WO2021111837A1

WO2021111837A1 - Device

Info

Publication number: WO2021111837A1
Application number: PCT/JP2020/042227
Authority: WO
Inventors: 康裕森泉; 壮眞壁; 祐介斉藤
Original assignee: 株式会社ベックス
Priority date: 2019-12-02
Filing date: 2020-11-12
Publication date: 2021-06-10
Also published as: JP2023007509A

Abstract

The present invention addresses the problem of providing a device which enables a high-temperature pressurization treatment, is unlikely to be broken, and can be used for cell fusion or electroporation.　A resin is used as a base material of the device, and electrodes are inserted into electrode insertion grooves formed on the base material and are held in the electrode insertion grooves. In this manner, the distance between the electrodes can be kept approximately constant even when the frequency of a high-temperature pressurizing treatment is increased. Because a resin is used as the material for the base material, the possibility of breakage due to the application of an impact during handling can be reduced compared with a glass.

Description

device

The disclosure in this application relates to a device used for cell fusion or electroporation (hereinafter, may be simply referred to as "device"). In particular, the present invention relates to a device that can be repeatedly subjected to a high temperature pressurization process by an autoclave.

Cell fusion that forms one hybrid cell from two or more cells is known. In addition to the protoplast-PEG method, cell fusion is also known as an electrical stimulation method. An electroporation method is also known in which the permeability of a cell membrane is increased by applying an electric pulse to a cell to introduce a molecule such as DNA into a cell of a eukaryote or a prokaryote.

It is necessary to prevent contamination when performing cell fusion or electroporation by electrical stimulation. Therefore, disposable devices are known. On the other hand, when the device is reused, it is necessary to prevent contamination, so that it is generally necessary to perform a high-temperature pressurization treatment.

As a device capable of high-temperature pressurization, these plate electrodes are adhered and fixed to a support plate except for a cell suspension accommodating portion so that a pair of edges of the plate electrodes are parallel to each other, and the cells are suspended. A thin transparent glass plate is adhered to the plate electrode as the bottom plate of the turbid liquid accommodating portion, and a cell fusion in which an insulator is interposed in the facing gap between the edges of the plate electrode in the portion excluding the cell suspension accommodating portion. Chambers are known (see Patent Document 1).

Japanese Unexamined Patent Publication No. 63-84476

When performing cell fusion, apply electricity while observing with an inverted microscope. Therefore, the conventional cell fusion chamber described in Patent Document 1 uses transparent glass. However, when glass is used, there is a problem that it is damaged by an impact during handling.

Further, Patent Document 1 also describes that a transparent resin has been conventionally used as a material other than transparent glass. By the way, in order to improve the efficiency of cell fusion and electroporation, it is necessary to optimize the electricity application conditions. As for the electricity application conditions, it is necessary to adjust the current value and the voltage value applied to the electrodes according to the conductivity of the solution filled between the electrodes, the distance between the electrodes, and the type of cells, bacteria, and the like. However, the present inventors have newly discovered a problem that the experiment cannot proceed according to the set electrical conditions when the conventional device is repeatedly subjected to high-temperature pressurization treatment.

The disclosure of this application is made to solve the above problems. And after diligent examination,
(1) As described in Patent Document 1, a conventional device is manufactured by adhering components using an adhesive that can withstand high-temperature pressure treatment, or a device that uses silicon in combination with an adhesive. Was making,
(2) However, as the number of high-temperature pressurization treatments increases, the adhesive deteriorates and the bonded portion is likely to be damaged, and silicon becomes soft when the high-temperature pressurization treatment is performed. Due to the change in the distance between the electrodes, cell fusion or electroporation cannot be performed according to the set electrical application conditions.
(3) Since resin is easier to process than glass, the number of high-temperature pressurization treatments can be increased by using resin as the base material of the device and inserting and holding the electrodes in the electrode insertion grooves formed in the base material. Even so, the distance between the electrodes can be kept almost the same.
(4) Since resin is used as the base material, the risk of damage due to impact during handling can be reduced compared to glass.
Was newly found.

That is, the purpose of the disclosure of this application is to provide a device that can be subjected to high temperature pressurization treatment and has a low risk of damage.

The disclosure in this application relates to the following devices used for cell fusion or electroporation.

