Flow path device, electroporation method, and electroporation device

Abstract

The present invention provides a flow path device and an electroporation method that have high productivity. Provided is a flow path device that is used for electroporation and that, by applying an electric field via an electrode pair to a suspension which contains biological matter and a bioactive substance, introduces the bioactive substance into the biological matter. The flow path device comprises: a main flow path through which a suspension flows; a sheath liquid flow path through which flows only a sheath liquid that is a liquid other than the suspension and which causes the sheath liquid to flow into the main flow path; and an electrode pair which apply an electric field to the suspension flowing through the main flow path. The sheath liquid flow path has a sheath liquid expansion part that expands the flow path of the sheath liquid in the width direction, and a first flow path that is downstream of the sheath liquid expansion part and that is continuous with the sheath liquid expansion part. The total of the length of the first flow path and the length of the sheath liquid expansion part is not less than 0.8 times the width of the main flow path between the electrode pair.

Description

Fluid device, electroporation method, and electroporation apparatus

[0001] The present invention relates to a flow channel device, electroporation method, and electroporation apparatus used in electroporation, which involves introducing a bioactive substance into a bio-derived substance by applying an electric field to a suspension containing a bio-derived substance and a bioactive substance while the suspension is flowed through a main channel. In particular, the present invention relates to a flow channel device, electroporation method, and electroporation apparatus that define the relationship between the width of the sheath liquid channel and the width of the main channel.

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

[0003] In contrast to the standard static cuvette method described above, a continuous liquid delivery type of electroporation has been proposed, as shown in Patent Documents 1 and 2, in which a suspension containing bio-derived materials and bioactive substances is circulated through a flow channel in which electrode pairs are installed.

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

[0005] The inventors are developing a unique continuous production platform and flow channel device for electroporation. To scale up production, the inventors attempted to enlarge the flow channel of the flow channel device. However, this resulted in decreased productivity, including a decline in the efficiency of introducing bioactive substances into bio-derived materials, a problem that was not present with conventional microscale flow channel devices. The object of the present invention is to provide a flow channel device, electroporation method, and electroporation apparatus with high productivity.

[0006] The above objective can be achieved with the following configuration. Invention [1] is a flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, and comprises a main flow channel through which the suspension flows, a sheath liquid flow channel through which only a sheath liquid other than the suspension flows and which allows the sheath liquid to flow into the main flow channel, and an electrode pair that applies an electric field to the suspension flowing in the main flow channel, wherein the sheath liquid flow channel comprises a sheath liquid expansion section that expands the sheath liquid flow channel in the width direction, and a first flow channel located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, and the sum of the length of the first flow channel and the length of the sheath liquid expansion section is 0.8 times or more the width of the main flow channel between the electrode pair.

[0007] Invention [2] is a flow channel device according to claim 1, the flow channel device according to invention [1], wherein the main flow channel is provided with a suspension inlet for introducing a suspension into the main flow channel, the sheath liquid flow channel is located downstream of the first flow channel and further includes a direction changing section for changing the flow direction of the sheath liquid in the first flow channel, a second flow channel located downstream of the direction changing section, and a sheath liquid confluence port located downstream of the second flow channel for introducing the sheath liquid into the main flow channel body, the sheath liquid confluence port being provided downstream of the suspension inlet and upstream of the electrode pair. Invention [3] is a flow channel device according to invention [1] or [2], wherein the first flow channel and the second flow channel have different thicknesses. Invention [4] is a flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, and comprises a main flow channel through which the suspension flows, a sheath liquid flow channel through which only a sheath liquid other than the suspension flows and which allows the sheath liquid to flow into the main flow channel, and an electrode pair that applies an electric field to the suspension flowing in the main flow channel, wherein the main flow channel comprises a suspension inlet for allowing the suspension to flow into the main flow channel, and a sheath liquid confluence port provided downstream of the suspension inlet and upstream of the electrode pair for allowing the sheath liquid to flow into the main flow channel, and the sheath liquid flow channel has sections of different thicknesses.

[0008] Invention [5] is a flow channel device according to any one of Inventions [1] to [4], wherein the width of the main flow channel is 3 mm or more. Invention [6] is a flow channel device according to any one of Inventions [1] to [5], wherein the first flow channel has a thickness of 2 mm or less. Invention [7] is a flow channel device according to any one of Inventions [4] to [6], wherein there is a direction changing section between the first flow channel and the second flow channel that changes the flow direction of the sheath liquid in the first flow channel. Invention [8] is a flow channel device according to any one of Inventions [4] to [7], further comprising a sheath liquid expanding section arranged upstream of the first flow channel that expands the sheath liquid flow channel in the width direction. Invention [9] is a flow channel device according to Invention [1] or [4], wherein the sheath liquid flow channel is a straight flow channel and is arranged at an inclination with respect to the main flow channel.

[0009] Invention

[10] is a flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, wherein the flow channel has a main flow channel through which the suspension flows, a sheath liquid flow channel through which only a sheath liquid other than the suspension flows and which allows the sheath liquid to flow into the main flow channel, and an electrode pair that applies an electric field to the suspension flowing in the main flow channel, the sheath liquid flow channel has a sheath liquid expansion section that expands the flow channel of the sheath liquid in the width direction, and a first flow channel located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, the width of the main flow channel between the electrode pair is 3 mm or more, the width of the first flow channel is 3 mm or more, the thickness of the first flow channel is 2 mm or less, and the total length of the length of the first flow channel and the length of the sheath liquid expansion section is 0.8 times or more the width of the main flow channel between the electrode pair. Invention

[11] is an electroporation method comprising the step of supplying a suspension at a rate of 5 mL / min or more to a flow channel device described in any one of Inventions [1] to

[10] , and applying an electric field to the suspension with an electrode pair to introduce a bioactive substance into a bio-derived material. Invention

[12] is an electroporation apparatus having a flow channel device described in any one of Inventions [1] to

[10] .

[0010] According to the present invention, a highly productive flow channel device, electroporation method, and electroporation apparatus can be provided.

[0011] This is a schematic diagram showing an electroporation apparatus having a flow channel device according to an embodiment of the present invention. This is an enlarged view of the main part of the flow channel device according to an embodiment of the present invention. This is a schematic diagram showing an example of the main flow channel of the flow channel device according to an embodiment of the present invention. This is a schematic diagram showing a first example of the sheath liquid flow channel of the flow channel device according to an embodiment of the present invention. This is a schematic diagram showing a calculation model and numerical calculation results of the flow velocity of the sheath liquid flow channel of the flow channel device according to an embodiment of the present invention. This is a schematic diagram showing a second example of the sheath liquid flow channel of the flow channel device according to an embodiment of the present invention. This is a schematic diagram showing a third example of the sheath liquid flow channel of the flow channel device according to an embodiment of the present invention. This is a schematic cross-sectional view for explaining the operation of the flow channel device in an electroporation apparatus having a flow channel device according to an embodiment of the present invention. This is a schematic diagram showing a type (reference example) of sheath liquid flow channel.

[0012] Hereinafter, based on the preferred embodiments shown in the accompanying drawings, the flow path device, the electroporation method, and the electroporation device of the present invention will be described in detail. The drawings described below are exemplary for explaining the present invention, and are simplified or the like for explaining the present invention, and the present invention is not limited to the drawings shown below. In the following, "~" indicating a numerical range includes the numerical values described on both sides. For example, when ε is a numerical value ε α ~ numerical value ε β it means that the range of ε is the numerical value ε α and the numerical value ε β and the range including, and in mathematical symbols, ε α ≦ ε ≦ ε β . Also, for a specific angle, parallel and perpendicular, and vertical, etc., if there is no particular description, it includes the error range generally acceptable in the corresponding technical field. Further, regarding length, width, thickness, etc., if there is no particular description, it includes the error range generally acceptable in the corresponding technical field. Also, regarding temperature, flow rate, electrical conductivity, conductivity, volume fraction, etc., if there is no particular description, it includes the error range generally acceptable in the corresponding technical field.

[0013] The flow path device 12 (see FIG. 1) is a device used for EP that introduces a bioactive substance into a biological origin by applying an electric field to a suspension Q (see FIG. 8) containing a biological origin and a bioactive substance by an electrode pair 22 (see FIG. 1). The electroporation device 10 (see FIG. 1) has the flow path device 12. Regarding the flow path device 12, which will be described in detail later, it has a main flow path 20 through which the suspension Q flows, sheath liquid flow paths 40 and 42 through which only the sheath liquid s, which is a liquid other than the suspension Q (see FIG. 8), flows and the sheath liquid s is made to flow into the main flow path 20, and an electrode pair 21 that applies an electric field to the suspension Q flowing through the main flow path 20. The sheath liquid flow paths 40 and 42 have sheath liquid expanding portions 30b and 32b that expand the flow path of the sheath liquid s in the width direction, and first flow paths 30c and 32c that are downstream of the sheath liquid expanding portions 30b and 32b and are continuous with the sheath liquid expanding portions 30b and 32b. The length L of the first flow paths 30c and 32c １The total length Ls of the sheath liquid expansion parts 30b and 32b and the length Ld is 0.8 times or more the width of the main flow path 20 between the electrode pairs 22. Further, in the flow path device 12, the main flow path 20 includes a suspension liquid inlet 34a for allowing the suspension liquid Q to flow into the main flow path 20, and sheath liquid confluence ports 30f and 32f provided on the downstream side of the suspension liquid inlet 34a and on the upstream side of the electrode pair 22 for allowing the sheath liquid s to flow into the main flow path 20. The sheath liquid flow paths 40 and 42 have sections with different thicknesses. Further, in the flow path device 12, the width of the main flow path 20 between the electrode pairs 22 is 3 mm or more, and the width W １ (see FIG. 4) of the first flow paths 30c and 32c is 3 mm or more, and the thickness t １ (see FIG. 2) of the first flow paths 30c and 32c is 2 mm or less.

[0014] (First Example of Electroporation Device) Hereinafter, the flow path device and the electroporation device will be described more specifically by taking the electroporation device 10 having the flow path device 12 shown in FIG. 1 as an example. FIG. 1 is a schematic diagram showing an electroporation device having a flow path device according to an embodiment of the present invention. FIG. 2 is an enlarged view of a main part of the flow path device according to an embodiment of the present invention. The electroporation device (hereinafter referred to as an EP device) 10 shown in FIG. 1 includes a flow path device 12, a power supply unit 14, a control unit 15, and pumps 16a, 16b, 16c, and 16d. The flow path device 12 has a main flow path 20 through which the suspension liquid Q flows and an electrode pair 22 that applies an electric field to the suspension liquid Q flowing through the main flow path 20. The main flow path 20 is, for example, a linear flow path. The main flow path 20 has a cross-sectional shape in a direction orthogonal to the extending direction D Ｌ that is, for example, a quadrilateral with all interior angles of 90°. The above-described cross-sectional shape of the main flow path 20 is not limited to a quadrilateral with all interior angles of 90°.

[0015] The flow path device 12 shown in FIG. 1 is arranged such that, for example, the extending direction D Ｌ of the linear main flow path 20 is orthogonal to the horizontal plane Hp. The main flow path 20 extends in the vertical direction z. The extending direction D ＬThe vertical direction z is perpendicular to the horizontal plane Hp, and the angle with respect to the horizontal plane Hp is 90°, however, the vertical direction z of the main flow channel 20 is allowed to be ±10° from 90°.

