless otherwise specified, "flow electroporation" refers to flow electroporation using a sheath flow. Conventional flow electroporation involves reducing the thickness of the suspension flow as thin as possible. For example, Patent Document 2 exemplifies a preferred embodiment in which flow electroporation is performed with the suspension flow thickness, i.e., the spacing between sheath flows, set to 50 to 100 μm. Patent Document 2 states that this allows for a monolayer flow in which the biological material (cells) passing between the electrodes does not overlap in the direction of electrode separation. Conventional flow electroporation is thought to enable highly efficient treatment by reducing the thickness of the suspension flow, thereby uniforming the flow of the biological material between the electrodes and uniforming the electric field applied to the biological material.

[0020] However, as a result of investigation, the present inventors have found that, conversely, more efficient flow electroporation can be achieved by significantly increasing the thickness of the suspension flow.

[0021] In flow electroporation, the diffusion of biologically derived substances and bioactive substances contained in the suspension forms a diffusion region between the sheath flow and the suspension flow, where the biologically derived substances and bioactive substances are diffused. When the thickness of the suspension flow is set to 50 to 100 μm, as in Patent Document 2, the diffusion region is thicker relative to the thickness of the suspension flow. As a result, the diffusion of biologically derived substances and bioactive substances has a significant effect on flow electroporation, resulting in reduced introduction efficiency and bioactive substance utilization efficiency. On the other hand, when the thickness of the suspension flow is set to approximately 500 μm, the thickness of the diffusion region relative to the thickness of the suspension flow is smaller, reducing the effect of diffusion, but reducing the uniformity of the electric field in the suspension flow. As a result, the introduction efficiency and bioactive substance utilization efficiency are lower than when the thickness of the suspension flow is set to 50 to 100 μm, as in Patent Document 2.

[0022] In contrast, by significantly increasing the thickness dm of the suspension flow to 1 mm or more, the effects of diffusion of biologically derived substances and the like can be suppressed. In other words, because the diffusion region is formed by diffusion, the thickness of the diffusion region is not affected by the thickness dm of the suspension flow. Therefore, by increasing the thickness dm of the suspension flow to 1 mm or more, the thickness of the uniform suspension m, which is not affected by diffusion relative to the thickness of the diffusion region, can be sufficiently increased, making it possible to process a large amount of suspension m. In addition, by increasing the thickness dm of the suspension flow to 1 mm or more, good electric field uniformity can be achieved. In other words, in electroporation, the strength of the electric field applied to the biologically derived substances varies depending on the number of biologically derived substances present in the inter-electrode direction. For example, as in Patent Document 2, when the thickness of the suspension flow is sufficiently narrow, such as approximately 50 μm, the probability of biologically derived substances overlapping in the inter-electrode direction is low, and the electric field applied to each biologically derived substance is approximately constant, resulting in good electric field uniformity. In contrast, when the thickness of the suspension flow is approximately 500 μm, the number of biologically derived substances present in the direction between the electrodes varies depending on the position in the flow direction, resulting in differences in the electric field applied to each biologically derived substance and poor electric field uniformity. In contrast, by making the thickness dm of the suspension flow sufficiently thick, at 1 mm or more, the number of biologically derived substances present in the direction between the electrodes at each position in the flow direction can be made uniform, resulting in a nearly constant electric field applied to each biologically derived substance and good electric field uniformity. As a result, the electroporation method of the present invention enables more efficient flow electroporation than conventional flow electroporation using a thinner suspension flow.

[0023] In the electroporation method of the present invention, as in the flow channel device of the present invention described below, a restricted region where only the suspension flows is provided upstream of the position where the sheath flow and the suspension flow join in the flow channel 104, and the thickness of the restricted region, i.e., the thickness of the suspension flow flowing in the restricted region, is set to 1 to 10 mm. In other words, in the electroporation method of the present invention, a suspension flow having a thickness of 1 to 10 mm in the electrode separation direction is formed in the restricted region, and the sheath flow is joined to both sides of the suspension flow in the electrode separation direction downstream of the restricted region. For example, in the flow channel device shown in FIG. 2 described below, the suspension flow channel 30a serves as the restricted region, and the distance d in this region in the electrode separation direction is set to 1 to 10 mm. It is sufficient that only a portion of the suspension flow channel 30a serves as the restricted region, and preferably only a portion downstream of the restricted region. Furthermore, in the flow channel device 50 shown in FIG. 7 described below, the flow channel between the convex portions 64a and 64b in the main flow channel 74 serves as the restricted region (hereinafter, this region will also be referred to as the suspension flow channel), and the distance d in this region in the electrode separation direction is set to 1 to 10 mm. The suspension flow path in FIG. 7 may have only a portion thereof as a regulated region, and only a portion downstream of the regulated region with a distance d of 1 to 10 mm. This configuration of the electroporation method of the present invention enables highly efficient flow electroporation by suitably setting the thickness dm of the suspension flow in flow electroporation to 1 to 10 mm (slightly less than 1 mm to slightly less than 10 mm). Preferably, the thickness dm of the suspension flow between the electrodes, i.e., between electrode 106 and electrode 108, is set to 1 to 10 mm, enabling highly efficient flow electroporation. Note that if the thickness dm of the suspension flow is sufficiently large, at 1 mm or more, the diffusion region between the sheath liquid and the suspension occurring between the electrodes will be very thin, although this depends on the distance from the supply port. In the following description, the thickness of the regulated region in the direction of separation between the electrodes will also be simply referred to as the "thickness of the regulated region."

[0024] In the electroporation method of the present invention, if the thickness of the restriction region is less than 1 mm, disadvantages such as the inability to perform efficient processing due to the significant influence of diffusion of biological substances and the like will occur. Conversely, if the thickness of the restriction region exceeds 10 mm, disadvantages such as the required applied voltage becoming higher, the power supply becoming larger, heat generation becoming more likely (cooling efficiency decreasing), and the likelihood of discharge due to electric field concentration will occur will occur. In the present invention, the thickness of the restriction region is preferably 1 to 8 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. The lower limit of the thickness of the restriction region is more preferably 2 mm, and even more preferably 3 mm.

[0025] In the present invention, when the suspension flow and the sheath flow are joined, it is preferable that the difference in flow velocity between them is small. Specifically, when the suspension flow and the sheath flow are joined, the flow velocity of the sheath 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%, i.e., constant velocity. Furthermore, when the suspension flow and the sheath flow are joined, it is preferable that the angle formed by the suspension flow and the sheath flow is small. Specifically, when the suspension flow and the sheath flow are joined, the angle formed by the suspension flow and the sheath flow is preferably 90° or less, more preferably 60° or less, even more preferably 45° or less, even more preferably 30° or less, and most preferably 0°, i.e., parallel.

[0026] In the electroporation method of the present invention, there is no limitation on the thickness ds of the sheath flow of the sheath fluid s. However, highly efficient flow electroporation can be achieved by setting the thickness ds (on one side) of the sheath flow in the direction of separation between the electrodes constituting the electrode pair to 0.1 mm or more. The thickness ds is preferably 0.2 mm or more, more preferably 0.3 mm or more, and particularly preferably 0.5 mm or more. The upper limit is as follows: Here, the total thickness of the sheath flow at the time of merging with the suspension flow is preferably equal to or less than the thickness of the suspension flow in the regulated region. In other words, the total thickness of the sheath flow at the time of merging with the suspension flow is preferably equal to or less than the thickness of the suspension flow in the regulated region. In other words, the dilution rate of the suspension m downstream of the electrode pair is preferably equal to or less than 2 times. In the case of the flow path device 10 shown in FIG. 2, the total thickness of the sheath fluid flow paths 26a and 26b is preferably equal to or less than the thickness of the suspension flow path 30a. In the case of the flow channel device 50 shown in FIG. 7, the total height of the convex portions 64a and 64b is preferably equal to or less than the gap d between the convex portions 64a and 64b.