[1] A device used for cell fusion or electroporation.
The device is
With the base material
Solution container and
With the 1st and 2nd electrodes,
The first electrode insertion groove and the second electrode insertion groove,
Including
The base material is
Formed of resin,
It has a first surface of the base material and a second surface of the base material which is a surface opposite to the first surface of the base material.
The solution container is
Formed from the first surface of the base material toward the second surface of the base material,
The first wall surface and
A second wall surface formed so as to be substantially equal to the first wall surface,
On the bottom and
Have,
The first electrode insertion groove is formed from the bottom surface toward the second surface of the base material so as to be substantially parallel to the first wall surface.
The second electrode insertion groove is formed from the bottom surface toward the second surface of the base material so as to be substantially parallel to the second wall surface.
The first electrode and the second electrode are
It has a first surface of the electrode and a second surface of the electrode which is a surface opposite to the first surface of the electrode.
The first electrode is
The first surface of the electrode faces the first wall surface and the first surface of the insertion groove of the first electrode insertion groove.
So that the second surface of the electrode cannot move in the direction of the second wall surface.
Inserted and held in the first electrode insertion groove,
The second electrode is
The first surface of the electrode faces the second wall surface and the first surface of the insertion groove of the second electrode insertion groove.
So that the second surface of the electrode cannot move in the direction of the first wall surface.
A device that is inserted and held in the second electrode insertion groove.
[2] (1) One end of the first electrode has a first stretched portion that extends from the solution accommodating portion to the inside of the base material.
The base material includes a first stretched portion insertion hole for inserting and holding the first stretched portion.
The first stretched portion is inserted and held in the first stretched portion insertion hole so as to be immovable in a substantially vertical direction with respect to the first surface of the electrode and the second surface of the electrode.
And / or
(2) One end of the second electrode has a second stretched portion that extends from the solution accommodating portion to the inside of the base material.
The base material includes a second stretched portion insertion hole for inserting and holding the second stretched portion.
The second stretched portion is inserted and held in the insertion hole of the second stretched portion so as to be immovable in the substantially vertical direction with respect to the first surface of the electrode and the second surface of the electrode in the above [1]. Described device.
[3] (1) The other end of the first electrode has a third stretched portion extending from the solution accommodating portion to the outside of the base material.
The base material includes a third stretched portion through hole for penetrating and holding the third stretched portion.
The third stretched portion is penetrated and held in the through hole of the third stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode.
And / or
(2) The other end of the second electrode has a fourth stretched portion that extends from the solution accommodating portion to the outside of the base material.
The base material includes a fourth stretched portion through hole for penetrating and holding the fourth stretched portion.
The fourth stretched portion is penetrated and held through the through hole of the fourth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode [1] or [2]. ] The device described in.
[4] (1) The other end of the first electrode has a fifth stretched portion extending from the solution accommodating portion to the base material.
The base material is
A through hole for the fifth stretched portion for penetrating and holding the fifth stretched portion,
The first accommodating portion accommodating the end portion of the fifth extending portion and
Including
The fifth stretched portion is penetrated and held in the through hole of the fifth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode.
And / or
(2) The other end of the second electrode has a sixth stretched portion extending from the solution accommodating portion to the base material.
The base material is
A through hole for the sixth stretched portion for penetrating and holding the sixth stretched portion,
A second accommodating portion accommodating the end portion of the sixth extending portion and
Including
The sixth stretched portion is penetrated and held through the through hole of the sixth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode [1] or [2]. ] The device described in.
[5] The device according to the above [1] or [2], wherein a protrusion protruding from the first surface of the base material is formed on the first electrode and / or the second electrode.
[6] A step portion is formed on the outer peripheral portion of the first surface of the base material, and a step portion is formed.
The device according to any one of the above [1] to [5], wherein the step portion is formed so as to have a step in a direction opposite to the second surface of the base material.

The device disclosed in this application can keep the distance between the electrodes substantially the same even if the number of high-temperature pressurization treatments increases. In addition, the risk of breakage due to impact during handling is reduced compared to devices using glass.