[0016] The flow channel device 12 is composed of, for example, a first substrate 30, a second substrate 32, a first introduction section 36, a second introduction section 38, and a flow channel substrate 34 provided between the first substrate 30 and the second substrate 32. The first introduction section 36, the first substrate 30, the flow channel substrate 34, the second substrate 32, and the second introduction section 38 are stacked in that order. The main flow channel 20 described above is provided on the flow channel substrate 34. In the flow channel device 12, it is preferable to have O-rings as sealing members (not shown) to prevent leakage of the suspension Q (see Figure 8) and sheath liquid s (see Figure 8) between the first introduction section 36 and the first substrate 30, between the first substrate 30 and the flow channel substrate 34, between the flow channel substrate 34 and the second substrate 32, and between the second substrate 32 and the second introduction section 38.

[0017] The electrode pair 22 has a first electrode 23 and a second electrode 24 arranged opposite each other. The first electrode 23 has an electrode surface 23a, which is a plane. The second electrode 24 has an electrode surface 24a, which is a plane. The electrode surface 23a of the first electrode 23 and the electrode surface 24a of the second electrode 24 are arranged opposite each other and parallel to each other. The space 25 between the opposing electrode surface 23a of the first electrode 23 and the electrode surface 24a of the second electrode 24 is the space between the electrode pair 22. The electrode pair 22 is arranged opposite each other across a stacked flow of suspension Q and sheath liquid s, and the first electrode 23 and the second electrode 24 of the electrode pair 22 are in direct contact with the sheath liquid s. The first electrode 23 is incorporated into the first substrate 30, and the second electrode 24 is incorporated into the second substrate 32, thereby forming the electrode pair 22. The electrode surface 23a of the first electrode 23 and the electrode surface 24a of the second electrode 24 each constitute the inner surface 20c of the main flow channel 20. For example, when EP is performed, if the suspension Q and the sheath liquid s are supplied to the main flow channel 20, the electrode pair 22 comes into contact with the sheath liquid s. In the flow channel device 12 shown in Figure 1, the electrode pair 22 is located in the main flow channel body portion 21b of the main flow channel 20.

[0018] The flow channel device 12 has a suspension supply port 17 that supplies a suspension Q to the main flow channel 20, and an outlet port 18 that discharges the suspension Q, to which an electric field has been applied, to the outside of the flow channel device 12. In the main flow channel 20, the suspension supply port 17 side is the upstream side, and the outlet port 18 side is the downstream side. A tube 19 is connected to the outlet port 18. The tube 19 is made of a flexible material such as a silicone tube.

[0019] The main channel 20 comprises, for example, an enlarged section 21a and a main channel body section 21b located downstream of the enlarged section 21a and continuous with the enlarged section 21a. The enlarged section 21a is located at one upstream end 20a of the main channel 20. A suspension supply port 17 for supplying suspension Q to the main channel 20 is provided on the surface 12a of the channel device 12 at one end 20a of the main channel 20. The suspension supply port 17 is provided in the first introduction section 36. The first introduction section 36 is provided with a through hole 36a extending in the stacking direction Ds. The first substrate 30 is provided with a through hole 30a that communicates with the through hole 36a of the first introduction section 36 and the main channel 20 and extends in the stacking direction Ds. The suspension supply port 17 communicates with the uppermost part of the main channel 20 through the through hole 36a of the first introduction section 36 and the through hole 30a of the first substrate 30. The through-hole 36a of the first introduction section 36 and the through-hole 30a of the first substrate 30 constitute the suspension Q supply path 37. The opening connecting the through-hole 30a and the main flow path 20 is the suspension inlet 34a. Suspension Q flows into the main flow path 20 through the suspension inlet 34a. In the flow path device 12 shown in Figure 1, the suspension inlet 34a communicates with the enlarged section 21a of the main flow path 20. For example, the shape of the opening of the suspension inlet 34a is circular.

[0020] An outlet 18 for discharging the suspension Q to the outside of the flow channel device 12 is provided on the back surface 12b of the flow channel device 12 from the other downstream end 20b of the main flow channel 20. The outlet 18 is provided on the second substrate 32. The main flow channel 20 and the outlet 18 are connected by a straight discharge passage 32a that penetrates the second substrate 32 and the second introduction section 38. The discharge passage 32a extends, for example, in a direction parallel to the horizontal plane Hp. The opening connecting the discharge passage 32a and the main flow channel 20 is the suspension outlet 34b. The suspension outlet 34b has, for example, a circular opening.

[0021] The suspension Q is supplied from the suspension supply port 17 and flows into the main flow path 20 through the through-hole 36a, the through-hole 30a, and the suspension inlet 34a. The suspension Q flows through the main flow path 20 along the direction Df shown in Figure 1 and is discharged from the outlet 18 via the suspension outlet 34b and the discharge passage 32a. The direction Df shown in Figure 1 is the direction from upstream to downstream of the main flow path 20, and is the direction from below to above in the vertical direction z. The sheath liquid s is also discharged from the outlet 18 together with the suspension Q.

[0022] Main flow path 20 extension direction D Ｌ However, this is the direction Df in which the suspension Q flows within the main channel 20. In the flow channel device 12, the direction Dd of the suspension Q being delivered between the electrode pair 22 is from below to above in the vertical direction z. The suspension Q flows within the main channel 20 in the direction from below to above in the vertical direction z. The width direction Dw of the main channel 20 (see Figure 3) is parallel to the direction x which is perpendicular to the vertical direction z, and the extension direction D of the main channel 20. Ｌ It is also a direction perpendicular to the vertical direction z and direction x. Direction y is defined as the direction perpendicular to the vertical direction z and direction x. The suspension supply port 17 is provided on the surface 12a (see Figure 1) of the flow channel device 12, and the discharge port 18 is provided on the back surface 12b (see Figure 1) of the flow channel device 12. However, the configuration is not limited to this, and it is also possible to provide the discharge port 18 on the surface 12a of the flow channel device 12 and the suspension supply port 17 on the back surface 12b of the flow channel device 12. The main flow channel 20 will be described in detail later.

[0023] A pipe 26 is connected to the suspension supply port 17. A mixer 27 is provided on the pipe 26. For example, a culture device 28 is connected to the pump 16a of the EP device 10. A pump 16a is provided between the mixer 27 and the culture device 28. A tank 29 is connected to the mixer 27. A pump 16b is provided between the mixer 27 and the tank 29. The mixer 27 mixes the culture medium and the bioactive substance. The mixture of the culture medium and the bioactive substance is called suspension Q, and suspension Q includes the culture medium. The culture device 28 contains a culture medium (not shown) containing biologically derived material and the culture medium. In the culture device 28, for example, cell culture is performed, and a culture medium containing cells and the culture medium is obtained. The bioactive substance is stored in the tank 29, for example, in a dispersed state in liquid. The configuration of the tank 29 is not particularly limited as long as it can store the bioactive substance.

[0024] Note that the mixer 27, culture device 28, and tank 29 do not constitute the EP device 10. In Figure 1, the culture device 28 is shown as the culture medium supply unit, but a tank for storing the culture medium removed from the culture device 28 may also be used. The suspension Q is pumped by the pump 16a from the bottom to the top in the vertical direction z through the main flow path 20, and the suspension Q is continuously pumped between the electrode pair 22 in the direction from the bottom to the top in the vertical direction z.

[0025] The flow channel device 12 uses a suspension Q and a sheath liquid s, and delivers the suspension Q by sandwiching it between the sheath liquid s, thus having two sheath liquid flows. In the flow channel device 12, the suspension Q is continuously delivered between the electrode pair 22 by sandwiching it between the sheath liquid s, and an electric field is applied to the suspension Q by the electrode pair 22 to perform EP, which introduces bioactive substances into biologically derived materials.

[0026] The flow channel device 12 uses a suspension Q and a sheath liquid s, and delivers the suspension Q by sandwiching it between the sheath liquid s, thus having two sheath liquid flows. In the flow channel device 12, the suspension Q is continuously delivered between the electrode pair 22 by sandwiching it between the sheath liquid s, and an electric field is applied to the suspension Q by the electrode pair 22 to perform EP, which introduces bioactive substances into biologically derived materials.

[0027] The flow channel device 12 has two sheath liquid channels 40 and 42 through which only the sheath liquid s, which is a liquid other than the suspension Q, flows, and which cause the sheath liquid s to flow into the main channel body 21b of the main channel 20. It has a first supply port 43a and a second supply port 43b for supplying the sheath liquid s to the main channel body 21b of the main channel 20. The first supply port 43a is provided in the first introduction section 36. The first introduction section 36 is provided with a supply path 36b that communicates with the first supply port 43a and extends in the stacking direction Ds. A sheath liquid expansion section 30b is provided in the first substrate 30 that communicates with the supply path 36b and expands the flow channel of the sheath liquid s in the width direction. The sheath liquid expansion section 30b extends in the vertical direction z. The opening that connects the supply path 36b of the sheath liquid s and the sheath liquid expansion section 30b is the sheath liquid inlet 31a. The first substrate 30 is provided with a second flow channel 30e, which is located downstream of the sheath liquid expansion section 30b and is continuous with the sheath liquid expansion section 30b. The second flow channel 30e is provided, for example, at an inclination with respect to the main flow channel 20.

[0028] As shown in Figures 1 and 2, between the sheath fluid expansion section 30b and the second flow path 30e, there is, for example, a first flow path 30c provided continuously with the sheath fluid expansion section 30b, and a direction changing section 30d provided continuously with the first flow path 30c. The direction changing section 30d changes the flow direction of the sheath fluid s in the first flow path 30c. The direction changing section 30d and the second flow path 30e are in communication, and the flow of the sheath fluid s is directed toward the second flow path 30e by the direction changing section 30d. For example, the first flow path 30c, the direction changing section 30d, and the second flow path 30e have the same width. The second flow path 30e and the main flow path body 21b are in communication, and the opening through which the second flow path 30e and the main flow path body 21b are in communication is the sheath fluid confluence port 30f for allowing the sheath fluid s to flow into the main flow path body 21b. The sheath liquid confluence port 30f connects the second flow path 30e and the main flow path body 21b, allowing the sheath liquid s to flow into the main flow path body 21b. The sheath liquid confluence port 30f is located downstream of the suspension inlet 34a and upstream of the electrode pair 21, and further downstream of the sheath liquid expansion section 30b. The supply path 36b of the first introduction section 36, the sheath liquid expansion section 30b of the first substrate 30, and the first flow path 30c form a substantially L-shaped flow path.

