Flow channel device and electroporation device

Abstract

The present invention provides a flow channel device with high productivity. Provided is a flow channel device which is used for electroporation, and which introduces a biologically active substance into a biologically derived material by applying, by means of an electrode pair, an electric field to a suspension that contains the biologically derived material and the biologically active substance. The flow channel device comprises: a main flow channel through which the suspension flows; and the electrode pair that applies the electric field to the suspension flowing through the main flow channel. The main flow channel comprises: a suspension inflow port through which the suspension flows into the main flow channel; an expanded part in which the flow channel is expanded in the width direction with the suspension inflow port used as the starting point; and a main flow channel body part that is disposed in the downstream of the expanded part and is continuous with the expanded part. A side surface of the expanded part does not have a bent part.

Description

Fluid flow devices and electroporation apparatus

[0001] The present invention relates to a flow channel device 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. More particularly, the present invention relates to a flow channel device and electroporation apparatus having a main channel with an expanding section that expands the flow channel in the width direction.

[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 objective of the present invention is to provide a flow channel device 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 with an electrode pair, and comprises a main flow channel through which the suspension flows, and an electrode pair for applying an electric field to the suspension flowing through the main flow channel, wherein the main flow channel comprises a suspension inlet for introducing the suspension into the main flow channel, an expansion section that expands the flow channel in the width direction starting from the suspension inlet, and a main flow channel body section located downstream of the expansion section and continuous with the expansion section, and the expansion section has no bends on its side surface.

[0007] Invention [2] is a flow channel device according to Invention [1], wherein there is no bend on the side surface upstream of the downstream end of the electrode pair in the main flow channel. Invention [3] is a flow channel device according to Invention [1] or [2], wherein the enlarged portion has a curved surface on its side surface, and the minimum radius of curvature of the curved surface is 1 / 5 or more the width of the main flow channel body between the electrode pair. Invention [4] is a flow channel device according to any one of Inventions [1] to [3], wherein the enlarged portion satisfies requirement 1 or 2. Requirement 1: A part of the side surface of the enlarged portion is flat. Requirement 2: The entire side surface of the enlarged portion is curved. Invention [5] is a flow channel device according to any one of Inventions [1] to [4], wherein only a sheath liquid, which is a liquid other than a suspension, flows through the sheath liquid channel, and the sheath liquid flows into the main channel body of the main channel, the sheath liquid channel having a sheath liquid expansion section that expands the sheath liquid channel in the width direction, a sheath liquid channel body located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, and a sheath liquid confluence port that connects the sheath liquid channel body and the main channel body and allows the sheath liquid to flow into the main channel body, the sheath liquid confluence port is located downstream of the sheath liquid expansion section and there is no bend on the side surface of the sheath liquid expansion section. Invention [6] is an electroporation apparatus having a flow channel device according to any one of Inventions [1] to [5].

[0008] According to the present invention, a highly productive flow channel device and electroporation apparatus can be provided.

[0009] 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 a first 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 second 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 third 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 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 the calculation model and simulation results of the flow velocity distribution of the main flow channel in Comparative Example 2. This is a schematic diagram showing the calculation model and simulation results of the flow velocity distribution of the main flow channel in Example 2. This is a schematic diagram showing the shape of the main flow channel of the flow channel device.

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

[0011] (First example of an electroporation apparatus) The flow channel device 12 (see Figure 1) is a device used in electroporation that introduces bioactive substances into a suspension Q (see Figure 7) containing bio-derived substances by applying an electric field to the suspension Q using an electrode pair 22 (see Figure 1). The electroporation apparatus 10 (see Figure 1) has the flow channel device 12. The flow channel device 12 will be described in detail later, but the main flow channel 20 (see Figure 1) through which the suspension flows comprises an enlarged section 21a (see Figures 1 and 3) and a main flow channel body section 21b (see Figures 1 and 3) located downstream of the enlarged section 21a and continuous with the enlarged section, and the enlarged section 21a has no bends on its side surface. By configuring the main flow channel 20 so that the enlarged section 21a has no bends on its side surface, stagnation of the suspension flow in the enlarged section 21a is less likely to occur. In suspensions containing bio-derived substances, stagnation of bio-derived substances 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 configuring the side of the enlarged section 21a without a bend, such a decrease can be suppressed. Furthermore, this configuration makes it easier to achieve a uniform flow velocity distribution of the suspension Q in the width direction in the main channel 20, allowing for uniform introduction efficiency of bioactive substances into bio-derived materials during EP, and thus increasing productivity. In addition, since the flow of suspension Q becomes less turbulent, the distance from the suspension inlet 34a to the electrode pair 22 can be shortened, suppressing the increase in size of the channel device 12. This also suppresses the increase in size of the electroporation apparatus 10.