1A to 1C are diagrams for explaining the device 1a according to the first embodiment. 2A to 2C are diagrams for explaining a modification 1 of the device 1a according to the first embodiment. 3A to 3C are diagrams for explaining the second modification of the device 1a according to the first embodiment. 4A to 4C are diagrams for explaining the device 1d according to the second embodiment. 5A to 5C are diagrams for explaining the device 1e according to the third embodiment. 6A to 6C are diagrams for explaining the device 1f according to the fourth embodiment. 7A to 7C are diagrams for explaining a modification 1 of the device 1f according to the fourth embodiment. FIG. 8 is a drawing substitute photograph, which is a photograph of the device produced in Example 1. FIG. 9 is a drawing substitute photograph, which is a photograph of the device of Comparative Example 1.

Hereinafter, the embodiment of the device will be described in detail with reference to the drawings. In the present specification, members having the same type of function are designated by the same or similar reference numerals. Then, the repeated description of the members with the same or similar reference numerals may be omitted.

In addition, the position, size, range, etc. of each configuration shown in the drawing may not represent the actual position, size, range, etc. for easy understanding. Therefore, the disclosure in this application is not necessarily limited to the position, size, range, etc. disclosed in the drawings.

(Definition of direction)
In the present specification, the direction in which the first electrode and the second electrode are inserted into the first electrode insertion groove and the second electrode insertion groove is defined as the Z direction. The Z direction is, for example, a vertical downward direction, but the Z direction is not limited to the vertical downward direction.

(First Embodiment of Device 1)
A first embodiment of the device 1 will be described with reference to FIG. FIG. 1A is a schematic top view of the device 1a according to the first embodiment. FIG. 1B is a schematic cross-sectional view of FIG. 1A in the XX'direction. FIG. 1C is a schematic cross-sectional view showing a state in which the electrode is separated from the base material in the XX'direction of FIG. 1A.

The device 1a according to the first embodiment includes a base material 2, a solution accommodating portion 3, a first electrode 4, a second electrode 5, a first electrode insertion groove 6, a second electrode insertion groove 7, and the like. At least include.

The base material 2 is made of resin and has a base material first surface 21 and a base material second surface 22 which is a surface opposite to the base material first surface 21.

The solution accommodating portion 3 has an opening 31 on the first surface 21 of the base material, and is formed in the direction from the first surface 21 of the base material to the second surface 22 of the base material. The solution accommodating portion 3 has a first wall surface 32, a second wall surface 33 formed so as to be substantially equal to the first wall surface 32, and a bottom surface 34. The solution accommodating portion 3 is covered with the base material 2 except for the opening 31 provided on the first surface 21 of the base material.

The first electrode 4 has an electrode first surface 41 and an electrode second surface 42 which is a surface opposite to the electrode first surface 41. Similarly, the second electrode 5 has an electrode first surface 51 and an electrode second surface 52 which is a surface opposite to the electrode first surface 51. As shown in FIG. 1C, the electrode second surface 42 of the first electrode 4 and the electrode second surface 52 of the second electrode 5 face each other.

The first electrode insertion groove 6 is formed for inserting and holding the first electrode 4. The first electrode insertion groove 6 has an insertion groove first surface 61 and an insertion groove second surface 62 which is a surface opposite to the insertion groove first surface 61. The first electrode insertion groove 6 is formed in the direction from the bottom surface 34 to the second surface 22 of the base material so as to be substantially parallel to the first wall surface 32. In the example shown in FIG. 1C, the insertion groove first surface 61 is the same plane extending from the first wall surface 32 (in other words, substantially the same plane as the first wall surface 32). Therefore, when the first electrode 4 is inserted into the first electrode insertion groove 6, the electrode first surface 41 of the first electrode 4 becomes the first wall surface 32 of the solution accommodating portion 3 and the insertion groove first of the first electrode insertion groove 6. It can be arranged along the surface 61 (so as to abut), and the first electrode 4 can be stably arranged. Note that FIG. 1C is merely an example, and there may be a slight step between the first wall surface 32 and the first surface 61 of the insertion groove of the first electrode insertion groove 6. In other words, the electrode first surface 41 of the first electrode 4 may be arranged so as to face the first wall surface 32 of the solution accommodating portion 3 and the insertion groove first surface 61 of the first electrode insertion groove 6.