[0029] The second supply port 43b is provided in the second introduction section 38. The second introduction section 38 is provided with a supply path 38b that communicates with the second supply port 43b and extends in the stacking direction Ds. The second substrate 32 is provided with a sheath liquid expansion section 32b that communicates with the supply path 38b and expands the flow path of the sheath liquid s in the width direction. The sheath liquid expansion section 32b extends in the vertical direction z. The opening that connects the supply path 38b of the sheath liquid s and the sheath liquid expansion section 32b is the sheath liquid inlet 31b. The second substrate 32 is provided with a second flow path 32e that is located downstream of the sheath liquid expansion section 32b and is continuous with the sheath liquid expansion section 32b. The second flow path 32e is provided, for example, at an inclination with respect to the main flow path 20. As shown in Figures 1 and 2, the configuration may include, for example, a first flow path 32c continuous with the sheath fluid expansion section 32b and a direction changing section 32d continuous with the first flow path 32c between the sheath fluid expansion section 32b and the second flow path 32e. The direction changing section 32d changes the flow direction of the sheath fluid s in the first flow path 32c. The direction changing section 32d and the second flow path 32e are in communication, and the flow of the sheath fluid s is directed toward the second flow path 32e by the direction changing section 32d. For example, the first flow path 32c, the direction changing section 32d, and the second flow path 32e have the same width. The second flow path 32e and the main flow path body 21b are in communication, and the opening through which the second flow path 32e and the main flow path body 21b are in communication is the sheath fluid confluence port 32f for allowing the sheath fluid s to flow into the main flow path body 21b. The sheath liquid confluence port 32f connects the second flow path 32e and the main flow path body 21b, allowing the sheath liquid s to flow into the main flow path body 21b. The sheath liquid confluence port 32f is located downstream of the suspension inlet 34a and upstream of the electrode pair 21, and further downstream of the sheath liquid expansion section 32b. The supply path 38b of the second introduction section 38, the sheath liquid expansion section 32b of the second substrate 32, and the first flow path 32c form a substantially L-shaped flow path. The sheath liquid inlet 31a and the sheath liquid inlet 31b have, for example, a circular opening. The second flow paths 30e and 32e are also referred to as the sheath liquid flow path body.

[0030] The through-hole 36a of the first introduction section 36, the sheath liquid expansion section 30b and second flow path 30e of the first substrate 30, the supply path 38b of the second introduction section 38, and the sheath liquid expansion section 32b and second flow path 32e of the second substrate 32 are arranged symmetrically with respect to the main flow path 20 which extends in the vertical direction z. The sheath liquid flow path 40 is a flow path through which only the sheath liquid s, which is a liquid other than the suspension Q, flows, and for example, it has a sheath liquid expansion section 30b that expands the flow path of the sheath liquid s in the width direction, a first flow path 30c arranged downstream of the sheath liquid expansion section 30b and continuous with the sheath liquid expansion section 30b, a direction changing section 30d, a second flow path 30e, and a sheath liquid confluence port 30f. Alternatively, the sheath liquid flow path 40 may have a configuration that includes a first supply port 43a and a supply path 36b. The sheath liquid flow path 42 is a flow path through which only the sheath liquid s, which is a liquid other than the suspension Q, flows. For example, it has a sheath liquid expansion section 32b that expands the flow path of the sheath liquid s in the width direction, a first flow path 32c located downstream of the sheath liquid expansion section 32b and continuous with the sheath liquid expansion section 32b, a direction changing section 32d, a second flow path 32e, and a sheath liquid confluence port 32f. Alternatively, the sheath liquid flow path 42 may have a second supply port 43b and a supply path 38b. In the main flow path 20, the region where the sheath liquid confluence port 30f and the sheath liquid confluence port 32f face each other is a confluence section 44 where the suspension flow and the sheath liquid flow merge. An electrode pair 22 is provided downstream of the confluence section 44. In the flow channel device 12, for example, on the first substrate 30, the electrode surface 23a of the first electrode 23, which constitutes the inner surface 20c of the main flow channel 20, and the sheath liquid confluence port 30f are arranged on the same plane. The sheath liquid s flows over the electrode surface 23a of the first electrode 23. Also, for example, on the second substrate 32, the electrode surface 24a of the second electrode 24, which constitutes the inner surface 20c of the main flow channel 20, and the sheath liquid confluence port 32f are arranged on the same plane. The sheath liquid s flows over the electrode surface 24a of the second electrode 24.

[0031] A pipe 26a is connected to the first supply port 43a. A pump 16c is connected to the first supply port 43a via the pipe 26a connected to the first supply port 43a. A tank 45 is connected to the pump 16c via the pipe 26c. A pipe 26b is connected to the second supply port 43b. A pump 16d is connected to the second supply port 43b via the pipe 26b connected to the second supply port 43b. A tank 45 is connected to the pump 16d via the pipe 26d.

[0032] (Main channel) The main channel 20 will be described in detail. Figure 3 is a schematic diagram showing an example of the main channel of a flow channel device according to an embodiment of the present invention. Figure 3 shows the contour shape of the main channel 20 as viewed from the suspension inlet 34a side. The contour shape of the main channel 20 shown in Figure 3 shows the two-dimensional shape of the side surface constituting the main channel 20. Also, in Figure 3, the suspension inlet 34a side is the upstream side of the main channel 20, and the extension direction D of the suspension inlet 34a Ｌ The opposite side of this is the downstream side of the main channel 20. As shown in Figure 3, the main channel 20 includes a suspension inlet 34a into which the suspension Q flows, an expanding section 21a that expands the channel in the width direction Dw starting from the suspension inlet 34a, and a main channel body 21b located downstream of the expanding section 21a and continuous with the expanding section 21a. The main channel 20 has a configuration in which there is no bend on the side surface 21c of the expanding section 21a.

[0033] In Figure 3, the main channel 20 has a contour shape that passes through the center of the suspension inlet 34a and extends in the direction D of the main channel 20. ＬThe shape is symmetrical with respect to a center line C parallel to the main channel, and the side surface of the main channel body 21b is straight. In Figure 3, the enlarged portion 21a of the main channel 20 has a flat side surface 21c. The side surface 21c extends linearly in the width direction Dw of the main channel 20, starting from the suspension inlet 34a. The connection portion 46 between the enlarged portion 21a and the main channel body 21b is made of a curved surface, and the enlarged portion 21a has a curved surface. The enlarged portion 21a and the main channel body 21b are connected continuously without any bends. The contour shape of the side surface 21c of the enlarged portion 21a shown in Figure 3 can be represented, for example, by a linear function. Figure 3 also shows a configuration where there are no bends in the connection portion between the enlarged portion 21a and the main channel body 21b. The side surface of the main channel body 21b is straight, and the main channel 20 has no bends on the side surface upstream of the furthest downstream part of the electrode pair 22.

[0034] In addition to the configuration shown in Figure 3, the side surface of the enlarged portion 21a, which does not have a bend on its side, may be composed of, for example, a single plane or a single curved surface. When the entire side surface of the enlarged portion 21a is composed of a single curved surface, the contour shape of the side surface of the enlarged portion 21a is an arc, and for example, the contour shape can be represented by a quadratic function. Note that the arc includes an elliptical arc. Also, when the entire side surface of the enlarged portion 21a is composed of multiple curved surfaces, for example, the side surface of the enlarged portion 21a can be composed by combining two curved surfaces. For example, the curved surfaces are connected such that the connection point between the curved surfaces is an inflection point. When the side surface of the enlarged portion 21a is composed of multiple curved surfaces, the contour shape can be represented by, for example, an arc, a cubic function, a trigonometric function, an inverse trigonometric function, or a sigmoid function. In the enlarged portion 21a, when the side surface without a bend is composed of multiple curved surfaces, mathematically, the multiple curved surfaces are in a relationship where the joints of the curved surfaces are tangently continuous, curvature continuous, or the tangential planes coincide. Furthermore, the main channel 20 is connected to the end of the curved surface that constitutes the side surface of the enlarged portion 21a and the main channel body portion 21b, and the tangent to the end of the side surface (not shown) coincides with the tangent to the side surface of the main channel body portion 21b (not shown). Therefore, the enlarged portion 21a and the main channel body portion 21b are connected without any bends at the connection point between the end of the curved surface that constitutes the side surface of the enlarged portion 21a and the main channel body portion 21b.

[0035] By making the main channel 20 an enlarged section 21a without a bend as shown in Figure 3 above, the stagnation of suspension Q in the enlarged section 21a is suppressed, improving productivity. Furthermore, the flow of suspension Q is less disturbed in the main channel body 21b, the flow velocity of suspension Q in the width direction of the main channel 20 becomes uniform, and during EP, bioactive substances can be uniformly introduced into the bio-derived material in the suspension flow, achieving high EP efficiency and increasing productivity. Since the amount of suspension Q that is delivered and discarded before the start of EP can be reduced, it also contributes to further improvement in productivity. The higher the cell density of suspension Q, the more the difference in introduction efficiency affects productivity. In this embodiment, which processes high-density suspension Q, productivity is further increased by the enlarged section 21a without a bend. By configuring the main channel 20 so that there is no bend on the side surface of the enlarged section 21a, stagnation of the suspension flow in the enlarged section 21a is less likely to occur. In suspensions containing bio-derived material, stagnation of bio-derived material occurs in the stagnation section, reducing productivity. More specifically, some cells remain in the retention area for a long time, leading to a decrease in cell viability and the accumulation and outflow of cell debris. As a result, EP efficiency and productivity decrease. By designing the expanded section 21a without a bend on its side surface, such a decrease can be suppressed. Furthermore, this configuration makes it easier for the flow velocity distribution of the suspension Q in the width direction of the main channel 20 to become uniform, allowing for uniform introduction efficiency of bioactive substances into bio-derived materials during EP, and thus increasing productivity. In addition, the flow of suspension Q becomes less turbulent, which is beneficial in the extension direction D of the main channel 20. Ｌ The distance from the suspension inlet 34a to the electrode pair 22 can be shortened, and the extension direction D of the flow channel device 12 ＬThis can suppress an increase in size. In addition, this can suppress an increase in the size of the electroporation apparatus 10. The uniformity of the flow velocity distribution of the suspension Q in the width direction of the main channel 20 can be confirmed, for example, by numerical analysis such as simulation, or by using a visualization device having the same shape as the main channel. The main channel 20 is not limited to a configuration having an enlarged portion 21a, and for example, as shown in Figure 3, the width of the main channel body portion 21b may be the same as the side surface 21g, and the overall contour shape of the main channel 20 may be rectangular. The contour shape of the main channel 20 can be obtained in the form of digital data by scanning the main channel 20 with a 2D scanner or a 3D scanner. If CAD (Computer Aided Design) data of the main channel 20 is available, the contour shape of the main channel 20 can be obtained in the form of digital data from the CAD data.

[0036] The width W of the main channel body 21b corresponds to the width of the main channel 20. The width W of the main channel body 21b is preferably 3 mm or more, and is more preferably 3 mm or more, 6 mm or more, 8 mm or more, 20 mm or more, 25 mm or more, and 60 mm or more, in that order. The upper limit of the width W of the main channel body 21b is 500 mm. The wider the main channel, the more uneven the flow velocity distribution becomes, which may reduce the EP efficiency, for example, the efficiency of introducing bioactive substances into bio-derived materials. However, in this embodiment, sufficient EP efficiency can be obtained even if the width of the main channel is wide. Also, the extension direction D of the enlarged portion 21a Ｌ The length is preferably 0.1 × width W or more, more preferably 0.3 × width W or more, and even more preferably 0.5 × width W or more. More specifically, 1 to 1000 mm is preferred. The lower limit depends on the width of the main flow path 20, but is preferably 3 mm, and more preferably 5 mm. The width W of the main flow path body 21b and the extension direction D of the enlarged portion 21a. Ｌ The lengths can be measured using calipers or similar tools, or they can be design values ​​from CAD data or similar sources.