[0012] 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, 16d. The flow path device 12 has a main flow path 20 through which the suspension Q flows, and an electrode pair 22 that applies an electric field to the suspension 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 which is, for example, a quadrangle with all interior angles being 90°. The above-described cross-sectional shape of the main flow path 20 is not limited to a quadrangle with all interior angles being 90°.

[0013] 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 Ｌ of the main flow path 20 is the vertical direction z orthogonal to the horizontal plane Hp, and the angle with respect to the horizontal plane Hp is 90°, but the vertical direction z of the main flow path 20 allows ±10° with respect to 90°.

[0014] 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 have approximately equal lengths in the vertical z direction. They are stacked in the order of the first introduction section 36, the first substrate 30, the flow channel substrate 34, the second substrate 32, and the second introduction section 38. 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 7) and sheath liquid s (see Figure 7) 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.

[0015] 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.

[0016] 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.

[0017] The main channel 20 comprises an enlarged portion 21a as described above, and a main channel body portion 21b located downstream of the enlarged portion 21a and continuous with the enlarged portion 21a. The enlarged portion 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 portion 36. The first introduction portion 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 portion 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 portion 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.

[0018] 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 and the second introduction section 38. 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 sheath liquid flow channel body 30e, which is located downstream of the sheath liquid expansion section 30b and is continuous with the sheath liquid expansion section 30b. The sheath liquid flow channel body 30e is, for example, provided at an inclination with respect to the main flow channel 20.

[0025] As shown in Figures 1 and 2, between the sheath fluid expansion section 30b and the sheath fluid flow path main section 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 and the sheath fluid flow path main section 30e are in communication, and the flow of the sheath fluid s is directed toward the sheath fluid flow path main section 30e by the direction changing section 30d. For example, the first flow path 30c, the direction changing section 30d, and the sheath fluid flow path main section 30e have the same width. The sheath fluid flow path main section 30e and the main flow path main section 21b are in communication, and the opening connecting the sheath fluid flow path main section 30e and the main flow path main section 21b is a sheath fluid confluence port 30f for allowing the sheath fluid s to flow into the main flow path main section 21b. The sheath liquid confluence port 30f connects the sheath liquid flow path body 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.

[0026] 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 sheath liquid flow path body section 32e that is located downstream of the sheath liquid expansion section 32b and is continuous with the sheath liquid expansion section 32b. The sheath liquid flow path body section 32e is provided, for example, at an inclination with respect to the main flow path 20. As shown in Figures 1 and 2, a configuration may be provided between the sheath fluid expansion section 32b and the sheath fluid flow path main section 32e, for example, having 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. The direction changing section 32d and the sheath fluid flow path main section 32e are in communication, and the flow of the sheath fluid s is directed toward the sheath fluid flow path main section 32e by the direction changing section 32d. For example, the first flow path 32c, the direction changing section 32d, and the sheath fluid flow path main section 32e have the same width. The sheath fluid flow path main section 32e and the main flow path main section 21b are in communication, and the opening connecting the sheath fluid flow path main section 32e and the main flow path main section 21b is the sheath fluid confluence port 32f for allowing the sheath fluid s to flow into the main flow path main section 21b. The sheath liquid confluence port 32f connects the sheath liquid flow path body 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 sheath liquid flow path body 30e and 32e are also referred to as the second flow path.

[0027] The through-hole 36a of the first introduction section 36, the sheath liquid expansion section 30b and sheath liquid flow path main section 30e of the first substrate 30, and the supply path 38b of the second introduction section 38, and the sheath liquid expansion section 32b and sheath liquid flow path main section 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 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 sheath liquid flow path main section 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 sheath liquid flow path main body section 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.