Further, the first electrode 4 is inserted and held in the first electrode insertion groove 6 so that the second electrode surface 42 cannot move in the direction of the second wall surface 33. In order to make the electrode second surface 42 of the first electrode 4 immovable in the direction of the second wall surface 33, the width of the first electrode 4 (distance between the electrode first surface 41 and the electrode second surface 42) and the first electrode The width of the insertion groove 6 (distance between the first surface 61 of the insertion groove and the second surface 62 of the insertion groove) may be substantially the same. In the present specification, "immovable" means that the distance between the first electrode 4 and the second electrode 4 does not change at all physically, and the set electrical conditions do not affect the experiment. If it is a change of, it is included in the concept of "immobility". For example, the change in the distance between the first electrode 4 and the second electrode 4 (the distance between the second electrode surface 42 and the second electrode surface 52) is, for example, 10% or less, 8% or less, as compared with the time of fabrication. If it is 6% or less, 4% or less, 2% or less, or 1% or less, it is included in the scope of "immobility" in the present specification.

The second electrode insertion groove 7 is formed to insert and hold the second electrode 5. The second electrode insertion groove 7 has an insertion groove first surface 71 and an insertion groove second surface 72 which is a surface opposite to the insertion groove first surface 71. The second electrode insertion groove 7 is formed in the direction from the bottom surface 34 to the second surface 22 of the base material so as to be substantially parallel to the second wall surface 33. In the example shown in FIG. 1C, the insertion groove first surface 71 is the same plane extending from the second wall surface 33 (in other words, substantially the same plane as the first wall surface 32). Therefore, when the second electrode 5 is inserted into the second electrode insertion groove 7, the electrode first surface 51 of the second electrode 5 becomes the second wall surface 33 of the solution accommodating portion 3 and the insertion groove first of the second electrode insertion groove 7. It can be arranged along the surface 71 (so as to abut), and the second electrode 5 can be stably arranged. Note that FIG. 1C is merely an example, and there may be a slight step between the second wall surface 33 and the insertion groove first surface 71 of the second electrode insertion groove 7. In other words, the electrode first surface 51 of the second electrode 5 may be arranged so as to face the second wall surface 33 of the solution accommodating portion 3 and the insertion groove first surface 71 of the second electrode insertion groove 7.

Further, the second electrode 5 is inserted and held in the second electrode insertion groove 7 so that the second electrode surface 52 cannot move in the direction of the first wall surface 32. In order to make the electrode second surface 52 of the second electrode 5 immovable in the direction of the first wall surface 32, the width of the second electrode 5 (distance between the electrode first surface 51 and the electrode second surface 52) and the second electrode The width of the insertion groove 7 (the distance between the first surface 71 of the insertion groove and the second surface 72 of the insertion groove) may be substantially the same. "Immovable" is as defined above.

Since the first electrode 4 and the second electrode 5 are made of metal, they are not easily deformed. Therefore, by inserting and holding the first electrode 4 in the first electrode insertion groove 6 and inserting and holding the second electrode 5 in the second electrode insertion groove 7, the second surface of the electrodes facing each other in the solution accommodating portion 3 The distance between the 42 and the second surface 52 of the electrode is difficult to change. In the example shown in FIG. 1C, the first electrode 4 and the second electrode 5 are cantilevered in the first electrode insertion groove 6 and the second electrode insertion groove 7, respectively. The higher the ratio of the cantilevered portion, the more stable the first electrode 4 and the second electrode 5 are held. Therefore, the length of the first electrode 4 and the second electrode 5 in the Z-axis direction and the first The relationship between the depths of the electrode insertion groove 6 and the second electrode insertion groove 7 in the Z-axis direction may be appropriately adjusted within a range in which the object disclosed in the present specification can be achieved. The following numerical values are merely examples and are not limited, but for example, when the length of the first electrode 4 (second electrode 5) in the Z-axis direction is 1, the first electrode insertion groove 6 (third electrode 5) is used. The length of the portion to be inserted into the two-electrode insertion groove 7) (in other words, the depth of the first electrode insertion groove 6 and the second electrode insertion groove 7) is 0.05 to 0.5 and 0.07 to 0. It can be 0.3, 0.1 to 0.2.

The device 1a according to the first embodiment may optionally additionally insert one or more spacers 35 into the solution accommodating portion 3 so as to abut the electrode second surface 42 and the electrode second surface 52. .. By inserting the spacer 35 into the solution accommodating portion 3, the distance between the second electrode surface 42 and the second electrode surface 52 becomes more difficult to change, so that the length of the first electrode 4 (second electrode 5) in the Z-axis direction is longer. The ratio of the length of the portion to be inserted into the first electrode insertion groove 6 (second electrode insertion groove 7) can be made smaller than that in the above example. The spacer 35 is not particularly limited as long as it is a non-conductive material, and for example, the same material as the base material 2 described later can be used.