[0027] In electroporation, when an electric field is applied, pores open in the membrane (or shell) of the biologically-derived material, allowing the bioactive substance to enter the biologically-derived material through electrophoresis. The pores formed in the membrane of the biologically-derived material gradually close over time. However, if the liquid components (osmotic pressure, ion concentration, etc.) outside the biologically-derived material suddenly change before the pores close, for example, due to operations such as diluting the suspension, the movement of ions and medium through the pores can cause a sudden change in the component ratio within the biologically-derived material, as well as damage such as deformation and expansion. As a result, for example, in the case of substance introduction into cells, the viability of the biologically-derived material can decrease, potentially reducing the introduction efficiency and utilization efficiency of the bioactive substance during electroporation. If the thickness of the sheath flow ds is greater than the thickness of the suspension flow dm and the dilution rate of the suspension m is high, for example, in the case of substance introduction into cells, the viability of the biologically-derived material can decrease (become damaged), resulting in low EP efficiency. As a result of our research, we have confirmed that, for example, when diluting a cell suspension with a medium sheath flow, a significant decrease in cell viability is often observed at a dilution rate of 2x or more. In contrast to this, by making the total thickness of the sheath flow at the time of merging with the suspension flow equal to or less than the thickness of the suspension flow liquid in the regulated region, i.e., by making the dilution rate of the suspension m equal to or less than three times, more preferably equal to or less than two times, it becomes possible to suppress the inconvenience caused by a large amount of sheath liquid s being mixed into the biological substance, such as a decrease in the viability of the biological substance in the case of substance introduction into cells, and to perform efficient processing.

[0028] In order to more suitably obtain this effect, the total thickness of the sheath flow at the time of merging with the suspension flow is more preferably 0.8 times or less, and even more preferably 0.5 times or less, the thickness of the suspension flow in the regulated region. That is, in the present invention, the dilution ratio of the suspension m downstream of the electrode pair is more preferably 1.8 times or less, and even more preferably 1.5 times or less. The thickness (one side) of the sheath flow at the time of merging with the suspension flow is preferably 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.5 mm or more.

[0029] Alternatively, as shown in FIG. 9 (described later), an outlet for discharging sheath fluid s (sheath flow) may be provided downstream of the electrode pair, and the sheath fluid s may be recovered from this outlet, thereby achieving a dilution rate of the suspension m downstream of the electrode pair of no more than 2 times. This configuration can also be used in the embodiment shown in FIG. 1 (described above). According to this method, even when the thickness of the sheath flow is greater than the thickness of the suspension gas flow, the dilution rate of the suspension treated by the electroporation method of the present invention can be maintained at no more than 2 times. Note that, in the present invention, the method of setting the total thickness of the sheath flow (ds × 2) to no more than the thickness dm of the suspension flow and the method of recovering the sheath fluid s downstream of the electrode pair may be used in combination.

[0030] In the electroporation method of the present invention, there is no limit to the concentration of the biologically derived substance in the suspension m. Here, in the present invention, it is preferable that the volume fraction of the biologically derived substance in the suspension m be 10% or more. In electroporation, by increasing the concentration of the biologically derived substance in the suspension, the amount of biologically derived substance processed can be increased compared to when the same amount of a low-concentration suspension is introduced. For example, doubling the concentration allows for twice the processing, thereby improving the electroporation throughput and the utilization efficiency of the biologically derived substance. In addition, in electroporation, as described above, the bioactive substance is incorporated into the biologically derived substance by electrophoresis. Therefore, even if the concentration of the biologically derived substance is increased, there is no need to increase the amount of the bioactive substance accordingly, and the utilization efficiency of the bioactive substance can also be improved.

[0031] In the present invention, by setting the volume fraction of the biologically derived material in suspension m to 10% or more, the above-mentioned effects can be preferably achieved and efficient processing can be performed. The volume fraction of the biologically derived material in suspension m is more preferably 20% or more, even more preferably 30% or more, particularly preferably 40% or more, and of these, 50% or more is preferred. On the other hand, in the present invention, the volume fraction of the biologically derived material in suspension m is preferably 70% or less. If the suspension concentration is too high, the viscosity of the cell suspension will increase rapidly, potentially clogging the flow path. However, by setting the volume fraction of the biologically derived material in suspension m to 70% or less, clogging of the flow path caused by an excessively high suspension concentration can be preferably prevented. The volume fraction of the biologically derived material in suspension m is more preferably 65% ​​or less, even more preferably 60% or less.

[0032] The electroporation method of the present invention is basically the same as known flow electroporation, except that the thickness of the suspension flow is set to 1 to 10 mm. Therefore, there are no limitations on the suspension and sheath liquid, or on the various processing conditions.

[0033] <Suspension> In the present invention, the suspension is a known suspension used in electroporation, which contains a biologically derived substance, a bioactive substance, and a medium.

[0034] (Biologically derived products) Biologically derived products are not particularly limited, but specific examples include cells, organelles, intracellular granules, vesicles, and bacteria. Among these, cells are preferred, animal cells are more preferred, mammalian cells are even more preferred, and human or Chinese hamster-derived cells are most preferred, due to the superior effects of the present invention. Specific examples of cells include human T cells, HEK (Human Embryonic Kidney) 293 cells, A549, SF9, EB66, Daudi, Hela, Vero, MDCK, BHK (Baby Hamster Kidney), CHO (Chinese Hamster Ovary), NS0, SP2 / 0, and hybridomas. In terms of pharmaceutical production, gene transfer into HEK293 cells and CHO cells is most commonly used.

[0035] (Bioactive Substances) Bioactive substances are substances such as DNA, RNA, and proteins that, when introduced into a biologically derived substance, exert some effect on the biologically derived substance. Examples of bioactive substances include plasmid DNA, linear DNA, mRNA, and proteins. Among these, plasmid DNA, mRNA, and linear DNA are preferred, with plasmid DNA being particularly preferred. The concentration of the bioactive substance in the suspension is preferably 1 to 1000 μg / mL, more preferably 2.5 to 500 μg / mL, and even more preferably 10 to 200 μg / mL. The concentration of the bioactive substance in the suspension relative to the medium in the suspension is preferably 10 to 500 μg / mL. The concentration of the bioactive substance per biologically derived substance in the suspension is preferably 20 pg / unit or less, more preferably 5 pg / unit or less, even more preferably 1 pg / unit or less, and particularly preferably 0.5 pg / unit or less. The lower the concentration of the bioactive substance per biologically derived substance in the suspension, the less the amount of the bioactive substance used per biologically derived substance can be, and therefore the lower the cost can be.

[0036] <Sheath Liquid> The sheath liquid used in the sheath flow can be any liquid other than a suspension. Among these, a preferred example of the sheath liquid is a culture medium used for a suspension. This culture medium is preferably a culture medium used for growing biological substances such as cells.

[0037] <Voltage> The voltage is set so that the electric field applied to the suspension between the electrodes has the desired field strength, taking into account the thickness and conductivity of the suspension flow, the thickness and conductivity of the sheath flow, etc. Note that the electrical resistance of the electrodes is preferably sufficiently lower than the resistance of the suspension and sheath fluid, as this may result in a decrease in the transfer efficiency and utilization efficiency of the bioactive substance during electroporation. The optimal value of the electric field applied to the suspension varies depending on the type and size of the biologically derived material, but is typically approximately 100 to 3500 V / cm, preferably approximately 500 to 3000 V / cm, more preferably approximately 750 to 2500 V / cm, and even more preferably approximately 1000 to 2000 V / cm. The voltage is preferably a pulse voltage. Alternatively, a bipolar pulse may be used to uniformly induce electrode reactions. Examples of electrode reactions include gas generation due to electrolysis and electrode deterioration. A bipolar pulse is a pulse voltage in which positive and negative voltages are alternately applied.