[0028] 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.

[0029] (Main channel) The main channel 20 will be described in detail. Figure 3 is a schematic diagram showing a first example of the main channel of the flow channel device according to an embodiment of the present invention, Figure 4 is a schematic diagram showing a second example of the main channel of the flow channel device according to an embodiment of the present invention, and Figure 5 is a schematic diagram showing a third example of the main channel of the flow channel device according to an embodiment of the present invention. Figures 3 to 5 show 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 Figures 3 to 5 shows the two-dimensional shape of the side surface constituting the main channel 20. Also, in Figures 3 to 5, 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 flow path 20. As shown in Figure 3, the main flow path 20 includes a suspension inlet 34a into which the suspension Q flows, an expanded section 21a that expands the flow path in the width direction Dw starting from the suspension inlet 34a, and a main flow path body section 21b that is located downstream of the expanded section 21a and is continuous with the expanded section 21a. The main flow path 20 is configured such that there are no bends on the side surface 21c of the expanded section 21a. The expanded section 21a without bends on its side surface preferably satisfies either requirement 1 or 2 below. Requirement 1: A part of the side surface of the expanded section is flat. Requirement 2: The entire side surface of the expanded section is curved. The expanded section 21a without bends on its side surface may, for example, be composed of a single flat surface or a single curved surface. Alternatively, the expanded section 21a without bends on its side surface may be composed of multiple curved surfaces. When a side surface without a bend is composed of multiple curved surfaces, mathematically, the multiple surfaces are tangentially continuous, curvature-continuous, or have coincident contact planes at their joints.

[0030] The main flow path 20 will be described more specifically. The main flow paths 20 in FIGS. 3 to 5 all have a contour shape that is symmetric with respect to a center line C passing through the center of the suspension liquid inlet 34a and parallel to the extending direction D of the main flow path 20, and the side surface of the main flow path main body portion 21b is linear. The enlarged portion 21a of the main flow path 20 in FIG. 3 has a side surface 21c formed of a flat surface. The side surface 21c extends linearly in the width direction Dw Dw Dw of the main flow path 20 starting from the suspension liquid inlet 34a. The connection portion 46 between the enlarged portion 21a and the main flow path main body portion 21b is formed of a curved surface portion, and the enlarged portion 21a has a curved surface portion. The enlarged portion 21a and the main flow path main body portion 21b are continuously connected without a bent portion. The contour shape of the side surface 21c of the enlarged portion 21a shown in FIG. 3 is represented by, for example, a linear function. Ｌ In the main flow path 20 shown in FIG. 4, the entire side surface 21d of the enlarged portion 21a is formed of a single curved surface. The contour shape of the side surface 21d of the enlarged portion 21a is an arc, and for example, the contour shape is represented by a quadratic function. Note that the arc includes an elliptical arc. In the main flow path 20 shown in FIG. 4, the end 21f of the curved surface constituting the side surface 21d of the enlarged portion 21a is connected to the main flow path main body portion 21b, and the tangent line (not shown) of the end 21f of the side surface 21d coincides with the tangent line (not shown) of the side surface of the main flow path main body portion 21b. Therefore, the enlarged portion 21a and the main flow path main body portion 21b are continuously connected without a bent portion.

[0031] The main channel 20 shown in Figure 5 is composed of multiple curved surfaces across the entire side surface 21e of the enlarged portion 21a. For example, the side surface 21e of the enlarged portion 21a is composed of two curved surfaces 47a and 47b. 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 21e of the enlarged portion 21a shown in Figure 5 can be represented, for example, by a circular arc, a cubic function, a trigonometric function, an inverse trigonometric function, or a sigmoid function. In the main channel 20 shown in Figure 5, the end 21f of the curved surface 47b constituting the side surface 21e of the enlarged portion 21a is connected to the main channel body 21b, and the tangent to the end 21f of the curved surface 47b coincides with the tangent to the side surface of the main channel body 21b. Therefore, the enlarged portion 21a of the curved surface 47b and the main channel body 21b are connected continuously without any bends. Figures 3 to 5 show a configuration in which there are no bends at the connection between the enlarged section 21a and the main channel body section 21b. The side surface of the main channel body section 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.