[0037] (Sheath Liquid Flow Channel) Figure 4 is a schematic diagram showing a first example of a sheath liquid flow channel of a flow channel device according to an embodiment of the present invention. Figure 4 shows the contour shape of the sheath liquid flow channels 40 and 42 as viewed from the sheath liquid inlet 31a and 31b side. The contour shape of the sheath liquid flow channels 40 and 42 shown in Figure 4 shows the two-dimensional shape of the side surface constituting the sheath liquid flow channels 40 and 42. As shown in Figures 2 and 4, the sheath liquid flow channels 40 and 42 have a length L of the first flow channels 30c and 32c. １ The total length Ls of the sheath liquid expansion sections 30b and 32b, including Ld, is 0.8 times or more the width of the main flow path 20 between the electrode pair 22. That is, 0.8 ≤ Ls / W. By satisfying these conditions, the velocity distribution in the width direction of the sheath liquid flow becomes uniform between the electrode pair, allowing for the uniform introduction of bioactive substances into the bio-derived material in the suspension flow during EP, achieving high EP efficiency and high productivity. If the velocity distribution in the width direction of the sheath liquid flow is non-uniform, the velocity distribution in the width direction of the suspension flow becomes non-uniform, or the three-layered flow of the sheath liquid flow, suspension flow, and sheath liquid flow becomes unstable, resulting in decreased EP efficiency and reduced productivity. Also, widening the width of the main flow path tends to make the velocity distribution in the width direction non-uniform, worsening EP efficiency. However, by setting 0.8 ≤ Ls / W, even if the width of the main flow path is widened, the velocity distribution in the width direction of the sheath liquid flow path can be made uniform, and high EP efficiency can be achieved. In other words, by setting the flow path device 12 to 0.8 ≤ Ls / W, the EP processing rate can be improved and productivity can be increased.

[0038] Furthermore, it was confirmed by numerical calculation that the total length Ls mentioned above is 0.8 times or more the width of the main flow path 20 between the electrode pair 22, and that the velocity distribution in the width direction of the sheath fluid flow path is uniform. Ansys 2023 R2 (software name) from Ansys, Inc. was used for the numerical calculation. The physical properties of the sheath fluid were set to be the same as water, with a viscosity of 1 mPa·s (cp). For the numerical calculation, a calculation model was used in which the width of the sheath fluid flow path was 25 mm, the thickness was 0.5 mm, and the length Ld of the sheath fluid expansion section was 12.5 mm. Figure 5 shows the calculation model 50 in which the velocity of the sheath fluid flow path was determined. The calculation model 50 is a numerically calculated model created using the above-mentioned Ansys 2023 R2 (software name). The sheath fluid expansion section 50b of the calculation model 50 shown in Figure 5 has the configuration shown in Figure 6, which will be described later. The calculation model 50 shown in Figure 5 has an inlet section 51a corresponding to the sheath fluid inlet, a sheath fluid expansion section 50b corresponding to the sheath fluid expansion section, and a first flow path section 50c corresponding to the first flow path. The side surface 51e of the sheath fluid expansion section 50b has a circular arc contour shape.

[0039] Numerical calculation results show that the length G of the region corresponding to the first flow path is as shown in Figure 5. １It was confirmed that the velocity distribution becomes uniform at a length of 7.5 mm. That is, the velocity distribution becomes uniform when the total length Ls is 20 mm (= 12.5 mm + 7.5 mm), and the velocity distribution remained stable even in regions beyond that. When the width of the main channel is 25 mm, 0.8 W = Ls, satisfying 0.8 ≤ Ls / W. The line Gs shown in Figure 5 indicates the point where the total length Ls is 20 mm. In numerical calculations of velocity, different velocities within a certain range are displayed in specific colors. For example, the fastest velocity is displayed in red, and the slowest velocity is displayed in blue. Since the velocity is displayed in specific colors, the velocity distribution is displayed as color unevenness. The uniformity of the velocity distribution was judged from this color unevenness. In Figure 5, the velocity is displayed in grayscale from white to black, with the slower the velocity becoming darker and closer to black, and the faster the velocity becoming lighter and closer to white. In Figure 5, the flow velocity distribution is represented by variations in color intensity, with smaller differences in intensity indicating a smaller flow velocity distribution. As shown in Figure 5, near the inlet 51a, the difference in intensity is particularly large when viewed in the width direction Dw, and the flow velocity distribution in the width direction Dw is unstable in the region where 0.8 > Ls / W, i.e., on the side of the inlet 51a beyond the line Gs shown in Figure 5. Similarly, it was confirmed that the flow velocity distribution stabilizes when 0.8 ≤ Ls / W is satisfied, even when the width of the main channel is 6 mm, 8 mm, and 20 mm.

[0040] As described above, the total length Ls is 0.8 times or more the width of the main flow path 20 between the electrode pair 22, but preferably 1 time or more, more preferably 1.2 times or more, even more preferably 1.4 times or more, with an upper limit of, for example, 10 times. If the total length Ls is long, the flow path device 12 becomes larger, so it is preferable that the total length Ls be short. If the thickness of the first flow path is thick, the distance over which the flow velocity distribution in the width direction of the sheath liquid flow becomes uniform tends to be longer. For this reason, it is preferable that the thickness of the first flow path be thin. The total length Ls is the length Ld of the sheath liquid expansion portion and the length L of the first flow path. １ This is the total length of the sheath fluid expansion section Ld and the length of the first flow path L. １ These can be actual measured values ​​using calipers or similar tools, or design values ​​such as CAD data.

[0041] The sheath fluid expansion portions 30b and 32b of the sheath fluid passages 40 and 42 are not particularly limited, but it is preferable that there are no bends on the side surface 31c (see Figure 4) of the sheath fluid expansion portions 30b and 32b. The contour shape of the sheath fluid passages 40 and 42 shown in Figure 4 is the same as the contour shape of the main passage 20 shown in Figure 3. The contour shape of the sheath fluid passages 40 and 42 in Figure 4 passes through the center of the sheath fluid inlets 31a and 31b, and the extension direction D of the sheath fluid expansion portions 30b and 32b, the first passages 30c and 32c, and the direction changing portions 30d and 32d of the sheath fluid passages 40 and 42. Ｌ The shape is symmetrical with respect to the center line Cs parallel to the first channel 30c, 32c and the side surfaces of the direction changing sections 30d, 32d are straight. Also, in Figure 4, the sheath liquid inlets 31a, 31b are on the upstream side of the sheath liquid channels 40, 42, and the extension direction D of the sheath liquid inlets 31a, 31b Ｌ The opposite side of this is the downstream side of the sheath fluid passages 40 and 42. Since the sheath fluid passage 40 and the sheath fluid passage 42 have the same configuration, the sheath fluid passage 40 will be described as a representative example, and the description of the sheath fluid passage 42 will be omitted.

[0042] The sheath fluid channel 40 has a flat side surface 31c of the sheath fluid expansion section 30b. For example, as shown in Figure 4, the sheath fluid expansion section 30b extends in the width direction Dws of the first channel 30c (second channel 30e) starting from the sheath fluid inlet 31a. The connection section 31d between the sheath fluid expansion section 30b and the first channel 30c is made of a curved surface, and the sheath fluid expansion section 30b and the first channel 30c are connected continuously without any bends. The width direction Dws is the extension direction D in which the sheath fluid channel 40 extends. Ｌｓ It is in a direction perpendicular to the given direction.

[0043] The configuration of the sheath fluid expansion portions 30b and 32b of the sheath fluid passages 40 and 42 is not limited to the configuration shown in Figure 4 above. For example, the entire side surface of the sheath fluid expansion portions 30b and 32b of the sheath fluid passages 40 and 42 can be composed of a single curved surface or multiple curved surfaces. Here, Figure 6 is a schematic diagram showing a second example of the sheath fluid passage of the flow channel device according to an embodiment of the present invention. Figure 7 is a schematic diagram showing a third example of the sheath fluid passage of the flow channel device according to an embodiment of the present invention. In Figures 6 and 7, the same components as those in the sheath fluid passages 40 and 42 shown in Figure 4 are denoted by the same reference numerals, and their detailed descriptions are omitted. Figures 6 and 7 show the contour shape of the sheath fluid passages 40 and 42 as viewed from the sheath fluid inlet 31a and 31b side. The contour shape of the sheath fluid passages 40 and 42 shown in Figures 6 and 7 shows the two-dimensional shape of the side surface constituting the sheath fluid passages 40 and 42.

[0044] In Figures 6 and 7, the contour shape of the sheath fluid channels 40 and 42 is such that it passes through the center of the sheath fluid inlets 31a and 31b, and extends in the direction D of the sheath fluid expansion sections 30b and 32b, the first channels 30c and 32c, and the direction changing sections 30d and 32d of the sheath fluid channels 40 and 42. Ｌ The shape is symmetrical with respect to a center line Cs parallel to it. In the sheath fluid channels 40 and 42 shown in Figure 6, the entire side surface 31e of the sheath fluid expansion section 30b and 32b is composed of a single curved surface. The contour shape of the side surface 31e of the sheath fluid expansion section 30b and 32b is an arc, and for example, the contour shape can be represented by a quadratic function. Note that elliptical arcs are also included in the arc. In the sheath fluid channels 40 and 42 shown in Figure 6, the end 31g of the curved surface constituting the side surface 31e of the sheath fluid expansion section 30b and 32b is connected to the first channels 30c and 32c, and the tangent (not shown) of the end 31g of the side surface 31e coincides with the tangent (not shown) of the side surface of the first channels 30c and 32c. For this reason, the sheath fluid expansion section 30b and 32b and the first channels 30c and 32c are connected continuously without any bends.

[0045] The sheath fluid channels 40 and 42 shown in Figure 7 are composed of multiple curved surfaces on the entire side surface 31f of the sheath fluid expansion sections 30b and 32b. For example, the side surface 31f of the sheath fluid expansion sections 30b and 32b is composed of two curved surfaces 47a and 47b combined. The curved surfaces 47a and 47b are connected such that the connection point 47c between them is an inflection point. The contour shape of the side surface 31f of the sheath fluid expansion sections 30b and 32b shown in Figure 7 can be represented, for example, by a cubic function, a trigonometric function, an inverse trigonometric function, or a sigmoid function. In Figure 7, the sheath fluid channels 40 and 42 are connected to the end 31g of the curved surface 47b that constitutes the side surface 31f of the sheath fluid expansion section 30b and 32b, and the first channels 30c and 32c are connected to this end 31g of the curved surface 47b. The tangent to the end 31g of the curved surface 47b coincides with the tangent to the side surface of the first channels 30c and 32c. Therefore, the sheath fluid expansion section 30b and 32b of the curved surface 47b and the first channels 30c and 32c are connected continuously without any bends.