Since it is preferable that the base material 2 does not deform even after repeated high-temperature pressure treatment (about 120 ° C., about 2 atm) and can be observed with a microscope, there is no particular limitation as long as it is a transparent resin. Examples thereof include, but are not limited to, polymethylpentene (PMP), polyarylate (PAR), polyallyl sulphon (PASF), polysulfone (PSU), polycarbonate (PC) and the like.

The first electrode 4 and the second electrode 5 are not particularly limited as long as they are metals generally used in the fields of cell fusion and electroporation. For example, stainless steel, platinum, aluminum, gold, carbon and the like can be mentioned.

In the device 1a, for example, the base material 2 is machined to form the solution accommodating portion 3, the first electrode insertion groove 6 and the second electrode insertion groove 7, and the first electrode 4 and the second electrode 5 prepared separately are formed. It may be produced by inserting and holding it. Alternatively, injection molding or a 3D printer may be used instead of cutting.

When the device 1a according to the first embodiment is used, the solution accommodating portion 3 is filled with (1) cells fused with the conductive solution in the case of cell fusion, and (2) electroporation. In the case, it is filled with a conductive solution and an introductory substance such as cells and DNA. Then, electricity may be applied while the electrodes of the power supply device (not shown) are in contact with the first electrode 4 and the second electrode 5.

The device 1a according to the first embodiment has an electrode second surface 42 and a second electrode of the first electrode 4 facing each other in the solution accommodating portion 3 without using the adhesive used in the conventional device fabrication. The distance between the electrode 5 and the second surface 52 of the electrode 5 is difficult to change. Therefore, even if the device 1a is subjected to a high-temperature pressurization treatment, the electrical conditions are unlikely to change. Further, as compared with a device using glass, there is an effect that the risk of breakage due to an impact during handling is reduced.

(Modification 1 of device 1a according to the first embodiment)
A modification 1 of the device 1a according to the first embodiment will be described with reference to FIG. FIG. 2A is a schematic top view of the device 1b according to the first modification. 2B is a schematic cross-sectional view of FIG. 2A in the X1-X1'direction, and FIG. 2C is a schematic cross-sectional view of FIG. 2A in the X2-X2' direction.

The device 1b according to the first embodiment will be described in detail below, but the description will focus on points different from those of the first embodiment, and will be a repetitive description of the matters already explained in the first embodiment. Is omitted. Therefore, it goes without saying that the matters explained in the first embodiment can be adopted even if they are not explicitly explained in the embodiment of the device 1b according to the first embodiment. Further, in the modified examples 2 and 3, the second to fourth embodiments, and the other embodiments, which will be described later, the points different from the described embodiments will be mainly described, and the explained matters will be omitted. However, it is similar that the items explained can be adopted.

In the device 1b according to the modified example 1 shown in FIG. 2, a first protruding portion 43 is formed in order to facilitate connecting the electrode of the power supply device to the first electrode 4, and similarly, the second protruding portion 5 is formed on the second electrode 5. It is the same as the device 1a according to the first embodiment except that the portion 53 is formed. When the device 1b according to the first modification is used, for example, it may be sandwiched between the alligator clips of the electrodes of the power supply device, so that the effect of improving the operability can be obtained. If the first protruding portion 43 and the second protruding portion 53 are too close to each other, the alligator clip or the like may come into contact with each other. Therefore, it is desirable to form the first protruding portion 43 and the second protruding portion 53 at distant positions rather than opposite positions. In the example shown in FIG. 2, the first protruding portion 43 and the second protruding portion 53 are formed, but any one of the first protruding portion 43 and the second protruding portion 53 may be formed.

(Modification 2 of device 1a according to the first embodiment)
A modification 2 of the device 1a according to the first embodiment will be described with reference to FIG. FIG. 3A is a schematic top view of the device 1c according to the second modification. FIG. 3B is a schematic cross-sectional view of FIG. 3A in the X1-X1'direction.