[0032] By making the main channel 20 an enlarged section 21a without bends as shown in Figures 3 to 5 above, the stagnation of suspension Q in the enlarged section 21a is suppressed, improving productivity. Furthermore, the flow of suspension Q becomes less turbulent 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 bends. Furthermore, since the flow of suspension Q becomes less turbulent, the main channel 20 extends in the direction D 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 Ｌ This helps to suppress the increase in size. Furthermore, 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.

[0033] Furthermore, in the configuration of the enlarged portion 21a of the main channel 20 shown in Figures 3 to 5 above, it is preferable that the minimum radius of curvature r of the curved portion is 1 / 5 or more of the width W of the main channel body portion 21b between the electrode pair 22. That is, it is preferable that (W / 5) ≤ r. If (W / 5) ≤ r, the flow velocity of the suspension Q in the width direction of the main channel 20 becomes uniform over an even shorter distance, thereby further increasing productivity. The minimum radius of curvature r of the curved portion described above is the smallest radius of curvature among the radii of curvature of the curved portion, and the radius of curvature of the suspension inlet 34a (starting point) into which the suspension Q flows into the main channel 20 is not included in the minimum radius of curvature r. The minimum radius of curvature r is obtained by acquiring the contour shape of the main channel 20 and measuring the radius of curvature of the curved portion of the contour shape. The smallest radius of curvature among the measured radii of curvature of the curved portion is taken as the minimum radius of curvature. 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 two-dimensional scanner or a three-dimensional scanner. Alternatively, 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. 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 2 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, and 60 mm or more. The upper limit of the width W of the main channel body 21b is 500 mm. While a wider main channel can lead to a more uneven flow velocity distribution and a decrease in EP efficiency, such as the efficiency of introducing bioactive substances into biological materials, in this embodiment, sufficient EP efficiency can be obtained even with a wide main channel. Furthermore, 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.

[0034] (Sheath fluid flow path) The configurations of the sheath fluid flow paths 40 and 42 are not particularly limited, but similar to the main flow path 20, it is preferable that there is no bending portion on the side surface 31c (see FIG. 6) of the sheath fluid expansion portions 30b and 32b. FIG. 6 is a schematic diagram showing a first example of the sheath fluid flow path of the flow path device according to the embodiment of the present invention. FIG. 6 shows the contour shapes of the sheath fluid flow paths 40 and 42 as viewed from the sheath fluid flow inlet 31a, 31b side. The contour shapes of the sheath fluid flow paths 40 and 42 shown in FIG. 6 show the two-dimensional shape of the side surfaces constituting the sheath fluid flow paths 40 and 42. The contour shapes of the sheath fluid flow paths 40 and 42 shown in FIG. 6 are the same as the contour shape of the main flow path 20 shown in FIG. 3. The sheath fluid flow paths 40 and 42 in FIG. 6 have a contour shape that passes through the centers of the sheath fluid inlets 31a and 31b, and extends in the extending direction D of the sheath fluid expansion portions 30b and 32b, the first flow paths 30c and 32c, and the direction conversion portions 30d and 32d of the sheath fluid flow paths 40 and 42 Ｌ and is symmetric with respect to the center line Cs parallel to it, and the side surfaces of the first flow paths 30c and 32c and the direction conversion portions 30d and 32d are linear. Also, in FIG. 6, the sheath fluid inlet 31a, 31b side is the upstream side of the sheath fluid flow paths 40 and 42, and the opposite side in the extending direction D of the sheath fluid inlets 31a and 31b Ｌ is the downstream side of the sheath fluid flow paths 40 and 42. Since the sheath fluid flow path 40 and the sheath fluid flow path 42 have the same configuration, the sheath fluid flow path 40 will be described representatively, and the description of the sheath fluid flow path 42 will be omitted.