[0038] <Pulse Width> The optimum value of the pulse width varies depending on the type of biological substance, etc., but is usually 0.1 to 100 ms (milliseconds), preferably about 1 to 10 ms.

[0039] <Pulse Period (Pulse Interval)> The pulse period is preferably synchronized with the time it takes for the biologically derived substance to pass through the electrodes (electrode length L). For example, a pulse voltage is applied 1 to 5 times, preferably once, on average while the biologically derived substance passes through the electrodes (electrode length L). The time it takes for the biologically derived substance to pass through the electrodes is determined by the flow rate and the cross-sectional area of ​​the channel (channel width W of the main channel × distance D between the electrodes). Specifically, the pulse period is preferably an integer multiple of the time it takes for the biologically derived substance to pass through the electrodes.

[0040] The method for producing a useful substance of the present invention includes the electroporation method of the present invention described above. In the method for producing a useful substance according to the present invention, preferably, the biologically derived substance is a cell, and the bioactive substance is a plasmid DNA. As the plasmid DNA, a plasmid DNA encoding the useful substance is used. The plasmid DNA is introduced into cells by the electroporation method of the present invention, and the cells into which the plasmid DNA has been introduced are cultured, thereby causing the cells to produce the useful substance.

[0041] In the present invention, the type of useful substance is not particularly limited, but is preferably a protein or a virus. Examples of useful substances include recombinant polypeptide chains, recombinant secreted polypeptide chains, antigen-binding proteins, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, bispecific antibodies, Fc fusion proteins, fragmented immune immunoglobulins, single-chain antibodies (scFv), and non-enveloped viruses. More specifically, non-enveloped viruses include adeno-associated viruses, adenoviruses, lentiviruses, baculoviruses, and retroviruses. Non-enveloped viruses are known in the art and are described in International Publication No. WO 2015 / 005430, which is incorporated herein by reference.

[0042] Useful materials are preferably human antibodies, humanized antibodies, chimeric antibodies, murine antibodies, and adeno-associated viruses.

[0043] The useful substance can be recovered and purified. The useful substance can be recovered by simply recovering the culture medium, or by using a filter or centrifuge to recover the liquid from which at least a portion of the cells have been removed. Known methods can be used without particular limitations. To improve the purity of the useful substance, change the solvent, or change the form, for example, by powdering, the culture medium or the liquid can be subjected to further processing. When the useful substance is a virus, the virus particles produced within the cells are present in the culture medium or within the cells (inside the nuclei). Extraction from within the cells (destruction and dissolution of cell membranes and nucleic acids) can be performed by freeze-drying or by adding a surfactant (e.g., Triton X) and stirring. Separation of the virus from cell debris can be performed by centrifugation or depth filtration.

[0044] If necessary, the useful substance can be purified by a purification process. The obtained useful substance can be purified to a high purity. Separation and purification of the useful substance can be performed using separation and purification methods commonly used for proteins or viruses. For example, useful substances can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and electrofocusing, but are not limited to these. When the useful substance is a virus, the virus may be purified by TFF (concentration / buffer exchange), affinity chromatography, and AEX (anion exchange chromatography) treatment, etc. This allows for the removal of cell debris, cell-derived nucleic acids, and cell-derived proteins. Alternatively, a commercially available virus purification kit (such as an AAV purification kit) may be used. For example, the AAVpro (registered trademark) Purification Kit Maxi / Midi manufactured by Takara Bio Inc. can be used.

[0045] The concentration of the useful substance obtained above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA), etc. Furthermore, when the useful substance is an antibody, the antibody titer can also be measured using a commercially available analytical instrument such as Roche's Cedex Bio. When the useful substance is a virus, the virus titer can be measured by a conventional method known to those skilled in the art. For example, after culturing, the cell culture medium is recovered, the cells are disrupted by freezing and thawing, and the supernatant is recovered by centrifugation. MgCl2 and Benzonase are added to the recovered supernatant and a reaction is carried out to digest gDNA (genomic DNA) and remaining plasmids. The sample containing the target virus obtained above can be used as a ddPCR (Droplet Digital PCR) sample, and the number of genome copies in the virus can be measured by ddPCR to determine the virus titer (vg / mL).

[0046] A cross section of an example of a flow channel device of the present invention for carrying out the electroporation method of the present invention is conceptually shown in Figure 2. The flow channel device 10 shown in Figure 2 is basically configured to have a first substrate 12, a second substrate 14, and a flow channel substrate 16 provided between the first substrate 12 and the second substrate 14. An electrode 18 is incorporated into the first substrate 12, and an electrode 20 is incorporated into the second substrate, forming an electrode pair.

[0047] The electrodes 18 and 20 are made of, for example, a metal material. While the metal material is not particularly limited, from the viewpoint of thermal conductivity, suitable examples include molybdenum (Mo), niobium (Nb), tungsten (W), and titanium (Ti), with Mo and Nb being more preferred. Various materials can be used for the substrates as long as they are sufficiently resistant to the suspension and sheath liquid and can ensure the necessary rigidity. Examples of materials for forming the substrates include resin materials such as polystyrene, polyether ether ketone (PEEK), fluorine-based resins such as polytetrafluoroethylene, polydimethylsiloxane (PDMS), polycarbonate, and polypropylene.

[0048] FIG. 3 shows a top view of the flow channel device 10. The cross-sectional view of the flow channel device 10 shown in FIG. 2 is a cross-section taken along line II-II in FIG. 3. The top view is a view of the flow channel device 10 viewed in the stacking direction of the first substrate 12, the second substrate 14, and the flow channel substrate 16. As shown in FIGS. 2 and 3, the flow channel device 10 has a suspension inlet channel 24 for introducing the suspension, a main channel 30, an outlet 32 ​​for discharging the suspension treated by flow electroporation, and sheath fluid inlet channels 26a and 26b for introducing the sheath fluid. The main channel 30 is formed between the first substrate 12 and the second substrate 14. In the flow channel device 10, the liquid flows from left to right in the drawing. The width of the main channel is preferably 2 mm or more. In the present invention, the width of the main channel refers to the length (maximum value) of the main channel in the direction perpendicular to the spacing direction of the electrodes. This also applies to the embodiment shown in FIG. 7, which will be described later.

[0049] The sheath fluid inlet channel 26a is connected to the sheath fluid flow path 28a shown at the bottom in the figure. The sheath fluid flow path 28a is a flow path parallel to the flow direction of the liquid in the main flow path 30 and opens upstream of, preferably immediately upstream of, the electrode 20. The sheath fluid inlet channel 26b is connected to the sheath fluid flow path 28b shown at the top in the figure. The sheath fluid flow path 28b is a flow path parallel to the flow direction of the liquid in the main flow path 30 and opens upstream of, the electrode 20, preferably at the same position as the upstream end of the electrode 20 in the flow direction. Hereinafter, this position will also be referred to as "immediately upstream." The suspension inlet channel 24 is connected to the suspension flow path 30a between the sheath fluid flow paths 28a and 28b. That is, the suspension flow path 30a is formed by the sheath fluid flow paths 28a and 28b and opens upstream of, preferably immediately upstream of, the electrode pair. The open end of this suspension flow path 30a serves as a supply port in the flow path device of the present invention, which supplies the suspension to the main flow path 30. Therefore, the main flow path 30 is formed by three flow paths, namely, the sheath liquid flow path 28a, the suspension flow path 30a, and the sheath liquid flow path 28b, on the upstream side of the electrode pair (electrodes 18 and 20).