[0046] In the sheath liquid channels 40 and 42, as shown in Figures 4 to 7 above, by configuring the sheath liquid expansion sections 30b and 32b without bends on their sides, the flow of the sheath liquid s in the second channels 30e and 32e becomes less turbulent, and the flow velocity of the sheath liquid s in the width direction of the sheath liquid channels 40 and 42 becomes uniform over a short distance. As a result, during EP, the three-layered flow of the sheath liquid flow, suspension flow, and sheath liquid flow is stabilized, and an electric field can be appropriately applied to the suspension Q. Consequently, the introduction efficiency and the utilization efficiency of bioactive substances are increased, improving productivity. Furthermore, since the thickness of the sheath liquid flow and the thickness of the suspension Q become uniform between the electrode pair 22, the amount of suspension Q discarded is reduced, and a highly productive EP can be performed. The uniformity of the sheath liquid flow velocity distribution in the width direction of the sheath liquid channels 40 and 42 can be confirmed, for example, by numerical analysis such as simulation, or by using a visualization device with a shape similar to that of the sheath liquid channels. Furthermore, the contour shapes of the sheath fluid channels 40 and 42 can be obtained in digital data form by scanning the sheath fluid channels 40 and 42 with a 2D scanner or a 3D scanner. Alternatively, if CAD data of the sheath fluid channels 40 and 42 is available, the contour shapes of the sheath fluid channels 40 and 42 can be obtained in digital data form from the CAD data. The width W of the first channels 30c and 32c. １ The width of the first flow channels 30c and 32c is preferably 3 mm or more, and more preferably in the order of 3 mm or more, 6 mm or more, 8 mm or more, 20 mm or more, 25 mm or more, and 60 mm or more. １ The upper limit is 500 mm. The width W of the first flow channels 30c and 32c. １ This can be an actual measurement using calipers or similar equipment, or it can be a design value such as CAD data.

[0047] The sheath fluid channels 40 and 42 are configured to have sections with different thicknesses. When the thickness of the sheath fluid channels 40 and 42 is thick, the distance required to achieve a uniform velocity distribution in the width direction of the sheath fluid tends to be longer. Therefore, by providing sections with thinner thicknesses, the length of the sheath fluid channels can be adjusted and shortened. In other words, by configuring the sheath fluid channels 40 and 42 to have sections with different thicknesses, the length of the channels required to achieve a uniform velocity distribution in the width direction of the sheath fluid channels 40 and 42 can be shortened, thereby suppressing the need to increase the size of the flow channel device 12.

[0048] More specifically, in the sheath fluid channels 40 and 42, the thickness t of the first channels 30c and 32c １ And the thickness t of the second channels 30e and 32e ２ It is preferable that it be different from the above. In this case, the thickness t of the first flow channels 30c and 32c １ <Thickness t of the second flow channels 30e and 32e> ２ However, the thickness t of the first channels 30c and 32c １ > Thickness t of the second flow channels 30e and 32e ２ However, this is also acceptable. The first channels 30c and 32c have the function of making the flow velocity distribution uniform in the width direction. The second channels 30e and 32e have the function of making the flow velocity distribution uniform in the thickness direction. Thickness t of the first channels 30c and 32c １ > Thickness t of the second flow channels 30e and 32e ２ In this case, the length L of the first channels 30c and 32c is used to make the flow velocity distribution in the width direction of the sheath fluid channels 40 and 42 uniform. １ This can shorten the length and make the flow channel device 12 smaller. Note that the thickness t of the second flow channels 30e and 32e ２ If the first channels 30c and 32c can be made thin enough to create a uniform flow velocity distribution in the width direction, then a configuration without these channels is also possible.

[0049] Thickness t of the first channel 30c, 32c １ The thickness is preferably 2 mm or less, more preferably 1 mm or less, and particularly preferably 0.5 mm or less. The lower limit is 0.1 mm. Here, the thickness of the main channel 20 corresponds to the distance D between the electrode pairs shown in Figure 1. Thickness t of the first channels 30c and 32c １The thickness of the second channels 30e and 32e is thinner than the thickness of the main channel 20. ２ Due to processing constraints, the thickness t of the first flow channels 30c and 32c is 2 mm or more. １ Similarly, a thinner thickness is preferable, and the preferred range is the same. Thickness t of the second flow channels 30e, 32e ２ The upper limit is, for example, 4 mm. Thickness t of the first flow channels 30c, 32c １ And the thickness t of the second channels 30e and 32e ２ For example, t １ <t ２ In the case of t １ <t ２ / 2, t １ <t ２ It can also be set to / 4. Thickness t of the first channels 30c, 32c １ , and the thickness t of the second flow channels 30e, 32e ２ These can be actual measured values ​​using calipers or similar tools, or design values ​​such as CAD data.

[0050] In the flow channel device 12, there are no restrictions on the angle formed by the suspension flow and the sheath liquid flow when the suspension flow and the sheath liquid flow are merged, that is, the angle formed by the main flow channel 20 and the second flow channels 30e and 32e, but it is preferable that it be small. Specifically, the extension direction D of the main flow channel 20. Ｌ Furthermore, the angle formed by the extending direction of the second flow path 30e and the extending direction of the second flow path 32e is preferably 90° or less, more preferably 60° or less, even more preferably 45° or less, and even more preferably 30° or less. Also, when the suspension flow and the sheath liquid flow are joined, it is preferable that the difference in flow velocity between the two is small. Specifically, when the suspension flow and the sheath liquid flow are joined, the flow velocity of the sheath liquid flow is preferably ±10% or less of the flow velocity of the suspension flow, more preferably ±5% or less, even more preferably ±3% or less, and most preferably ±0%, that is, the suspension flow and the sheath liquid flow are at the same velocity.

[0051] The first electrode 23 of the electrode pair 22 is electrically connected to the power supply unit 14 by wiring 14a. The second electrode 24 is electrically connected to the power supply unit 14 by wiring 14b. In Figure 1, to show the connection between the power supply unit 14 and the first electrode 23 and the second electrode 24, wiring 14a is shown passing through the first introduction section 36 and the first substrate 30, and wiring 14b is shown passing through the second introduction section 38 and the second substrate 32. For example, an outlet window (not shown) for the first electrode 23 is provided in the first introduction section 36 and the first substrate 30 to expose the first electrode 23. A connection terminal (not shown) is electrically connected to the exposed portion (not shown) of the first electrode 23, and then wiring 14a is electrically connected to the connection terminal. Also, an outlet window (not shown) for the second electrode 24 is provided in the second introduction section 38 and the second substrate 32 to expose the second electrode 24. A connection terminal (not shown) is electrically connected to the exposed portion (not shown) of the second electrode 24, and then a wire 14b is electrically connected to the connection terminal. In this way, the first electrode 23 and the wire 14a are electrically connected, and the second electrode 24 and the wire 14b are electrically connected. A voltage is applied to the electrode pair 22 by the power supply unit 14. That is, the power supply unit 14 applies a voltage to the opposing first electrode 23 and second electrode 24. The configuration of the power supply unit 14 is not particularly limited, as long as it can apply a pulse voltage to the electrode pair 22 and adjust the pulse width and pulse period. For example, a pulse power supply can be used as the power supply unit 14.

[0052] The control unit 15 is connected to the pumps 16a to 16d and the power supply unit 14. The control unit 15 controls the operation of the pumps 16a to 16d. The control unit 15 also adjusts the magnitude, pulse width, and pulse period of the voltage applied by the power supply unit 14 to the electrode pair 22. The control unit 15 also adjusts the timing of the operation of the pumps 16a to 16d and the timing of the voltage application by the power supply unit 14 to the electrode pair 22. The control unit 15 may be configured as a computer that functions by executing a program, or as a dedicated device configured with a dedicated circuit. The control unit 15 may be configured to be located separately from the flow path device 12. It may also be configured to be remotely controlled by the control unit 15. The configuration of remote control by the control unit 15 is not particularly limited and known configurations can be used. The above-mentioned computer and dedicated device have, for example, a processor. The processor may be configured as one or more hardware components, and the type of hardware is not limited. For example, a processor may consist of programmable logic devices such as a CPU (Central Processing Unit), MPU (Micro Processing Unit), FPGA (Field Programmable Gate Array), dedicated circuits for performing specific processing such as an ASIC (Application Specific Integrated Circuit), and hardware such as a GPU (Graphic Processing Unit) or NPU (Neural Processing Unit).

[0053] Here, Figure 8 is a schematic cross-sectional view illustrating the operation of the flow channel device in an electroporation apparatus having a flow channel device according to an embodiment of the present invention. In Figure 8, the same components as those in the flow channel device 12 shown in Figure 1 are denoted by the same reference numerals, and their detailed descriptions are omitted. Also, in Figure 8, the power supply unit 14, wiring 14a, 14b, control unit 15, pumps 16a to 16d, tubes 19, piping 26, 26a to 26d, mixer 27, culture apparatus 28, tank 29, and tank 45 shown in Figure 1 are omitted from the illustration.

[0054] As conceptually shown in Figure 8, when suspension Q and sheath liquid s are supplied to the flow channel device 12, upstream of the electrode pair 22, sheath liquid flows consisting of sheath liquid s are formed on both sides in the opposite direction to the suspension flow of suspension Q, forming a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, and this three-layered flow flows between the electrode pair 22. In the flow channel device 12, suspension Q and sheath liquid s are continuously supplied between the electrode pair 22, i.e., into the space 25 described above, in a state where they form a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow. By applying, for example, a pulsed electric field to the suspension Q in the state of the three-layered flow using the first electrode 23 and the second electrode 24, processing by continuous liquid supply type EP using sheath liquid flow as described above can be performed. In this case, the thickness Dm of the suspension flow is maintained at 1 to 10 mm at least between the electrode pair 22 and at the upstream end of the electrode pair 22. After processing by EP, the sheath liquid s and suspension Q are discharged from the outlet 18.

[0055] As described above, the sheath liquid s supplied from the first supply port 43a slopes downstream and flows into the main flow path 20 via the sheath liquid confluence port 30f. On the other hand, the sheath liquid s supplied from the second supply port 43b slopes downstream and flows into the main flow path 20 via the sheath liquid confluence port 32f. As described above, the thickness of the suspension flow 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 liquid flow less than or equal to the thickness of the suspension flow, the dilution ratio of the suspension after EP can be reduced to 2 times or less.

[0056] Furthermore, there is no limit to the thickness ds of the sheath liquid flow, but the thickness of the sheath liquid flow at least between the electrode pair 22 and at the upstream end of the electrode pair 22 is preferably 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.5 mm or more. By making the sum of the thicknesses of the two sheath liquids less than or equal to the thickness Dm of the suspension flow, the dilution ratio of the suspension after continuous liquid delivery EP can be reduced to 2 times or less. For this reason, it is preferable that the sum of the thicknesses ds of the two sheath liquid flows be less than or equal to the thickness Dm of the suspension flow. The thickness Dm of the suspension flow is the maximum length of the suspension flow in the stacking direction Ds of the main flow channel 20. The thickness ds of the sheath liquid flow is the maximum length of the sheath liquid flow in the stacking direction Ds of the main flow channel 20. The thickness Dm of the suspension flow (see Figure 8) and the thickness ds of the sheath liquid flow (see Figure 8) are adjusted by the flow rate of the suspension Q and the flow rate of the sheath liquid s. More specifically, the thickness of the suspension flow can be measured using a visualization device (not shown) having a shape similar to that of the flow channel device. The suspension and water are flowed through the visualization device at predetermined flow rates. Since the suspension and water have different colors, the thickness of the suspension flow can be measured. Based on the flow rates of the suspension and water and the information on the thickness of the suspension flow at that time, the thickness of the suspension flow can be adjusted by changing the flow rates of the suspension flow and the sheath liquid flow. Note that the configuration of the EP apparatus may be the one described in PCT / JP / 2025 / 016650 or International Publication No. 2024 / 248133.