As shown in FIGS. 3A and 3B, the device 1c according to the second modification has the first embodiment except that the step portion 23 is formed on the outer peripheral portion of the first surface 21 of the base material of the base material 2. This is the same as the device 1a. When conducting an experiment using the device 1, the solution accommodating portion 3 may be filled with a solution and then covered with a cover glass or the like. Even when the device 1a according to the first embodiment is used, it is possible to cover it with the cover glass, but the cover glass may deviate from the first surface 21 of the base material. On the other hand, in the device 1c according to the modified example 2 shown in FIG. 3, the step portion 23 has a step in the direction opposite to the second surface 22 of the base material (in other words, the direction from the second surface 22 of the base material toward the opening 31). Since it is formed so as to have a cover glass, the cover glass is less likely to slip when covered with the cover glass.

The step portion 23 does not come off when covered with the cover glass, but it is desirable to form the step portion 23 so that it can be easily attached and detached. In the example shown in FIG. 3A, the pair of stepped portions 23 are formed so as to be substantially parallel along the outer circumference of the first surface 21 of the base material in the longitudinal direction, but the distance between the pair of stepped portions 23 is the device. Both ends (L1) in the longitudinal direction are shorter than the distance (L2) at the center of 1c. Further, a step portion 23 is not formed on a part of both end portions. Therefore, the cover glass attached to the device 1c is hard to come off, but when it is removed, a finger is put on the end portion of the cover glass, so that the cover glass can be easily removed.

The example shown in FIG. 3A is only an example of the step portion 23. Other embodiments may be used as long as they cannot be removed when covered with the cover glass, but can be formed so that they can be easily attached and detached. For example, although not shown, a plurality of stepped portions 23 may be arranged at predetermined intervals so that the distance between the stepped portion 23 and the stepped portion 23 is shorter than the length of the corresponding side of the cover glass.

Further, the examples shown in FIGS. 3A and 3B show an example in which one step portion 23 is formed on the first surface 21 of the base material. Alternatively, as shown in FIG. 3C, the stepped portion 23 may be formed in a plurality of stages. For example, in the case of the device 1b according to the deformability 1 shown in FIG. 2, a first protruding portion 43 is formed on the first electrode 4, and a second protruding portion 53 is formed on the second electrode 5. Therefore, the first stepped portion 23a higher than the first protruding portion 43 and the second protruding portion 53 may be formed first, and the second stepped portion 23b may be formed on the outer periphery of the first stepped portion 23a. In the example shown in FIG. 3C, the cover glass can be attached without contacting the first protruding portion 43 and the second protruding portion 53. In the case of the example shown in FIG. 3C, an electrode insertion hole 24 for inserting an electrode of the power supply device may be formed in the first step portion 23a and / or the second step portion 23b.

The step portion 23 (first step portion 23a and second step portion 23b) may be formed at the same time when the base material 2 is cut or injection molded. Alternatively, it may be formed separately from the base material 2 and fixed with screws or the like.

When the device 1c related to the deformability 2 is used, the convenience of using the cover glass is also improved.

(Device 1d according to the second embodiment)
The device 1d according to the second embodiment will be described with reference to FIG. FIG. 4A is a schematic top view of the device 1d according to the second embodiment. 4B is a schematic cross-sectional view of FIG. 4A in the X1-X1'direction, and FIG. 4C is a schematic cross-sectional view of FIG. 4A in the X2-X2' direction. The cross-sectional view of FIG. 4A in the XX'direction is the same as that of FIG. 1B, and is therefore omitted.

The device 1d according to the second embodiment has a first stretching portion 44 extending from the solution accommodating portion 3 to the inside of the base material 2 at one end of the first electrode 4. Further, the base material 2 is formed with a first stretched portion insertion hole 25a for inserting and holding the first stretched portion 44. The first stretched portion 44 is inserted into the first stretched portion insertion hole 25a so as to be immovable in a substantially vertical direction (direction indicated by an arrow in FIG. 4B) with respect to the electrode first surface 41 and the electrode second surface 42.・ Retained. FIG. 4B shows a state in which the first stretched portion 44 is inserted into the first stretched portion insertion hole 25a, but in order to prevent the first stretched portion 44 from moving in the direction indicated by the arrow in FIG. 4B. In the same manner as in the example shown in FIG. 1C, the width of the first stretched portion 44 (distance between the electrode first surface 41 and the electrode second surface 42) and the width of the first stretched portion insertion hole 25a (in the arrow of FIG. 4B). The distances in the indicated directions) may be substantially the same.