[0050] In such a flow path device 10, the flow path substrate 16 may be formed by various known methods. As an example, as shown in FIGS. 4 and 5 , the flow path substrate 16 may be formed from five flow path plates, namely, a first flow path plate 16a to a fifth flow path plate 16e. The first flow path plate 16a, which is the top layer in the figure, is a plate having an opening corresponding to the sheath fluid inlet channel 26a, an opening corresponding to the suspension inlet channel 24, and openings corresponding to the sheath fluid flow path 28b and the main flow path 30 at the top of the figure. The second flow path plate 16b, which is the second from the top in the figure, is a plate having an opening corresponding to the sheath fluid inlet channel 26a, an opening corresponding to the suspension inlet channel 24, and an opening corresponding to the main flow path 30. The third flow path plate 16c, which is the third from the top in the figure, is a plate having an opening corresponding to the sheath fluid inlet channel 26a and an opening corresponding to the main flow path 30. The fourth flow path plate 16d, which is the fourth from the top in the figure, is a plate having an opening corresponding to the sheath fluid inlet channel 26a and an opening corresponding to the main flow path 30. The fifth flow path plate 16e, which is the fifth from the top in the figure, is a plate member having an opening corresponding to the sheath liquid inlet path 26a at the bottom in the figure and an opening corresponding to the main flow path 30.

[0051] In the flow path device 10, the suspension m flows into the suspension inlet channel 24, flows through the suspension channel 30a in the main channel 30, and reaches the outlet 32 ​​from the main channel 30. On the other hand, the sheath fluid s flows into the sheath fluid channels 28a and 28b. The sheath fluid s that flows into the sheath fluid channel 28a flows into the sheath fluid channel 28a at the bottom in the figure in the main channel 30, and reaches the outlet 32 ​​from the main channel 30. On the other hand, the sheath fluid s that flows into the sheath fluid channel 28b flows into the sheath fluid channel 28b at the top in the figure in the main channel 30, and reaches the outlet 32 ​​from the main channel 30.

[0052] As described above, sheath fluid flow path 28a is a flow path parallel to the direction of liquid flow in main flow path 30 and opens upstream of electrode 20. Sheath fluid flow path 28b is a flow path parallel to the direction of liquid flow in main flow path 30 and opens upstream of electrode 20. Furthermore, suspension flow path 30a is formed by sheath fluid flow paths 28a and 28b and opens upstream of the electrode pair. Therefore, upstream of the electrode pair, as conceptually shown in Figure 6, sheath flows made of sheath fluid s are formed on both sides of the suspension flow made of suspension m in the direction of separation between the electrodes, i.e., on the upper and lower sides in the figure, and a three-layer liquid flow of sheath flow / suspension flow / sheath flow is formed.

[0053] Therefore, by applying a pulsed electric field using electrodes 18 and 20 while a sheath flow / suspension flow / sheath flow is formed, flow electroporation using the sheath flow can be performed as described above.

[0054] As described above, the suspension flow path 30a is formed by the sheath fluid flow paths 28a and 28b and opens upstream, preferably immediately upstream, of the electrode pair. Therefore, the thickness of the suspension flow in the suspension flow path 30a in the direction separating the electrodes is determined by the distance d between the sheath fluid flow paths 28a and 28b. In other words, the sheath fluid flow paths 28a and 28b act as a means for restricting the thickness of the suspension flow through the suspension flow path 30a. Furthermore, the sheath fluid flow paths 28a and 28b are flow paths parallel to the flow direction of the liquid in the main flow path 30.

[0055] That is, in the illustrated example of the flow path device 10, the suspension flow path 30a serves as a regulating region that regulates the thickness of the suspension flow before it merges with the sheath flow. Therefore, in the flow path device 10, the distance d between the sheath fluid flow paths 28a and 28b is set to 1 to 10 mm, thereby setting the thickness of the suspension flow in the electrode separation direction through the suspension flow path 30a to 1 to 10 mm. As described above, this allows the flow path device 10 to control the thickness of the suspension flow to 1 to 10 mm in the three-layer flow of sheath flow / suspension flow / sheath flow in flow electroporation. As described above, the thickness of the suspension flow in this regulating region, i.e., the distance d, is preferably 1 to 8 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. Furthermore, by making the total thickness of the sheath fluid flow path 28a and the sheath fluid flow path 28b less than the distance d between the sheath fluid flow path 28a and the sheath fluid flow path 28b, i.e., the thickness of the suspension flow, the total thickness of the sheath flow can be made less than the thickness of the suspension flow, and the dilution rate of the suspension after flow electroporation can be made less than two times.

[0056] Figure 7 conceptually shows a cross section of another example of a flow channel device of the present invention for carrying out the electroporation method of the present invention. The flow channel device 50 shown in Figure 7 is basically configured to have a first substrate 52, a second substrate 54, a first introduction section 56, a second introduction section 58, and a flow channel substrate 64 provided between the first substrate 52 and the second substrate 54. An electrode 60 is incorporated into the first substrate 52, and an electrode 62 is incorporated into the second substrate, forming an electrode pair.

[0057] As shown in FIG. 7, the flow path device 50 has a suspension inlet channel 70 for introducing the suspension, a main flow path 74, an outlet 72 for discharging the processed suspension that has undergone flow electroporation, and sheath fluid inlet channels 76a and 76b for introducing the sheath fluid.

[0058] The main channel 74 is formed in a substantially straight line between the first substrate 52 and the second substrate 54. In the channel device 50, too, liquid flows from left to right in the drawing.

[0059] The suspension inlet channel 70 is formed by penetrating the first introduction part 56 and the first substrate 52 in the up-down direction in the figure, and opens at the upstream-most part of the main channel 74. In the main channel 74 (channel substrate 64), convex parts 64a and 64b are provided from the upstream-most part toward downstream, protruding in the direction in which the electrodes in the electrode pair are spaced apart, i.e., in the direction in which electrodes 60 and 62 are spaced apart.