[0057] (First example of electroporation method) The first example of the electroporation method (EP method) is a continuous liquid delivery type EP, for example, the EP device 10 shown in Figure 1 is used. The first example of the EP method is a method of introducing a bioactive substance into a bio-derived substance by continuously delivering a suspension Q containing a bio-derived substance and a bioactive substance, and a sheath liquid s, between the electrode pair 22, i.e., in the space 25 described above, to the main flow path 20 such that the suspension Q is sandwiched between the sheath liquid s, and applying an electric field to the suspension Q with the electrode pair 22. In the first example of the EP method, the culture medium in the culture device 28 is drawn up by the pump 16a of the EP device 10, and the bioactive substance in the tank 29 is drawn up by the pump 16b, and without changing the culture medium of the culture medium with an EP buffer, the culture medium in the culture device 28 and the bioactive substance in the tank 29 are mixed in the mixer 27 to form a suspension Q. Pump 16a causes suspension Q to flow from piping 26 through suspension supply port 17, through hole 36a of the first introduction section 36, and through hole 30a of the first substrate 30 into the main flow path 20 from suspension inlet 34a. At this time, suspension Q is supplied to the main flow path 20 of the flow path device 12 at a rate of 5 mL (milliliters) / min or more. Pump 16c causes sheath liquid s in tank 45 to flow from piping 26a through the first supply port 43a and through the sheath liquid flow path 40 into the main flow path 20 from sheath liquid confluence port 30f. Pump 16d causes sheath liquid s in tank 45 to flow from piping 26b through the second supply port 43b and through the sheath liquid flow path 42 into the main flow path 20 from sheath liquid confluence port 32f. At the confluence section 44, the suspension flow and the sheath liquid flow merge to form a three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, and this layered flow flows continuously between the electrode pair 22 in a vertical direction from bottom to top. At this time, the power supply unit 14 applies, for example, a pulse voltage to the electrode pair 22 to perform EP. In the EP method, as described above, the flow channel device 12 has a configuration in which there is no bend on the side surface 21c of the enlarged portion 21a of the main flow channel 20, so productivity can be increased. In addition, the flow velocity of the suspension Q in the width direction of the main flow channel 20 becomes uniform over a short distance, so that the efficiency of introducing bioactive substances into bio-derived materials can be properly controlled during EP.Furthermore, since the sheath liquid flow channels 40 and 42 have no bends on the side surfaces 31c of the sheath liquid expansion sections 30b and 32b, the three-layered flow of sheath liquid flow, suspension flow, and sheath liquid flow is stable during EP. The thickness of the sheath liquid flow and the suspension Q become uniform between the electrode pair 22, and an electric field can be properly applied to the suspension Q. As a result, the introduction efficiency and the utilization efficiency of bioactive substances are increased, and productivity is improved.

[0058] The EP method may include, for example, a culture step in which a culture medium containing a biological substance and culture medium is obtained by cell culture before performing EP, and a mixing step in which the culture medium and a bioactive substance are mixed to form a suspension.

[0059] The contour shape of the enlarged portion 21a of the main channel 20 described above can be determined, for example, by using an optimization calculation that finds the shortest length over which the velocity distribution of the suspension Q in the width direction of the main channel 20 is uniform. Similarly, the contour shapes of the sheath liquid enlarged portions 30b and 32b of the sheath liquid channels 40 and 42 described above can also be determined, for example, by using an optimization calculation that finds the shortest length over which the velocity distribution of the sheath liquid s in the width direction of the sheath liquid channels 40 and 42 is uniform. In the optimization calculation, for example, a condition is set that there are no bends on the side surface of the enlarged portion. Multiple shapes that meet this condition are set using the Monte Carlo method. For example, if the contour shape is represented by a function, a function representing the contour shape is set, and multiple shapes that meet the condition are set by changing the parameters of the function. Also, an evaluation criterion for uniform velocity distribution is set. For each set shape, the velocity is calculated using numerical analysis, and the length over which the velocity distribution is uniform is determined using the evaluation criterion described above. For example, among the set shapes, the one with the shortest length over which the velocity distribution is uniform is selected as the contour shape. In this way, the flow velocity can be calculated while changing the shape to suit the conditions, and the contour shape can be explored. The method for setting the suitable shape is not limited to the Monte Carlo method, and known methods can be used as appropriate, for example, experimental design can be used. Examples of conditions for optimization calculation include the viscosity of the suspension and the viscosity of the sheath fluid, in addition to the absence of bends on the side surface of the enlarged section mentioned above. The contour shape of the enlarged section 21a of the main channel 20 can be set by a person, and the flow velocity can be calculated using numerical analysis for each contour shape. Then, a person can look at the results of the numerical calculation of the flow velocity for each contour shape and explore the contour shape that has the shortest length over which the flow velocity distribution of the suspension Q in the width direction of the main channel 20 is uniform. The contour shapes of the sheath fluid enlarged sections 30b and 32b of the sheath fluid channels 40 and 42 can also be explored by a person in the same way as the contour shape of the enlarged section 21a of the main channel 20 mentioned above. The parts of the EP device will be described below.

[0060] [Distance D between electrode pairs] The distance D between electrode pairs 22 (the distance between the electrode surface 23a of the first electrode 23 and the electrode surface 24a of the second electrode 24) is preferably 1 to 10 mm. The lower limit of the distance D between electrode pairs is more preferably 2 mm, and even more preferably 3 mm. The upper limit of the distance D between electrode pairs is more preferably 6 mm, and even more preferably 5 mm. If the distance D between electrode pairs becomes too large, an excessive electric field will be generated, causing heat generation, electric field concentration at the electrode ends, boiling or discharge, which may reduce the survival rate or introduction efficiency. Furthermore, a distance D between electrode pairs of 1 to 3 mm is even more preferable because it suppresses the generation of heat generation and discharge, and also suppresses the reduction in the survival rate or introduction efficiency of biologically derived substances such as cells due to heat generation or discharge. Note that the distance D between electrode pairs corresponds to the thickness of the flow channel substrate 34 (see Figure 1). The distance D between electrode pairs is also called the electrode gap or gap. The above-mentioned distance D between electrode pairs can be measured by measuring the length of the relevant portion using calipers or a micrometer.

[0061] <Electrode Pair> The first electrode 23 and the second electrode 24 constituting the electrode pair are made of, for example, a metallic material or a carbon material. Specifically, a configuration similar to that of the electrodes described in International Publication No. 2023 / 157673 can be adopted.

[0062] (Thickness) The thickness of the first electrode 23 and the second electrode 24 are not particularly limited, but are preferably 0.5 to 10 mm, and more preferably 1 to 5 mm. The thickness of the first electrode 23 and the second electrode 24 can both be measured using a caliper or micrometer.

[0063] <Configuration of the flow channel device> The materials constituting the first introduction section 36, first substrate 30, flow channel substrate 34, second substrate 32, and second introduction section 38 of the flow channel device 12 in Figure 1 are not particularly limited. Various materials can be used as these components, for example, as long as they have sufficient resistance to suspensions, or suspensions and sheath fluids, and can ensure the necessary rigidity.

[0064] <Suspension> The suspension to be subjected to electroporation (EP) contains biologically derived substances and bioactive substances. Furthermore, the suspension does not have to be one in which the culture medium has been replaced with EP buffer or to which EP buffer has been added. That is, the suspension does not contain EP buffer, nor does it substantially contain salt (e.g., sodium chloride), nor does it substantially contain poloxamer. The suspension preferably has an electrical conductivity of 2 mS / cm or more, more preferably 3 mS / cm or more, and even more preferably 4 mS / cm or more. Normally, before EP, the culture medium in the suspension is replaced with EP buffer, so the culture medium in the suspension is replaced with EP buffer, the electrical conductivity of the suspension decreases, and the generation of bubbles associated with the application of the electric field is reduced. A suspension with the above electrical conductivity is usually not usable for EP, but it can be used in this embodiment. The electrical conductivity (mS / cm) is the value obtained by measuring the suspension at a temperature of 25°C using an electrical conductivity meter. Electrical conductivity (mS / cm) is synonymous with electrical conductivity, and both electrical conductivity and electrical conductivity share the same unit. Methods for adjusting electrical conductivity include, for example, adjusting the volume fraction of biological substances contained in the suspension, and changing the type of culture medium.

[0065] (Biologically Derived Substances) Biologically derived substances are not particularly limited, but specific examples include cells, organelles, intracellular granules and vesicles, and bacteria. Among these, cells are preferred, animal cells are more preferred, mammalian cells are even more preferred, and human or Chinese hamster-derived cells are most preferred, for the reasons that the effects of the present invention are superior. Specific examples of cells include human T cells, HEK (Human Embryonic Kidney) 293, A549, SF9, EB66, Daudi, Hela, Vero, MDCK, BHK (Baby Hamster Kidney), CHO (Chinese Hamster Ovary), NS0, SP2 / 0, hybridomas, etc. In pharmaceutical manufacturing, gene transfer using HEK293 or CHO is most commonly used.

[0066] (Bioactive Substances) Bioactive substances are substances that, when introduced into biologically derived substances such as nucleic acids (e.g., DNA, RNA) and proteins, exert some effect on the biologically derived substances. Examples of nucleic acids include plasmids, linear DNA, and mRNA (messenger RNA), with plasmids being particularly preferred. The concentration of the bioactive substance in the suspension is preferably 1 to 1000 μg / mL, more preferably 2.5 to 500 μg / mL, and even more preferably 10 to 200 μg / mL. The concentration of the bioactive substance in the suspension relative to the culture medium is preferably 10 to 500 μg / mL.

[0067] There are no restrictions on the concentration of bio-derived substances in suspension Q, but it is preferable that the volume fraction of bio-derived substances in suspension Q be 20% or more. In EP, increasing the concentration of bio-derived substances in suspension Q allows for a larger processing volume of bio-derived substances compared to introducing the same amount of lower-concentration suspension. For example, if the concentration is doubled, twice the processing can be performed, improving the throughput of EP and the utilization efficiency of bio-derived substances. In addition, in EP, as described above, bioactive substances are incorporated into the bio-derived substances by electrophoresis. Therefore, even if the concentration of bio-derived substances is increased, it is not necessary to increase the amount of bioactive substances accordingly, thus improving the utilization efficiency of bioactive substances.

[0068] By having a volume fraction of biologically derived materials in suspension Q of 20% or more, the above-mentioned effects can be suitably obtained, and efficient processing can be achieved. Furthermore, if the concentration of suspension Q is too high, the viscosity of the cell suspension will increase rapidly, potentially causing the flow path to become clogged. However, by keeping the volume fraction of biologically derived materials in suspension Q to 70% or less, clogged flow paths caused by excessively high concentrations of suspension Q can be suitably prevented. The volume fraction of biologically derived materials in suspension Q is more preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. The upper limit of the volume fraction of biologically derived materials in suspension Q is more preferably 65%, and even more preferably 60%. The volume fraction of biologically derived materials in suspension Q is determined by measuring the average diameter and concentration (number of cells per unit volume) of the biologically derived materials using image analysis (e.g., Vi-CELL XR (Beckman Coulter), etc.), and calculating the volume fraction (= 4 ÷ 3 × (average diameter ÷ 2)). 3 The volume fraction can be calculated as (π × concentration × 100 (%)). Note that the volume fraction is calculated assuming that the biological material is spherical. More specifically, for example, if the average diameter of the biological material in suspension Q is 20 μm and the concentration is 40 × 10 ６ If the concentration is per mL (milliliters), the volume fraction of biologically derived substances in suspension Q can be calculated as 17%.