Further, the device 1d according to the second embodiment has a second stretching portion 54 extending from the solution accommodating portion 3 to the inside of the base material 2 at one end of the second electrode 5. Further, the base material 2 is formed with a second stretched portion insertion hole 25b for inserting and holding the second stretched portion 54. The second stretched portion 54 is inserted into the second stretched portion insertion hole 25b so as to be immovable in a substantially vertical direction (direction indicated by an arrow in FIG. 4C) with respect to the first surface 51 of the electrode and the second surface 52 of the electrode.・ Retained. Similar to the first stretched portion 44, in order to prevent the second stretched portion 54 from moving in the direction indicated by the arrow in FIG. 4C, the width of the second stretched portion 54 (the first electrode surface 51 and the second electrode). The distance of the surface 52) and the width of the second extending portion insertion hole 25b (distance in the direction indicated by the arrow in FIG. 4C) may be substantially the same.

In the example shown in FIG. 4A, the first stretched portion 44 and the second stretched portion 54 are formed at the ends of the first electrode 4 and the second electrode 5 in opposite directions. Alternatively, the first stretched portion 44 and the second stretched portion 54 may be formed at the ends of the first electrode 4 and the second electrode 5 in the same direction.

Further, in the example shown in FIG. 4A, an example in which one extension portion is formed at each end of the first electrode 4 and the second electrode 5 is shown. Alternatively, one stretched portion may be formed at one end of the first electrode 4 and the second electrode 5. Further, as an alternative, extension portions may be formed at both ends of the first electrode 4 and both ends of the second electrode 5.

The device 1d according to the second embodiment cannot move in the direction in which the distance between the first electrode 4 and the second electrode 5 changes even in the first stretched portion 44 and / or the second stretched portion 54. Is held in. Therefore, the device 1d according to the second embodiment has an effect that the electrical conditions are less likely to change than the device 1a according to the first embodiment even if the device 1d is subjected to high temperature and high pressure treatment.

(Device 1e according to the third embodiment)
The device 1e according to the third embodiment will be described with reference to FIG. FIG. 5 is a schematic top view of the device 1e according to the third embodiment. 5B is a schematic cross-sectional view of FIG. 5A in the X1-X1'direction, and FIG. 5C is a schematic cross-sectional view of FIG. 5A in the X2-X2' direction. The cross-sectional view of FIG. 5A in the XX'direction is the same as that of FIG. 1B, and is therefore omitted.

In the device 1e according to the third embodiment, the "first stretched portion 44" of the device 1d according to the second embodiment is referred to as the "third stretched portion 45", and the "second stretched portion 54" is referred to as the "fourth stretched portion". 55 ”,“ 1st stretched portion insertion hole 25a ”is read as“ 3rd stretched portion through hole 26a ”,“ 2nd stretched portion insertion hole 25b ”is read as“ 4th stretched portion through hole 26b ”, and“ 3rd stretched portion through hole 26b ” Similar to the device 1d according to the second embodiment, except that "45" is formed only on one end of the first electrode 4 and "fourth stretched portion 55" is formed only on one end of the second electrode 5. Is.

More specifically, the tips of the first stretched portion 44 and the second stretched portion 54 of the device 1d according to the second embodiment are not exposed to the outside of the base material 2. On the other hand, the third stretched portion 45 and the fourth stretched portion 55 of the device 1e according to the third embodiment are different in that they penetrate the base material 2 and are exposed to the outside of the base material 2.

Further, in the second embodiment, one end of the first stretched portion insertion hole 25a and the second stretched portion insertion hole 25b communicates with the solution accommodating portion 3, but the other end stays inside the base material 2. On the other hand, in the third embodiment, one end of the third stretched portion through hole 26a and the fourth stretched portion through hole 26b communicates with the solution accommodating portion 3, but the other end is the outer peripheral surface of the base material 2 (base material first). It differs in that it reaches the surface (the surface that intersects the surface 21 and the second surface 22 of the base material).

In the device 1e according to the third embodiment, the electrodes of the power supply device can be connected to the third stretched portion 45 and the fourth stretched portion 55 exposed to the outside of the base material 2. Therefore, as compared with the first and second embodiments, there is an effect that the connection with the electrode of the power supply device is easy.