[0060] The sheath liquid inlet channel 76a opens into the main channel 74 via a substantially L-shaped through-hole 56a in the first introduction section 56 and a through-hole 52a in the first substrate 52. In the example shown in FIG. 7 , the through-hole 56a and the through-hole 52a constitute the sheath liquid flow channel of the present invention. Here, the through-hole 52a is provided so as to be inclined downstream with respect to the flow direction of the liquid in the main channel 74, and opens immediately downstream of the convex portion 64a of the main channel 74. That is, the sheath liquid flow channel is provided so as to be inclined downstream with respect to the flow direction of the liquid in the main channel 74, and opens so that the sheath liquid flows downstream of the suspension supply port and upstream of the electrode pair. In the example shown in FIG. 7 , the region upstream of the main channel 74 through which only the suspension flows constitutes the suspension flow channel of the present invention, and the region sandwiched between the convex portions 64a and 64b in the downstream portion of the suspension flow channel constitutes the restriction region of the present invention. The through-hole 56a (part of the sheath fluid flow path) may widen in the width direction, for example, downstream from the bent position of the approximately L-shaped flow path, so that the sheath flow is uniform in the width direction. This also applies to the through-hole 58a described below. That is, the sheath fluid flow path preferably has a region where the flow path widens in the width direction toward downstream. As a result, even if the main flow path is wide, the flow velocity distribution downstream is uniform, allowing for uniform electroporation of the suspension. In this case, it is also preferable that the suspension flow path has a region where the flow path widens in the width direction toward downstream. This ensures a uniform flow velocity distribution in the suspension flow path as well. The through-hole 56a may be provided across the first introduction section 56 and the first substrate 52. On the other hand, the sheath fluid inlet path 76b opens into the main flow path 74 via a approximately L-shaped through-hole 58a provided in the second introduction section 58 and a through-hole 54a provided in the second substrate 54. Here, the through-hole 54a is provided so as to be inclined downstream with respect to the flow direction of the liquid in the main flow channel 74, and opens immediately downstream of the convex portion 64b of the main flow channel 74. Like the through-hole 56a and the through-hole 52a, the through-hole 58a and the through-hole 54a also constitute a sheath liquid flow path in the present invention. Therefore, the above description of the through-hole 56a and the through-hole 52a also applies to the through-hole 58a and the through-hole 54a.The through-hole 58a may be provided across the second introduction section 58 and the second substrate 54. Therefore, the downstream ends of the convex portions 64a and 64b serve as supply ports in the flow channel device of the present invention that supply the suspension to the main channel 74. The opening of the through-hole 52a to the main channel 74 and the opening of the through-hole 54a to the main channel 74 serve as supply ports for the sheath fluid to the main channel 74 in the flow channel device 50 of the present invention. It is preferable that the widths of the supply ports for the suspension and the supply ports for the sheath fluid are approximately the same.

[0061] In the flow channel device 50, the flow channel substrate 64 may be formed by any of various known methods. Similarly to the above-described flow channel substrate 16, the flow channel substrate 64 may be formed using a plurality of flow channel plates. As an example, the flow channel substrate 64 may be formed using three flow channel plates: one having a portion (non-opening) corresponding to the convex portion 64a and an opening corresponding to the main flow channel 74, another having an opening corresponding to the entire main flow channel 74, and another having a portion (non-opening) corresponding to the convex portion 64b and an opening corresponding to the main flow channel 74.

[0062] In the flow path device 50, the suspension m flows into the suspension inlet channel 70, flows from the most upstream portion into the main flow path 74, and then flows from the main flow path 74 to the outlet 72. On the other hand, the sheath fluid s flows into the sheath fluid inlet channel 76a and the sheath fluid inlet channel 76b. The sheath fluid s that flows into the sheath fluid inlet channel 76a flows into the main flow path 74 via the through-hole 56a in the first introduction section 56 and the through-hole 52a in the first substrate 52, and then flows from the main flow path 74 to the outlet 72. The sheath fluid s that flows into the sheath fluid inlet channel 76b flows into the main flow path 74 via the through-hole 58a in the second introduction section 58 and the through-hole 54a in the second substrate 54, and then flows from the main flow path 74 to the outlet 72.

[0063] As described above, the main flow channel 74 (flow channel substrate 64) has protrusions 64a and 64b extending from the most upstream portion toward downstream, protruding in the direction of separation between the electrodes in the electrode pair, i.e., the direction of separation between electrodes 60 and 62. In other words, in the region having protrusions 64a and 64b, the thickness of the suspension m is the distance between the protrusions 64a and 64b, i.e., thickness d. Furthermore, as described above, the through-hole 52a is provided so as to incline toward the downstream direction with respect to the flow direction of the liquid in the main flow channel 74, and opens immediately downstream of the protrusion 64a of the main flow channel 74. As described above, the region sandwiched between the protrusions is the restriction region in the present invention. Meanwhile, the through-hole 54a is provided so as to incline toward the downstream direction with respect to the flow direction of the liquid in the main flow channel 74, and opens immediately downstream of the protrusion 64b of the main flow channel 74. 7, the sheath liquid flow path is composed of through-hole 56a (58a) and through-hole 52a (54a), but only through-hole 52a (54a) is required as the sheath flow path, and the flow path configuration is designed appropriately depending on the device configuration. Therefore, upstream of the electrode pair, as conceptually shown in Figure 8, sheath flows composed of sheath liquid s are formed on both sides of the suspension flow composed of suspension m in the direction in which the electrodes are separated, i.e., on the upper and lower sides in the figure, and a three-layer liquid flow of sheath flow / suspension flow / sheath flow is formed, and a three-layer laminated flow flows between the electrode pair.

[0064] Therefore, in a state where a sheath flow / suspension flow / sheath flow is formed, a flow electroporation process using the sheath flow can be performed as described above by applying a pulsed electric field using electrodes 60 and 62. In this case, the thickness of the suspension flow becomes approximately equal to the thickness of the regulated region, and the thickness dm of the suspension flow is maintained at 1 to 10 mm at least between the electrode pair and at the upstream end of the electrode pair.

[0065] As described above, the sheath fluid s from the sheath fluid inlet channel 76a flows into the main channel 74 immediately downstream of the convex portion 64a, sloping downward. On the other hand, the sheath fluid s from the sheath fluid inlet channel 76b flows into the main channel 74 immediately downstream of the convex portion 64b, sloping downward. Furthermore, upstream of the inlet portion for the sheath fluid s, the main channel 74 is formed with the convex portions 64a and 64b, making the thickness of the main channel 74 in the electrode separation direction thinner than downstream of the inlet position for the sheath fluid s. Furthermore, the thickness of the main channel 74 in this region in the electrode separation direction, i.e., the thickness of the suspension flow, is determined by the distance between the convex portions 64a and 64b. Therefore, the convex portions 64a and 64b constitute a sheath flow forming means and also function as a regulating means for regulating the thickness of the suspension flow.

[0066] That is, in the illustrated flow path device 50, the region of the main flow path 74 where the convex portions 64a and 64b are formed serves as a regulating region that regulates the thickness of the suspension flow before merging with the sheath flow. Therefore, in the flow path device 70, the distance d between the convex portions 64a and 64b is set to 1 to 10 mm, thereby controlling the thickness of the suspension flow flowing through the regulating region to 1 to 10 mm. As described above, the flow path device 70 can thereby control the thickness of the suspension flow to 1 to 10 mm in the three-layer flow of sheath flow / suspension flow / sheath flow in flow electroporation. As described above, the thickness of the suspension flow in this regulating region, i.e., the distance d, is preferably 1 to 8 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. The distance d may be 2 to 10 mm or 3 to 10 mm. Furthermore, by making the total thickness of the convex portions 64a and 64b, i.e., the total height in the electrode separation direction, equal to or less than the distance d between the convex portions 64a and 64b, the total thickness of the sheath flow can be made equal to or less than the thickness of the suspension flow, and the dilution rate of the suspension after flow electroporation can be made equal to or less than two times.

[0067] In such a flow path device 50, there is no limitation on the angle formed between the suspension flow and the sheath flow when they are joined together, i.e., the angle formed between the main flow path 74 and the through-holes 52 a and 54 a, but the smaller the angle, the better. Specifically, as described above, the angle formed between the main flow path 74 and the through-holes 52 a and 54 a is preferably 90° or less, more preferably 60° or less, even more preferably 45° or less, and particularly preferably 30° or less.

[0068] As described above, in the present invention, an outlet for discharging the sheath fluid s (sheath flow) may be provided downstream of the electrode pair, and the sheath fluid s may be recovered from this outlet, thereby making the dilution rate of the suspension m downstream of the electrode pair 2-fold or less. As an example, as conceptually shown in Figure 9, which imitates the flow path device shown in Figure 7, a recovery path 52b for discharging the sheath fluid s from the sheath flow is provided downstream of the electrode 60 on the first substrate 52. On the other hand, a recovery path 54b for discharging the sheath fluid s from the sheath flow is provided downstream of the electrode 62 on the second substrate 54.