[0069] As described above, in the first example of the EP method, suspension Q is supplied at a rate of 1 mL / min or more. The upper limit of the flow rate of suspension Q is not particularly limited, but for example, it is 1000 mL / min. The flow rate of the suspension is more preferably 2 mL / min or more, even more preferably 3 mL / min or more, even more preferably 4 mL / min or more, even more preferably 5 mL / min or more, even more preferably 8 mL / min or more, even more preferably 10 mL / min or more, even more preferably 12 mL / min or more, even more preferably 15 mL / min or more, even more preferably 20 mL / min or more, even more preferably 25 mL / min or more, even more preferably 30 mL / min or more, and even more preferably 35 mL / min or more. The upper limit of the flow rate of the suspension by the pump is preferably 1000 mL / min.

[0070] As the culture medium, liquid culture media commonly used for culturing animal cells can be used. Various companies sell culture media optimized for cell culture, such as HEK293 based on PBS (phosphate-buffered saline). The culture medium contains, but is not particularly limited to, amino acids, salts, sugars (glucose, etc.), vitamins, hormones, growth factors, lipids, trace elements, etc. The pH (hydrogen ion concentration) of the culture medium is 6 to 8, preferably 6.8 to 7.6, and more preferably 7.2 to 7.6.

[0071] <Sheath Fluid> Any liquid other than a suspension can be used as the sheath fluid for the sheath fluid flow. Among these, culture media used for cell culture are preferred as the sheath fluid. As mentioned above, there are two sheath fluid flows. It is preferable that the flow rate of each of the two sheath fluid flows be 0.1 mL / min or more. It is preferable that the upper limit of the sheath fluid flow rate is less than or equal to the flow rate of the suspension. For example, the flow rate of the sheath fluid is adjusted by a pump that delivers the sheath fluid. Setting the sheath fluid flow rate to 0.1 mL / min or more is preferable because it effectively prevents the suspension from coming into contact with the electrode. It is also preferable to set the sheath fluid flow rate to less than or equal to the flow rate of the suspension because it suppresses the amount of sheath fluid used and suppresses the dilution of the suspension.

[0072] In electrophoresis (EP), when an electric field is applied, pores open in the membrane (or shell) of the bio-derived material, and bioactive substances enter the bio-derived material by electrophoresis. The pores formed in the membrane of the bio-derived material gradually close over time, but if the external liquid components (osmotic pressure, ion concentration, etc.) of the bio-derived material change abruptly due to operations such as dilution of the suspension before the pores close, the movement of ions and the medium through the pores can cause abrupt changes in the component ratio within the bio-derived material, as well as damage such as deformation and expansion. As a result, for example, in the case of introducing substances into cells, the viability of the bio-derived material may decrease, potentially lowering the introduction efficiency and utilization efficiency of bioactive substances in EP. If the thickness ds of the sheath liquid flow is greater than the thickness Dm of the suspension flow, and the dilution ratio of the suspension Q is high, for example, in the case of introducing substances into cells, the viability of the bio-derived material may decrease (it may be damaged), and the EP efficiency may decrease. The inventors' investigations revealed that, for example, when diluting a cell suspension with a culture medium sheath liquid stream, a significant decrease in cell viability was often observed when the dilution ratio was more than twofold. Therefore, as described above, by keeping the total thickness of the sheath liquid stream at the point of merging with the suspension stream below the thickness of the suspension stream, i.e., by keeping the dilution ratio of the suspension Q to twofold or less, it becomes possible to suppress the inconvenience caused by the mixing of a large amount of sheath liquid s with biological materials, such as a decrease in the viability of biological materials in the case of substance introduction into cells, and to perform efficient processing.

[0073] To more favorably obtain this effect, the total thickness of the sheath liquid flow at the time of junction with the suspension flow is preferably 1 time or less, more preferably 0.8 times or less, and even more preferably 0.5 times or less, compared to the thickness of the suspension liquid, as described above. That is, the dilution ratio of the suspension Q downstream of the electrode pair is preferably 2 times or less, more preferably 1.8 times or less, and even more preferably 1.5 times or less, as described above.

[0074] <Electroporation Voltage> The electroporation voltage is set so that the electric field applied to the suspension between the electrode pair reaches the desired electric field strength, taking into account the thickness of the suspension flow, the electrical conductivity of the suspension flow, the thickness of the sheath liquid flow, and the electrical conductivity of the sheath liquid flow. It is preferable that the electrical resistance of the electrodes is sufficiently lower than the resistance of the suspension and sheath liquid, as this may cause a decrease in the introduction efficiency and utilization efficiency of bioactive substances in electroporation. The optimal value of the electric field applied to the suspension varies depending on the type and size of the bio-derived material, but is usually around 100 to 2000 V / cm. The voltage is preferably a pulse voltage. In addition, a bipolar pulse (alternating positive and negative) may be used to homogenize the electrode reaction (gas generation due to electrolysis, electrode degradation, etc.). The period of the electric field applied to the suspension is preferably a pulse period.

[0075] <Pulse width> The optimal pulse width varies depending on the type of biological material, but is usually 0.1 to 100 ms (milliseconds), preferably 1 to 10 ms.

[0076] <Pulse Period (Pulse Interval)> It is preferable to synchronize the pulse period with an integer multiple of the pulse period to the time it takes for the biological material to pass through the electrodes (electrode length L (see Figure 1)). For example, while the biological material contained in the suspension passes through the electrodes (electrode length L), a pulse voltage is applied to the suspension between the electrodes 1 to 5 times, preferably 1 to 2 times, and more preferably only once. Applying a pulse voltage is equivalent to applying an electric field. From the viewpoint of ensuring that the next pulse voltage is applied after any bubbles generated between the electrodes have been removed, it is preferable to apply a pulse voltage once while the biological material is passing through the electrodes (electrode length L).

[0077] Here, the pulse period (pulse interval) is the period (time) from the point where a signal representing a pulse voltage transitions from a low level to a high level (higher in absolute value than the low level) and from a high level to a low level, through the low level, to the point where the signal transitions from low to high level again. Note that the high level of the signal in the pulse period is an absolute value and can be a positive value higher than the low level, or a negative value lower than the low level. The period (time) from the point where a signal representing a pulse voltage transitions from a low level to a high level, through the low level, to the point where the signal transitions from low to high level again is one pulse period. The absolute value of the high level of the signal representing the pulse voltage corresponds to the voltage value of the pulse voltage. The pulse voltage can be positive or negative, and in either case, it is considered a high level. A low level of the signal representing the pulse voltage represents a state where no pulse voltage is applied, for example, it corresponds to 0V. Furthermore, the time (period) between the starting point of the signal transition from a low level to a high level and the point in time when the signal transitions from a high level to a low level is called the pulse width. The pulse width corresponds to the period (time) during which the pulse voltage is applied. In one cycle of the pulse period, the period (time) other than the pulse width corresponds to the period (time) during which the pulse voltage is not applied.

[0078] The time it takes for a biological substance to pass through an electrode (electrode length L) has a distribution corresponding to the flow velocity distribution of the suspension in the channel, and is determined by the flow rate (flow velocity) of the suspension, the shape of the channel cross-section, the channel cross-sectional area, and the position within the channel cross-section. If the shape of the channel cross-section is a rectangle with all interior angles at 90°, the channel cross-sectional area is (electrode width) × (distance D between electrode pairs). The distance D between electrode pairs is shown in Figure 1. The time it takes for the biological substance to pass through the electrode (electrode length L) described above may be the average time it takes for the biological substance to pass through the electrode (electrode length L), or it may be the shortest time it takes for the biological substance to pass through the electrode (electrode length L) in the center of the channel where the flow velocity is fastest. The average time (Time A) for the biological substance to pass through the electrode (electrode length L) can be calculated by dividing the electrode length L by the average flow velocity vA (= flow rate / channel cross-sectional area) obtained from the flow rate of the suspension and the channel cross-sectional area (Time A = electrode length L / average flow velocity vA). Furthermore, the shortest time (Time M) for biological material to pass through the electrode (electrode length L) in the center of the flow path where the flow velocity is fastest can be calculated by determining the central flow velocity vM using visualization experiments such as flow path tracking methods or fluid simulations, and then dividing the electrode length L by the central flow velocity vM (Time M = electrode length L / central flow velocity vM). The average flow velocity vA is 0.03 to 14.3 cm / sec, preferably 0.2 to 5.7 cm / sec, and more preferably 0.5 to 2.5 cm / sec. The central flow velocity vM is 0.1 to 14.3 cm / sec, preferably 0.5 to 7.2 cm / sec, and more preferably 1 to 3.6 cm / sec.

[0079] Preferably, the pulse period Time I and the average time Time A for the bio-derived material to pass through the electrode (electrode length L) satisfy the following relationship: 0.3 × Time A ≤ Time I < Time A More preferably, the following relationship is satisfied: 0.4 × Time A × ≤ Time I ≤ 0.8 × Time A Even more preferably, the following relationship is satisfied: 0.5 × Time A ≤ Time I ≤ 0.7 × Time A By satisfying this relationship, the electric field applied to each bio-derived material tends to become uniform, the efficiency of introducing bioactive substances into the bio-derived material during EP can be made uniform, and productivity can be increased. The shortest time Time M is always shorter than the average time Time A. That is, the following equation is satisfied: Time M < Time A Preferably, the pulse period Time I and the shortest time Time M for the bio-derived material to pass through the electrode (electrode length L) satisfy the following relationship: Time I ≤ Time M Particularly preferably, the following relationship is satisfied: Time I = Time M The pulse period Time I is 75 milliseconds to 10 seconds, preferably 150 milliseconds to 2 seconds, and more preferably 300 milliseconds to 1 second. If the pulse period Time I becomes too small, heat generated when the pulse voltage is applied may accumulate, potentially reducing the survival rate or introduction efficiency. When the culture medium is not changed, the suspension contains more culture medium than normally EP, has higher electrical conductivity, and a higher total sodium ion content, resulting in a higher heat generation when an electric field is applied. The average time Time A is 75 milliseconds to 33.3 seconds, preferably 187.5 milliseconds to 5 seconds, and more preferably 429 milliseconds to 2 seconds. The shortest time Time M is 75 milliseconds to 10 seconds, preferably 150 milliseconds to 2 seconds, and more preferably 300 milliseconds to 1 second.

[0080] In the first example of the EP method described above, the EP method described in PCT / JP / 2025 / 016650 or International Publication No. 2024 / 248133 may be used. The contents of (1) cell pre-culture, (2) adjustment of cell concentration (concentration, etc.), (3) addition and mixing of nucleic acids, (4) electroporation (gene transfer), (5) culture for virus production (main culture), and (6) recovery and purification of the virus are incorporated herein by reference.