[0035] The sheath fluid flow path 40 is configured such that the side surface 31c of the sheath fluid expansion portion 30b is flat. For example, as shown in FIG. 6, starting from the sheath fluid inlet 31a, the sheath fluid expansion portion 30b extends in the width direction Dws of the first flow path 30c (the sheath fluid flow path main body portion 30e). The connection portion 31d between the sheath fluid expansion portion 30b and the first flow path 30c is configured by a curved surface portion, and the sheath fluid expansion portion 30b has a curved surface portion. The sheath fluid expansion portion 30b and the first flow path 30c are continuously connected without a bending portion. The width direction Dws is the extending direction D in which the sheath fluid flow path 40 extends ＬｓThis is in a direction perpendicular to the direction. Note that the configuration of the sheath liquid expansion sections 30b and 32b of the sheath liquid flow paths 40 and 42 is not limited to the configuration shown in Figure 6 above. For example, the configuration of the expansion section 21a shown in Figures 4 and 5 above can also be used. In the sheath liquid flow paths 40 and 42, by configuring the sheath liquid expansion sections 30b and 32b to have no bends on the side surfaces 31c, the flow of the sheath liquid s in the main body sections 30e and 32e becomes less turbulent, and the flow velocity of the sheath liquid s in the width direction of the sheath liquid flow paths 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 applied appropriately to the suspension Q. As a result, the introduction efficiency and the utilization efficiency of bioactive substances are increased, improving productivity. Furthermore, the uniformity of the velocity distribution of the sheath fluid in the width direction of the sheath fluid channels 40 and 42 can be confirmed, for example, by numerical analysis such as simulation, or by using a visualization device having a shape similar to that of the sheath fluid channels.

[0036] In the configuration of the sheath liquid expansion sections 30b and 32b of the sheath liquid flow paths 40 and 42, it is preferable that the minimum radius of curvature rs of the curved section is 1 / 5 or more of the width Ws of the sheath liquid flow path main section 30e. That is, it is preferable that (Ws / 5) ≤ rs. If (Ws / 5) ≤ rs, the flow velocity of the sheath liquid s in the width direction of the sheath liquid flow path becomes uniform over an even shorter distance, so the waste of sheath liquid s is reduced, and productivity can be further increased. In addition, the flow path device can be made smaller. Furthermore, due to the structure of the flow path device, the suspension inlet 34a of the suspension Q is upstream of the sheath liquid inlet 31a of the sheath liquid, so if the run-up distance of the sheath liquid is short, the run-up distance of the suspension Q is also short, and the waste of suspension Q can be reduced. The width Ws of the sheath liquid flow path main section 30e corresponds to the width of the sheath liquid flow path 40. The width Ws of the sheath fluid channel 40 is preferably 2 mm or more, and more preferably 3 mm or more, 6 mm or more, 8 mm or more, 20 mm or more, and 60 mm or more, in that order. The upper limit of the width Ws of the sheath fluid channel 40 is 500 mm. The minimum radius of curvature rs of the curved surface described above is the smallest radius of curvature among the radii of curvature of the curved surface, and the radius of curvature of the sheath fluid inlet 31a is not included in the minimum radius of curvature rs. The minimum radius of curvature rs is obtained by acquiring the contour shapes of the sheath fluid channels 40 and 42 and measuring the radius of curvature of the curved surface of the contour shape. The smallest radius of curvature among the measured radii of curvature of the curved surface is taken as the minimum radius of curvature. The contour shapes of the sheath fluid channels 40 and 42 can be obtained in the form of digital data by scanning the sheath fluid channels 40 and 42 with a two-dimensional scanner or a three-dimensional scanner. Furthermore, 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 from the CAD data in the form of digital data.

[0037] In the flow channel device 12, there are no restrictions on the angle formed between the suspension flow and the sheath liquid flow when the suspension flow and the sheath liquid flow are merged, that is, the angle formed between the main flow channel 20 and the sheath liquid flow channel body 30e and the sheath liquid flow channel body 32e, but a smaller angle is preferable. Specifically, the extending direction D of the main flow channel 20. ＬFurthermore, the angle formed by the extending direction of the sheath liquid channel body 30e and the extending direction of the sheath liquid channel body 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.

[0038] 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.

[0039] 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).

[0040] Here, Figure 7 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 7, 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 7, 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.

[0041] As conceptually shown in Figure 7, 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.

[0042] 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.

[0043] 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 7) and the thickness ds of the sheath liquid flow (see Figure 7) 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.