[0069] In this way, sheath fluid s is recovered from the sheath flow downstream of the electrode pair, and the sum of the thicknesses of the upper and lower sheath fluid flows after the sheath fluid s is recovered, i.e., the sum rds, can be made thinner than the thickness rdm of the suspension flow flow path. This makes it possible to reduce the dilution rate of the suspension treated by flow electroporation to no more than two times. Note that the thickness of the sheath fluid flow after recovery can be controlled, for example, by the thickness of the recovery path. Furthermore, according to this method, even if the thickness of the sheath flow at the time of merging is thicker than the thickness of the suspension flow between the restriction means, i.e., in the restriction region, the dilution rate of the suspension after flow electroporation can be reduced to no more than two times.

[0070] As described above, in the electroporation method of the present invention, flow electroporation is performed by applying a pulsed electric field to a suspension, as an example. To perform proper flow electroporation, a pulsed electric field with an appropriate voltage and pulse width (length of one pulse) must be applied depending on the concentration of the suspension, etc. The optimal conditions for applying such an electric field are predetermined based on the steady-state concentration of the suspension, the flow rate, the set frequency of the pulsed electric field, and other factors. However, the concentration of the suspension may fluctuate due to factors such as differences in cell proliferation from day to day, or fluctuations during continuous cell concentration or mixing of bioactive substances immediately before electroporation, which may prevent the desired cell concentration from being achieved. Fluctuations in the suspension may alter the optimal conditions, making it impossible to perform proper flow electroporation even when a pulsed electric field is applied under predetermined conditions. To address this issue, the present invention preferably performs feedback control by measuring the conductivity of the suspension supplied to the flow path device and adjusting the electric field applied by the electrode pair based on the measurement results.

[0071] An example of this is conceptually shown in FIG. 10 . In FIG. 10 , a flow path device 10 is used as an example. In the example shown in FIG. 10 , a suspension is supplied from a suspension container 80 to the flow path device 10 (suspension inlet channel 24) by a liquid supply pump 82, and flow electroporation is performed as described above. A measuring jig 84 is provided in the suspension flow path from the suspension container 80 to the flow path device 10. The measuring jig 84 measures, for example, the current waveform and voltage of the suspension passing through it and sends the results to a conductivity calculation unit 86. The conductivity calculation unit 86 calculates the conductivity σm′ of the suspension from the supplied current waveform or voltage and supplies the calculated value to a calculation / power supply control device 90. The measuring jig 84 and the conductivity calculation unit 86 constitute a flow cell type conductivity measurement device.

[0072] The calculation / power supply control device 90 first uses the supplied conductivity σm to calculate a correction value Ton′ for the pulse width of the voltage to be applied or a correction value V0′ for the voltage to be applied, and drives the pulse power supply 92 in accordance with this correction value.

[0073] Specifically, the pulse width set as the steady condition is Ton, the voltage set as the steady condition is V0, the assumed conductivity of the suspension is σm, the assumed conductivity of the sheath liquid is σs, the assumed thickness of the suspension flow is dm, and the assumed thickness of the sheath flow is ds. Then, the calculation / power supply control device 90 calculates a correction value Ton' for the pulse width of the pulsed electric field to be applied from the measured conductivity σm' of the suspension using the following formula: Ton' = Ton · (σm' · 2ds + σs · dm) / (σm · 2ds + σs · dm) Alternatively, the calculation / power supply control device 90 calculates a correction value V0' for the voltage of the pulsed electric field to be applied from the measured conductivity σm' of the suspension using the following formula: V0' = V0 · (σm' · 2ds + σs · dm) / (σm · 2ds + σs · dm)

[0074] Next, the calculation / power supply control device 90 drives the pulsed power supply 92 in accordance with the calculated pulse width correction value Ton' or the calculated voltage correction value V0'. The pulsed power supply 92 supplies a pulsed potential having a pulse width corresponding to the supplied correction value or a pulsed potential having a voltage corresponding to the supplied correction value to the electrode pair (electrodes 18 and 20) to perform flow electroporation. In the present invention, by performing such feedback control, appropriate flow electroporation can be performed even when the concentration of the suspension fluctuates.

[0075] In the example shown in Fig. 10, feedback control is performed online, but the present invention is not limited to this, and feedback control may also be performed offline. Fig. 11 shows an example of this. In the example shown in Fig. 11, instead of measuring the conductivity of the suspension in the suspension flow path from the suspension container 80 to the flow path device 10 using a flow cell-type conductivity measuring device, a conductivity calculation unit 86 measures the conductivity σm' of the suspension filled in the suspension container 80. As before, this measured value is sent to the calculation / power supply control device 90, which calculates a pulse width correction value Ton' or a voltage correction value V0', and drives the pulse power supply 92 accordingly, thereby performing feedback control.

[0076] Furthermore, feedback control may be performed by measuring the concentration of the suspension instead of the conductivity σm' of the suspension. That is, a suspension concentration measuring device is provided in place of the flow cell conductivity measuring device in FIG. 10 and the conductivity calculation unit in FIG. 11, and the measurement results of the suspension concentration are sent to the calculation / power supply control device 90. The calculation / power supply control device 90 determines the conductivity σm' of the suspension from the cell concentration of the suspension using a look-up table showing the relationship between cell concentration and conductivity or a logical formula. Thereafter, similarly, the calculated conductivity σm' of the suspension is used to calculate a pulse width correction value Ton' or a voltage correction value V0', and feedback control is performed by driving the pulse power supply 92 accordingly.

[0077] The electroporation apparatus of the present invention is an apparatus having a flow channel device of the present invention for carrying out the electroporation method of the present invention, and a pair of contacts for applying electricity to the electrodes of the flow channel device.

[0078] The electrodes included in the flow path device of the present invention described above do not need to be entirely exposed to the flow path of the liquid (suspension (sheath liquid)), but may have a flow path exposed portion that is exposed to the flow path and comes into contact with the liquid flow, and a non-exposed portion that is not exposed to the flow path. In this case, it is preferable that the non-exposed portion be a contact portion that comes into contact with a contactor to apply electricity to the electrode. For example, the electrode may be L-shaped, with one end of the L-shape serving as the flow path exposed portion and the other end serving as the contact portion.

[0079] As described above, an electroporation apparatus including a flow path device of the present invention has a pair of contacts for applying current to electrodes included in the flow path device. In one example of the flow path device of the present invention, a liquid flow path is formed by sandwiching a flow path forming plate having a groove that serves as the flow path between electrode holding plates. The electrode holding plate holds the electrode so that a portion of the electrode is an exposed portion of the flow path and a portion is a contact portion (non-exposed portion). The electrode holding plate has openings at positions corresponding to the contact portions for contact with the current-applying contacts. When contacting the pair of contacts with the electrodes, for example, one of the pair of contacts is approximately fixed in a contacting state with the contact portion of one of the pair of electrodes included in the flow path device. Next, the other contact fixed to the actuator is brought close to the contact portion of the other electrode of the flow path device and brought into contact with it. Thereafter, the other contact is fixed by a fixing means, and the pair of electrodes and the pair of contacts are maintained in contact with each other, thereby setting the flow path device in a flow electroporation apparatus.

[0080] The electroporation method, useful substance manufacturing method, flow path device, and flow electroporation apparatus of the present invention have been described in detail above. However, the present invention is not limited to the above-described embodiments, and various improvements or modifications may be made without departing from the spirit and scope of the present invention.