[0081] The fluid channel device, electroporation method, and electroporation apparatus of the present invention are suitably usable for the manufacture of gene therapy drugs and the like. The present invention is basically configured as described above. Although the fluid channel device, electroporation method, and electroporation apparatus of the present invention have been described in detail above, the present invention is not limited to the embodiments described above, and various improvements or modifications may be made without departing from the spirit of the present invention. All publications, patents, and patent applications referenced herein are incorporated herein by direct reference.

[0082] The features of the present invention will be further described in detail below with reference to examples. The materials, reagents, amounts and proportions of substances, and procedures shown in the following examples can be modified 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.

[0083] [Gene Introduction] Fluid channel devices with the configurations of Examples 1-4 and Type (Reference Example) were prepared, and genes were introduced into cells. The configurations of the fluid channel devices of Examples 1-4 and Type (Reference Example) are shown in Tables 1 and 2 below. In the fluid channel devices of Examples 1-4 and Type (Reference Example), the width of the main channel is the same as the width of the first channel and the width of the second channel, so the widths of the first channel and the second channel are omitted in Tables 1 and 2 below. Also, the "ratio" in Table 2 below is the ratio (Ls / W) of the width of the main channel between the electrode pair to the total length Ls of the length of the first channel and the length of the sheath fluid expansion section. In the fluid channel device of Type (Reference Example), the sheath fluid channel has the configuration shown in Figure 9. The sheath fluid channel 49 shown in Figure 9 is a straight channel with no expansion section and a constant width. The width Wm of the sheath fluid channel 49 is 2 mm. In the fluid channel device of Type (Reference Example), the width of the main channel is 2 mm. In Figure 9, components identical to those in the sheath fluid flow path 40 shown in Figure 4 are given the same reference numerals, and their detailed descriptions are omitted.

[0084] <Preparation of Suspension> Prepare a cell suspension (cell concentration: 120 Mcells / mL) of HEK293 cells (corresponding to a biological product) with an average diameter of 20 μm. The average cell diameter and cell concentration are measured using Vi-CELL XR (Beckman Coulter). Add Lonza's pmaxGFP plasmid (corresponding to a bioactive substance) to the obtained cell suspension to a concentration of 30 μg / mL. In this way, a suspension containing HEK293 cells, GFP plasmid, and culture medium is obtained. The cell volume fraction in the suspension is 50%, and the plasmid concentration is 30 μg / mL. From the cell concentration (120 Mcells / mL) and the average cell diameter (20 μm), assuming the cells are spherical, the cell volume fraction in the suspension is (= 4 ÷ 3 × (20 μm ÷ 2) 3 ×π × 120 Mcells / mL ÷ 10 6 Calculate the result of ×100 (%). <Genetic expression efficiency> The recovered suspension is 2M (×10 6 Dilute to cells / mL, then sow 2 mL into a well plate, CO ２The cells are placed in an incubator with a concentration of 8% and an ambient temperature of 37°C and cultured statically for 24 hours. The cell concentration X after 24 hours of culture is obtained using Vi-CELL XR (Beckman Coulter), and the proportion of gene-transfected cells Y is obtained using BD FACS Calibur (Becton Dickinson). Then, the gene transfection expression efficiency (= cell concentration X × cell proportion Y ÷ 2 Mcells / mL × 100) (%) is calculated as the ratio of gene-transfected cells obtained after 24 hours of culture to electroporated cells.

[0085] EP is performed using the flow channel devices of type 1 to 4. The thickness of the suspension flow is 2 mm, and the thickness of the sheath liquid flow is 0.5 mm (one side). In this example, the thickness of the suspension flow and sheath liquid flow refers to the thickness of the suspension flow and sheath liquid flow at the upstream end between the electrodes of the flow channel device. The sheath liquid is the culture medium of the suspension. The pulse voltage is 330 V, the voltage applied to the suspension flow is 1376 V / cm, and the pulse width is 3.5 ms. In the flow channel device of type 1, the dilution ratio of the suspension is 1.5, and the cell processing rate is 480 Mcells / min, and the cell processing rate increases with increasing width.

[0086] <Genetic expression efficiency> The recovered suspension was divided into 2M (x10) 6 Dilute to cells / mL, then sow 2 mL into a well plate, CO ２The cells were placed in an incubator with a concentration of 8% and an ambient temperature of 37°C and incubated statically for 24 hours. The cell concentration X after 24 hours of incubation was obtained using Vi-CELL XR (Beckman Coulter), and the proportion of gene-transfected cells Y was obtained using BD FACS Calibur (Becton Dickinson). Then, the gene transfer expression efficiency (= cell concentration X × cell proportion Y ÷ 2 Mcells / mL × 100) (%) was calculated as the ratio of gene-transfected cells obtained after 24 hours of incubation to electroporation-treated cells. <Results> The flow channel devices of Examples 1 to 4 were compared to the Type flow channel device, using the Type flow channel device as a standard. Compared to the Type flow channel device, the flow channel devices of Examples 1 to 4 maintained the same gene transfer efficiency as the Type flow channel device even when the width of the main flow channel (width of the sheath liquid flow channel) was widened. The results are indicated as "Equivalent to Standard" in Table 2 below. As described above, gene transfer efficiency is maintained and the cell processing volume increases, thus improving productivity.

[0087]

[0088]

[0089] [Flow velocity distribution simulation] The flow velocity distribution of the sheath liquid flow in the flow channel devices of Examples 1 to 4 was simulated using Ansys Fluent or Ansys 2023 R2 (software name) from Ansys, Inc. The viscosity of the sheath liquid was set to 1 mPa·s (cp). In all Examples 1 to 4, the total length of the first flow channel and the length of the sheath liquid expansion section was 0.8 times or more the width of the main flow channel (see Table 2), and it was confirmed that the flow velocity distribution in the width direction of the sheath liquid flow was uniform at the electrode. As a result, during EP, the three-layered flow of sheath liquid flow, suspension flow and sheath liquid flow is stable, EP is performed uniformly between electrode pairs, and productivity is increased.

[0090] 10 Electroporation apparatus (EP apparatus) 12 Flow channel device 12a Front surface 12b Back surface 14 Power supply unit 14a, 14b Wiring 15 Control unit 16a, 16b, 16c, 16d Pump 17 Suspension supply port 18 Discharge port 19 Tube 20 Main flow channel 20a One end 20b Other end 20c Inner surface 21a Enlarged section 21b Main flow channel body 21c Side surface 22 Electrode pair 23 First electrode 23a, 24a Electrode surface 24 Second electrode 25 Space 26, 26a, 26b, 26c, 26d Piping 27 Mixer 28 Culture apparatus 29 Tank 30 First substrate 30a Through hole 30b, 32b Sheath liquid enlarged section 30c, 32c First flow channel 30d, 32d Direction change section 30e, 32e Second flow path 30f, 32f Sheath liquid confluence port 31a, 31b Sheath liquid inlet 31c Side view 31d Connection section 31e, 31f Side view 31g Termination 32 Second substrate 32a Discharge path 34 Flow path substrate 34a Suspension inlet 34b Suspension outlet 36 First introduction section 36a Through hole 36b, 37, 38b Supply path 38 Second introduction section 40, 42, 49 Sheath liquid flow path 43a First supply port 43b Second supply port 44 Confluence section 45 Tank 46 Connection section 47a, 47b Curved surface 47c Connection point 50 Calculation model 50b Sheath liquid expansion section 50c First flow path section 51a Inlet section 51e Side part C, Cs Center line D Distance D Ｌ , D Ｌｓ Extension direction Dd, liquid delivery direction Df, direction Dm, thickness Ds, stacking direction Dw, Dws, width direction Hp, horizontal plane L, electrode length Q, suspension ds, thickness s, sheath liquid x, y direction z, vertical direction

Claims

1. A flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, wherein the flow channel has a main channel through which the suspension flows, a sheath liquid channel through which only a sheath liquid other than the suspension flows and which causes the sheath liquid to flow into the main channel, and an electrode pair that applies an electric field to the suspension flowing in the main channel, the sheath liquid channel having a sheath liquid expansion section that expands the sheath liquid channel in the width direction, and a first channel located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, and the sum of the length of the first channel and the length of the sheath liquid expansion section is 0.8 times or more the width of the main channel between the electrode pair.

2. The flow path device according to claim 1, wherein the main flow path is provided with a suspension inlet for introducing the suspension into the main flow path, the sheath liquid flow path is located downstream of the first flow path and further includes a direction changing unit for changing the flow direction of the sheath liquid in the first flow path, a second flow path located downstream of the direction changing unit, and a sheath liquid confluence port located downstream of the second flow path for introducing the sheath liquid into the main flow path body, the sheath liquid confluence port being provided downstream of the suspension inlet and upstream of the electrode pair.

3. The flow channel device according to claim 2, wherein the first flow channel and the second flow channel have different thicknesses.

4. A flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, the flow channel having a main channel through which the suspension flows, a sheath liquid channel through which only a sheath liquid other than the suspension flows and which allows the sheath liquid to flow into the main channel, and an electrode pair for applying an electric field to the suspension flowing in the main channel, wherein the main channel has a suspension inlet for allowing the suspension to flow into the main channel, and a sheath liquid confluence port provided downstream of the suspension inlet and upstream of the electrode pair for allowing the sheath liquid to flow into the main channel, and the sheath liquid channel has sections of different thicknesses.

5. The flow channel device according to claim 1 or 4, wherein the width of the main flow channel is 3 mm or more.

6. The flow channel device according to claim 1 or 4, wherein the first flow channel has a thickness of 2 mm or less.

7. The flow channel device according to claim 4, further comprising a direction changing section between the first flow channel and the second flow channel, which changes the flow direction of the sheath fluid in the first flow channel.

8. The flow path device according to claim 4, further comprising a sheath fluid expanding section disposed upstream of the first flow path and expanding the sheath fluid flow path in the width direction.

9. The flow path device according to claim 1 or 4, wherein the sheath fluid flow path is a straight flow path and is arranged at an inclination with respect to the main flow path.

10. A flow channel device for use in electroporation, which introduces a bioactive substance into a suspension containing a bio-derived substance by applying an electric field to the suspension using an electrode pair, wherein the flow channel comprises: a main flow channel through which the suspension flows; a sheath liquid flow channel through which only a sheath liquid other than the suspension flows and which causes the sheath liquid to flow into the main flow channel; and an electrode pair that applies an electric field to the suspension flowing in the main flow channel, wherein the sheath liquid flow channel comprises: a sheath liquid expansion section that expands the sheath liquid flow channel in the width direction; and a first flow channel located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, wherein the width of the main flow channel between the electrode pair is 3 mm or more; the width of the first flow channel is 3 mm or more; the thickness of the first flow channel is 2 mm or less; and the sum of the length of the first flow channel and the length of the sheath liquid expansion section is 0.8 times or more the width of the main flow channel between the electrode pair.

11. An electroporation method comprising the step of supplying a suspension at a rate of 5 mL / min or more to a flow channel device according to any one of claims 1, 4, and 10, and applying the electric field to the suspension with the electrode pair to introduce the bioactive substance into the bio-derived material.

12. An electroporation apparatus having a flow channel device according to any one of claims 1, 4, and 10.

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