[0044] (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 the suspension Q to flow from piping 26 through the suspension supply port 17, the through-hole 36a of the first introduction section 36, and the through-hole 30a of the first substrate 30 into the main flow path 20 through the suspension inlet 34a. Pump 16c causes the sheath liquid s in tank 45 to flow from piping 26a through the first supply port 43a and the sheath liquid flow path 40 into the main flow path 20 through the sheath liquid confluence port 30f. Pump 16d causes the sheath liquid s in tank 45 to flow from piping 26b through the second supply port 43b and the sheath liquid flow path 42 into the main flow path 20 through the 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 are no bends on the side surface 21c of the expanded 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 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 also have a configuration in which there are no bends on the side surface 31c of the expanded portions 30b and 32b of the sheath liquid, the three-layered flow of sheath liquid flow, suspension flow and sheath liquid flow is stabilized during EP.Between the electrode pair 22, the thickness of the sheath liquid flow and the thickness of the suspension Q become uniform, allowing the electric field to be applied appropriately to the suspension Q. As a result, the introduction efficiency and the utilization efficiency of bioactive substances are increased, improving productivity.

[0045] 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.

[0046] 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.

[0047] [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.

[0048] <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.

[0049] (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.

[0050] <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.

[0051] <Suspension> The suspension to be subjected to electroporation (EP) contains biologically derived substances and bioactive substances. Furthermore, the suspension does not necessarily 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.

[0052] (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.

[0053] (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.

[0054] There are no restrictions on the concentration of biologically derived substances in suspension Q, but it is preferable that the volume fraction of biologically derived substances in suspension Q be 20% or more. In EP, increasing the concentration of biologically derived substances in suspension Q allows for a larger processing volume of biologically derived substances compared to introducing the same amount of a lower-concentration suspension. In addition, in EP, as described above, bioactive substances are incorporated into the biologically derived substances by electrophoresis. Therefore, even if the concentration of biologically derived substances is increased, it is not necessary to increase the amount of bioactive substances accordingly, thus improving the utilization efficiency of bioactive substances.

[0055] 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)). 3The 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%.

[0056] 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.

[0057] 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.

[0058] <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.

[0059] 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.

[0060] 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.

[0061] <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.

[0062] <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.

[0063] <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).

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] The fluid channel device 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 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.

[0069] 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.

[0070] [Flow velocity distribution simulation 1] The flow velocity distribution of the cell suspension in the flow channel device is simulated using Ansys Fluent or Ansys 2023 R2 (software name) from Ansys, Inc. Examples 1 to 3 are V-shaped, semicircular, and arc shapes without bends, while Comparative Example 1 is almost the same shape as the V-shape of Example 1 but has a bend. (Comparative Example 1: With a bend) In the configuration where there is a bend on the side of the enlarged section 21a, cells accumulate at the bend, resulting in cells that do not flow. Since cells in suspension do not flow easily, such accumulation is likely to occur. (Examples 1 to 3: Without a bend) In all examples, it was confirmed that the flow velocity distribution of the suspension Q in the width direction in the main flow channel 20 becomes uniform over a short distance. Therefore, during EP, the efficiency of introducing bioactive substances into bio-derived materials can be made uniform, and productivity can be increased. In Example 2 (semicircular), it was confirmed that cell retention was suppressed at the bend, and that the cells in the suspension flowed smoothly throughout the entire channel. Specifically, the flow velocity distribution in the vertical direction was observed, and the flow velocity was fastest near the suspension inlet. As the cross-sectional area increases towards the main channel, the flow velocity gradually decreases in the widened section. After passing through the widened section, the flow velocity becomes constant, and towards the outlet, the flow velocity becomes almost uniform, and the flow velocity is also almost uniform in the width direction of the channel. As a result, the cell passage speed between the electrode pairs becomes uniform in the width direction, and the flow velocity distribution becomes uniform. A uniform electric field is applied between the electrode pairs, and because the cell flow velocity distribution is uniform, the EP (electron pulse) also becomes uniform, and productivity increases. If the flow velocity distribution is non-uniform, the voltage application may be insufficient where the flow velocity is high, which can lead to poor EP. Where the flow velocity is low, the voltage application becomes excessive, and the cell viability decreases. In other words, if the flow velocity distribution is non-uniform, productivity decreases. Furthermore, in the side of the enlarged section of Example 2, cell retention is suppressed, and the flow velocity in the width direction becomes uniform over a short distance. In Example 1 (V-shape without a bend) and Example 3 (arc), similar to Example 2 (semicircle), cell retention is suppressed on the side of the enlarged section, and the cells in the suspension flow smoothly. Also, similar to Example 2, the flow velocity distribution in the width direction of the suspension flow becomes uniform, and EP is performed uniformly.