[0081] The features of the present invention will be explained in more detail below with reference to examples. The materials, reagents, amounts and proportions of substances, and procedures shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

[0082] Example 1 Preparation of Suspension A cell suspension (cell concentration: 40 Mcells / mL) of cultured and expanded HEK293 cells (corresponding to a biologically derived substance) with an average diameter of 20 μm was prepared. As mentioned above, "mL" stands for milliliter. The average cell diameter and cell concentration were measured using a Vi-CELL XR (Beckman Coulter). Lonza's pmaxGFP plasmid (corresponding to a biologically active substance) was added to the obtained cell suspension to a concentration of 30 μg / mL. In this way, a suspension containing HEK293 cells, GFP plasmid, and medium was obtained. The cell volume fraction in the suspension was 17%, and the plasmid concentration was 30 μg / mL. From the cell concentration (40 Mcells / mL) and the average cell diameter (20 μm), assuming that the cells were spherical, the cell volume fraction in the suspension (= 4 ÷ 3 × (20 μm ÷ 2)) was calculated. 3 ×π×40 Mcells / mL÷10 6 × 100(%)) was calculated.

[0083] Flow electroporation was performed using a flow path device as shown in Figure 2. A suspension culture solution was used as the sheath fluid. The suspension flow thickness was 2 mm, the sheath flow thickness was 2 mm (one side), and the flow path width of the main flow path was 2 mm. Therefore, in this example, the dilution rate of the suspension was 3 times. Note that the flow path width of the main flow path is the length in the direction of electrode separation, i.e., the direction perpendicular to the thickness of the suspension flow, etc. The suspension flow rate was 4 mL / min, and the sheath fluid flow rate (one side) was 4 mL / min. Note that in this example, the suspension flow thickness refers to the thickness of the suspension flow in the suspension flow path of the flow path device, and the sheath flow thickness refers to the thickness of the sheath flow in the sheath fluid flow path of the flow path device. This also applies to the following examples.

[0084] Example 2 Flow electroporation was performed in the same manner as in Example 1, except that the sheath flow thickness was 0.5 mm (one side) and the pulse voltage was 381 mV. The sheath fluid flow rate (one side) was 1 mL / min. Therefore, in this example, the dilution ratio of the suspension was 1.5 times.

[0085] Example 3 Flow electroporation was performed in the same manner as in Example 1, except that a recovery channel was provided downstream of the electrode pair to recover sheath fluid from the sheath flow after passing through the electrode pair (see FIG. 9 ). The thickness of the sheath fluid recovery channel (on one side) was 1.5 mm. In other words, the thickness of the sheath flow after sheath fluid recovery was 0.5 mm. Therefore, in this example, the dilution rate of the suspension was 1.5 times.

[0086] Example 4 Flow electroporation was carried out in the same manner as in Example 2, except that the cell concentration in the suspension was 120 Mcells / mL, the cell volume fraction in the suspension was 50%, and the pulse voltage was 330 mV.

[0087] [Example 5] Flow electroporation was performed in the same manner as in Example 2, except that the cell concentration in the suspension was 140 Mcells / mL, the cell volume fraction in the suspension was 59%, and the pulse voltage was 330 mV. [Example 6] Flow electroporation was performed in the same manner as in Example 5, except that the conductivity of the suspension was measured while it was being fed into the flow path device, the pulse width of the pulsed electric field to be applied was feedback-controlled in accordance with the measurement results, and the pulse voltage was set to 319 mV, as shown in Figure 10. [Example 7] Flow electroporation was performed in the same manner as in Example 5, except that the conductivity of the suspension was measured while it was being fed into the flow path device, the pulse width of the pulsed electric field to be applied was feedback-controlled in accordance with the measurement results, and the pulse width was set to 3.39 mV, as shown in Figure 10.

[0088] Comparative Example 1 Flow electroporation was performed in the same manner as in Example 1, except that the thickness of the suspension flow was 0.1 mm, the thickness of the sheath flow (one side) was 0.1 mm, the width of the main flow channel was 0.2 mm, the flow rate of the suspension was 0.02 mL / min, the flow rate of the sheath fluid (one side) was 0.2 mL / min, and the pulse voltage was 35 mV. Therefore, in this example, the dilution rate of the suspension was 3 times.

[0089] Comparative Example 2 Flow electroporation was performed in the same manner as in Example 1, except that the suspension flow thickness was 0.5 mm, the sheath flow thickness was 0.1 mm (one side), the suspension flow rate was 0.02 mL / min, the sheath fluid flow rate (one side) was 0.2 mL / min, and the pulse voltage was 90 mV. Therefore, in this example, the dilution rate of the suspension was 1.4 times.

[0090] Comparative Example 3 Flow electroporation was performed in the same manner as in Example 1, except that a sheath flow was not formed, the cell concentration in the suspension was 120 Mcells / mL, the cell volume fraction in the suspension was 50%, and the pulse voltage was 275 mV. In this example, retention of cells (biologically derived material) in the suspension was observed near the wall surface of the main channel.

[0091] Comparative Example 4 Flow electroporation was performed in the same manner as in Example 1, except that the suspension flow thickness was 0.1 mm, the sheath flow thickness (one side) was 0.1 mm, the main channel width was 0.2 mm, the suspension flow rate was 0.02 mL / min, the sheath fluid flow rate (one side) was 0.2 mL / min, the cell concentration in the suspension was 120 Mcells / mL, the cell volume fraction in the suspension was 50%, and the pulse voltage was 24.7 mV. Therefore, in this example, the dilution rate of the suspension was 3 times. Note that in this example, the main channel became clogged during flow electroporation, and the process could not be completed.

[0092] The above examples are summarized in Table 1 below. In Table 1, "Plasmid concentration (in medium)" represents the concentration of GFP plasmid in the suspension relative to the medium in the suspension (calculated value). In Table 1, "Plasmid concentration per cell" represents the concentration of GFP plasmid in the suspension relative to the cells in the suspension (calculated value). In Table 1, "Cell processing amount" represents the cell processing amount (Mcells) per minute (calculated value).

[0093] [Evaluation] The gene transfer expression efficiency, throughput, and plasmid efficiency were evaluated as follows. <Gene transfer expression efficiency> The collected suspension was diluted with 2 M (×106 ) cells / mL, and then 2 mL was seeded in a well plate and incubated in CO ２ The cells were placed in an incubator with a cell concentration of 8% and an atmospheric temperature of 37°C, and statically cultured for 24 hours. The cell concentration X after 24 hours of culture was obtained using a Vi-CELL XR (Beckman Coulter), and the gene-transfected cell ratio Y was obtained using a BD FACS Calibur (Becton Dickinson). The gene transfection expression efficiency (= cell concentration X × cell ratio Y ÷ 2Mcells / mL × 100) (%) was calculated as the ratio of gene-transfected cells obtained after 24 hours of culture to electroporated cells. The gene transfection expression efficiency was evaluated based on the following evaluation criteria. The evaluation criteria are as follows. The results are shown in Table 1. A: 50% or more B: 35% or more, less than 50% C: 20% or more, less than 35% D: Less than 20%

[0094] <Throughput> Throughput was evaluated based on the amount of cells processed per minute as described above. The evaluation criteria are as follows. The results are shown in Table 1. A: 400 Mcells / min or more B: 100 Mcells / min or more, less than 400 Mcells / min C: 10 Mcells / min or more, less than 100 Mcells / min D: Less than 10 Mcells / min

[0095] <Plasmid Efficiency> Plasmid efficiency was evaluated based on the above-mentioned plasmid concentration per cell. The evaluation criteria were as follows. The results are shown in Table 1. A: Less than 0.5 pg / cell B: 0.5 pg / cell or more

[0096]

[0097] As shown in Table 1, according to the present invention, in flow electroporation, the thickness of the suspension flow is set to 1 mm or more, and favorable results are obtained in terms of gene transfer expression efficiency, throughput, and plasmid effect. Furthermore, as shown in Examples 2 and 3, the gene transfer expression efficiency can be further improved by setting the dilution rate of the suspension to 2x or less. Furthermore, as shown in Example 4, the gene transfer expression efficiency, throughput, and plasmid effect can all be improved by setting the cell volume fraction in the suspension to 20% or more. Furthermore, as shown in Examples 6 and 7, the gene transfer expression efficiency can be improved by performing feedback control using the conductivity of the suspension.