[0071] [Flow velocity distribution simulation 2] The flow velocity distribution of a cell suspension in the main channel of a flow channel device was simulated using Ansys 2023 R2 (software name) from Ansys, Inc. The physical properties of the cell suspension were set to a viscosity corresponding to 100 Mcells / mL and 10 mPa·s (cp). Figure 9 shows the simulation results for Example 2 (semicircular) without a bend. Comparative Example 2 shown in Figure 8 shows the simulation results for a comparative example of Example 2 (semicircular). (Comparative Example 2: with a bend) In a configuration where there is a bend on the side of the enlarged section 21a, cells accumulate at the bend, resulting in cells that do not flow. Since cells in suspension do not flow easily, such accumulation is likely to occur. Specifically, Figure 9 shows the calculation model and the simulation results of the flow velocity distribution in the main channel of Comparative Example 2. In Comparative Example 2, calculation model 110 (see Figure 8) with a main channel width of 25 mm and a thickness of 2 mm was used. The calculation model 110 is a numerically calculated model created using the Ansys 2023 R2 (software name) mentioned above. The calculation model 110 shown in Figure 8 has an inlet section 110a corresponding to the suspension inlet 34a (see Figure 4) and a main flow path body section 110b corresponding to the main flow path body section 21b (see Figure 4). The calculation model 110 has a configuration in which a corner section 111 is located at the end of the width direction Dw of the inlet section 110a. In the numerical calculation of flow velocity, different ranges of flow velocity are displayed in specific colors. For example, the fastest flow velocity is displayed in red, and the slowest flow velocity is displayed in blue. The flow velocity is displayed in specific colors, and the flow velocity distribution is displayed as color unevenness. The uniformity of the flow velocity distribution was judged from this color unevenness. In Figure 8, the flow velocity is displayed in grayscale from white to black, with the slower the flow velocity becoming darker and closer to black, and the faster the flow velocity becoming lighter and closer to white. In Figure 8, 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 8, in the calculation model 110 which has a corner 111 corresponding to a bend, the difference in intensity is particularly large near the corner 111, meaning that the flow velocity distribution is large and the flow velocity is slow at the corner 111, confirming that the cell suspension is stagnating there.

[0072] (Example 2: No Bending Section) Figure 9 shows the calculation model and simulation results of the velocity distribution of the main channel in Example 2. In Example 2, a calculation model 92 (see Figure 9) with a main channel width of 25 mm and a thickness of 2 mm was used. The calculation model 92 is a numerically calculated model created using the Ansys 2023 R2 (software name) mentioned above. The calculation model 92 shown in Figure 9 has an inlet section 92a corresponding to the suspension inlet 34a (see Figure 4) and a main channel body section 92b corresponding to the main channel body section 21b (see Figure 4). In the calculation model 92, the contour shape of the side portion 92d of the enlarged section 92c, which corresponds to the side portion 21d (see Figure 4) of the enlarged section 21a (see Figure 4), is semicircular. The calculation model 92 has a configuration in which there is no bending section on the side portion of the inlet section 92a. In Figure 9, the flow velocity is displayed on a grayscale from white to black. The slower the flow velocity, the darker the color becomes, approaching black, and the faster the flow velocity, the lighter the color becomes, approaching white. In Figure 9, the flow velocity distribution is represented by variations in color intensity, and the smaller the difference in intensity, the smaller the flow velocity distribution. As shown in Figure 9, the calculation model 92 has no bends on its sides, resulting in small differences in intensity. Furthermore, the difference in flow velocity is small on the side portion 92d of the enlarged portion 92c, and there are few areas with slow flow velocity. This confirms that the retention of the cell suspension is suppressed.