[0098] In contrast, in Comparative Examples 1 and 2, where the suspension flow thickness was less than 1 mm, the throughput was low, and in particular, Comparative Example 2, where the cell flow rate was increased, also had low gene transfer expression efficiency. Furthermore, in Comparative Example 3, where no sheath flow was used, cell flow retention was observed near the wall surface of the channel, resulting in low gene transfer expression efficiency. Furthermore, in Comparative Example 4, where the suspension flow thickness was less than 1 mm and the cell volume fraction was 50%, the channel became clogged despite the presence of a sheath flow.

[0099] Flow electroporation was performed in the same manner as in Examples 4 and 6, except that the thickness of the suspension flow was set to 1 mm, 3 mm, 5 mm, or 8 mm. Even in this case, sufficient gene transfer expression efficiency and throughput were obtained. Flow electroporation was performed in the same manner as in Examples 4 and 6, except that the sheath flow thickness (one side) was set to 0.1 mm or 1 mm. Even in this case, sufficient gene transfer expression efficiency and throughput were obtained. Flow electroporation was performed in the same manner as in Examples 4 and 6, except that the cell concentration was set to 50 Mcells / mL or 80 Mcells / mL. Even in this case, sufficient gene transfer expression efficiency and throughput were obtained. Flow electroporation was performed in the same manner as in Examples 4 and 6, except that the flow channel device shown in FIG. 7 was used. Even in this case, sufficient gene transfer expression efficiency and throughput were obtained. Flow electroporation was performed in the same manner as in Examples 4 and 6, except that the plasmid DNA introduced was three types of plasmid DNA for adeno-associated virus (AAV) production. Even in this case, sufficient gene transfer expression efficiency and throughput were obtained. Furthermore, good AAV production was achieved. From the above results, the effects of the present invention are clear.

[0100] It can be suitably used for the production of gene therapy drugs, etc.

[0101] 10, 50 Flow path device 12, 52, 100 First substrate 14, 54, 102 Second substrate 16, 64 Flow path substrate 16a First flow path plate 16b Second flow path plate 16c Third flow path plate 16d Fourth flow path plate 16e Fifth flow path plate 18, 20, 60, 62, 106, 108 Electrodes 24, 70 Suspension inlet channel 26a, 26b, 76a, 76b Sheath liquid inlet channel 28a, 28b Sheath liquid flow path 30, 74 Main flow path 30a Suspension flow path 32, 72 Outlet 52a, 54a, 56a, 58a Through hole 52b, 54b Recovery path 64a, 64b Convex portion 80 Suspension container 82 Liquid delivery pump 84 Measuring jig 86 Conductivity calculation unit 90 Calculation / power supply control device 92 Pulse power supply 104 Flow path m Suspension s Sheath liquid

Claims

1. In electroporation, in which an electric field is applied to a suspension containing a bio-derived substance and a bioactive substance using an electrode pair to introduce the bioactive substance into the bio-derived substance, A sheath flow is formed by a sheath liquid, which is a liquid other than the suspension, that flows together with the suspension and comes into contact with the electrodes constituting the electrode pair, and further, A regulated region is provided through which only a suspension flow consisting of the aforementioned suspension flows, the thickness of the electrodes constituting the electrode pair in the suspension flowing through the regulated region is set to 1 to 10 mm in the separating direction, and downstream of the regulated region, the suspension flow and the sheath flow are merged. An electroporation method in which a three-layered flow consisting of sheath flow / suspension flow / sheath flow flows between the electrode pair.

2. The electroporation method according to claim 1, wherein the total thickness of the sheath flow in the direction of separation of the electrodes when it merges with the suspension flow is less than or equal to the thickness of the suspension flow in the direction of separation of the electrodes in the regulated region.

3. The electroporation method according to claim 1, wherein the sheath liquid is recovered downstream of the electrode pair, thereby reducing the dilution ratio of the suspension in the mixture of the suspension and the sheath liquid after the recovery of the sheath liquid to 2 times or less.

4. The electroporation method according to any one of claims 1 to 3, wherein the volume fraction of the biologically derived substance in the suspension is 10% or more.

5. The electroporation method according to claim 4, wherein the volume fraction of the biologically derived substance in the suspension is 40% or more.

6. The electroporation method according to any one of claims 1 to 3, wherein the suspension is a cell suspension and the sheath liquid is a culture medium used when culturing the cells of the cell suspension.

7. The electroporation method according to any one of claims 1 to 3, wherein, prior to applying the electric field, the conductivity of the suspension or the concentration of the biological substance in the suspension is measured, and the electric field or pulse width applied by the electrode pair is adjusted according to the measurement result.

8. The electroporation method according to any one of claims 1 to 3, wherein the flow rate of the suspension is 1 mL / min or more.

9. The electroporation method according to any one of claims 1 to 3, wherein the flow rate of the sheath liquid is 0.1 mL / min or more.

10. In electroporation, in which an electric field is applied to a suspension containing a bio-derived substance and a bioactive substance using an electrode pair to introduce the bioactive substance into the bio-derived substance, A sheath flow is formed by a sheath liquid, which is a liquid other than the suspension, that flows together with the suspension and comes into contact with the electrodes constituting the electrode pair, and further, In at least a portion of the space between the electrode pairs, the thickness of the suspension flow consisting of the suspension is set to 1 to 10 mm in the direction of separation of the electrodes. An electroporation method in which a three-layered flow consisting of sheath flow / suspension flow / sheath flow flows between the electrode pair.

11. A method for producing a useful substance, comprising the electroporation method described in any one of claims 1 to 3.

12. A flow channel device for electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance and a bioactive substance by applying an electric field with an electrode pair, Main channel and A pair of electrodes that apply an electric field to the liquid flowing through the main channel, The main flow path has a supply port for supplying the suspension, A suspension channel through which the suspension flows, It has a sheath fluid channel through which only the sheath fluid, which is a liquid other than the aforementioned suspension, flows. The sheath fluid channel is positioned downstream of the supply port and upstream of the electrode pair, such that the sheath fluid flows into it. The distance between the electrodes constituting the electrode pair is set to 1 to 10 mm. The width of the main channel is 2 mm or more. The angle between the main flow path and the sheath fluid flow path is 60° or less. The aforementioned sheath fluid channel has a region where the channel is widened in the width direction toward the downstream side. A flow channel device in which a three-layered flow of sheath flow / suspension flow / sheath flow flows between the electrode pair in the main flow channel.

13. The flow channel device according to claim 12, further comprising a discharge port downstream of the electrode for discharging the sheath flow from the main flow channel.

14. The flow channel device according to claim 12, further comprising a discharge port downstream of the electrode for discharging the sheath flow and the treated suspension flow from the main flow channel.

15. The flow channel device according to claim 12, wherein the electrodes constituting the electrode pair are arranged spaced apart from the main flow channel.

16. An electroporation apparatus comprising a flow channel device according to claim 12 and a pair of contacts for energizing the electrodes of the flow channel device.