[0073] [Gene Transfer] Genes are transferred into cells using the following fluidic device. <Fluidic Devices> Fluidic devices are prepared with a distance of 3 mm between electrode pairs and main channel widths of 2 mm (hereinafter referred to as the 2 mm fluidic device), 6 mm, 8 mm, 20 mm, 60 mm, and 150 mm. In the 2 mm fluidic device, as shown in Figure 10, the main channel 100 is a straight channel with a constant width and no expansion section. Note that in Figure 10, the same reference numerals are used for components identical to the main channel 20 shown in Figure 3, and their detailed explanation is omitted. For fluidic devices other than the 2 mm fluidic device, three patterns are prepared for the side of the expansion section, as shown in Figures 3 to 5: a V-shape, a semicircle, and a circular arc, with no bends on the side.

[0074] <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 × 100 (%).

[0075] EP (Electron Processing) is performed using the flow channel devices of the Examples and Comparative Examples. The thickness of the suspension flow is 2 mm, and the thickness of the sheath liquid flow is 0.5 mm (on 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 2 mm flow channel device, 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.

[0076] <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 ２Cells 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 ratio of gene-transfected cells Y is obtained using BD FACS Calibur (Becton Dickinson). Then, the gene transfection efficiency (= cell concentration X × cell ratio Y ÷ 2 Mcells / mL × 100) (%) is calculated as the ratio of gene-transfected cells obtained after 24 hours of culture to electroporated cells. <Results> Compared to a 2 mm channel device, a channel device with a main channel width greater than 2 mm allows for uniform EP even with a wider main channel. In other words, even with a wider main channel, the same gene transfection efficiency as a 2 mm channel device is maintained, and the cell processing volume increases, thus improving productivity.

[0077] 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, 92c Enlarged section 21b, 92b, 110b Main flow channel body 21c, 21d, 21e Side surface 21f End 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 fluid expansion section 30c, 32c First flow path 30d, 32d Direction change section 30e, 32e Sheath fluid flow path main body section 30f, 32f Sheath fluid confluence port 31a, 31b Sheath fluid inlet 31c Side surface 31d Connection section 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 Sheath fluid 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 92, 110 Calculation model 92a, 110a Inlet part 92d Side part 100 Main channel 111 Corner 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 r, rs, minimum radius of curvature s, sheath fluid 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, the device comprising: a main flow channel through which the suspension flows; an electrode pair for applying an electric field to the suspension flowing through the main flow channel; the main flow channel comprising: a suspension inlet for introducing the suspension into the main flow channel; an expansion section that widens the flow channel in the width direction starting from the suspension inlet; and a main flow channel body section disposed downstream of the expansion section and continuous with the expansion section, wherein there is no bend on the side surface of the expansion section.

2. The flow channel device according to claim 1, wherein there is no bend on the side surface upstream of the downstream end of the electrode pair in the main flow channel.

3. The flow channel device according to claim 1, wherein the enlarged portion has a curved portion on its side surface, and the minimum radius of curvature of the curved portion is 1 / 5 or more the width of the main flow channel body portion between the electrode pair.

4. The flow path device according to claim 1, wherein the enlarged portion satisfies requirement 1 or 2. Requirement 1: A part of the side surface of the enlarged portion is flat. Requirement 2: The entire side surface of the enlarged portion is curved.

5. A flow path device according to claim 1, wherein only a sheath liquid, which is a liquid other than the suspension, flows through the sheath liquid flow path, and the sheath liquid flows into the main flow path body of the main flow path, the sheath liquid flow path comprises a sheath liquid expansion section that expands the flow path of the sheath liquid in the width direction, a sheath liquid flow path body located downstream of the sheath liquid expansion section and continuous with the sheath liquid expansion section, and a sheath liquid confluence port that connects the sheath liquid flow path body and the main flow path body and allows the sheath liquid to flow into the main flow path body, the sheath liquid confluence port is located downstream of the sheath liquid expansion section, and there is no bend on the side surface of the sheath liquid expansion section.

6. An electroporation apparatus having a flow channel device according to any one of claims 1 to 5.

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