In the example shown in FIG. 5, an example in which the third stretched portion 45 is formed on the first electrode 4 and the fourth stretched portion 55 is formed on the second electrode 5 is shown. Alternatively, only one of the third stretched portion 45 or the fourth stretched portion 55 may be formed. Further, the first stretched portion 44 is formed at the end portion of the first electrode 4 opposite to the third stretched portion 45, and the second stretched portion 44 is formed at the end portion of the second electrode 5 opposite to the fourth stretched portion 55. The portion 54 may be formed.

(Device 1f according to the fourth embodiment)
The device 1f according to the fourth embodiment will be described with reference to FIG. FIG. 6 is a schematic top view of the device 1f according to the fourth embodiment. 6B is a schematic cross-sectional view of FIG. 6A in the X3-X3'direction, and FIG. 6C is a schematic cross-sectional view of FIG. 6A in the X4-X4'direction. The cross-sectional view of FIG. 6A in the XX'direction is the same as that of FIG. 1B, the cross-sectional view of FIG. 6A in the X1-X1'direction is the same as that of FIG. 4B, and the cross-sectional view of FIG. Since it is the same as, it is omitted.

In the device 1f according to the fourth embodiment, the "first stretched portion 44" of the device 1d according to the second embodiment is referred to as the "fifth stretched portion 46", and the "second stretched portion 54" is referred to as the "sixth stretched portion". 56 ”,“ first stretched portion insertion hole 25a ”is read as“ fifth stretched portion through hole 27a ”, and“ second stretched portion insertion hole 25b ”is read as“ sixth stretched portion through hole 27b ”. Then, (1) a first accommodating portion 28a reaching the fifth stretched portion through hole 27a from the outer peripheral surface of the base material 2 (the surface intersecting the first surface 21 of the base material and the second surface 22 of the base material) is formed, and the fifth A point where the end portion of the stretched portion 46 is accommodated in the first accommodating portion 28a, and (2) a second accommodating portion 28b reaching the sixth extending portion through hole 27b from the outer peripheral surface of the base material 2 are formed, and the second accommodating portion 28b is formed. 6 It is the same as the device 1d according to the second embodiment except that the end portion of the stretched portion 56 is accommodated in the second accommodating portion 28b.

More specifically, the tips of the first stretched portion 44 and the second stretched portion 54 of the device 1d according to the second embodiment are embedded in the base material 2. Further, the tips of the third stretched portion 45 and the fourth stretched portion 55 of the device 1e according to the third embodiment penetrate the base material 2 and are exposed to the outside of the base material 2. On the other hand, in the device 1f according to the fourth embodiment, the fifth stretched portion 46 and the sixth stretched portion 56 penetrate a part of the base material 2, but the tip thereof is the first accommodating portion 28a formed on the base material 2. And it is housed in the second housing part 28b. Therefore, similarly to the third stretched portion 45 and the fourth stretched portion 55 of the device 1e according to the third embodiment, in addition to the effect that the connection with the electrode of the power supply device is easy, the fifth stretched portion 46 and the fifth stretched portion 46 and the third stretched portion 55. Since the tip of the 6-stretched portion 56 stays inside the base material 2, there is also an effect that the risk of being caught by other parts and being damaged during handling is reduced as compared with the third embodiment.

In the example shown in FIG. 6, an example in which the fifth stretched portion 46 is formed on the first electrode 4 and the sixth stretched portion 56 is formed on the second electrode 5 is shown. Alternatively, only one of the fifth stretched portion 46 or the sixth stretched portion 56 may be formed. Further, the first stretched portion 44 is formed at the end portion of the first electrode 4 opposite to the fifth stretched portion 46, and the second stretched portion 44 is formed at the end portion of the second electrode 5 opposite to the fifth stretched portion 56. The portion 54 may be formed.

(Other embodiments and modifications)
The above-mentioned first embodiment, modifications thereof, and second to fourth embodiments are merely examples, and may be appropriately modified without departing from the technical concept disclosed in the present specification. For example, any one or more embodiments described above (variants thereof) may be combined.

Further, in the examples shown in FIGS. 1 to 6, the first electrode 4 and the second electrode 5 are formed so as to have substantially the same height as the first surface 21 of the base material. Alternatively, the height of the first electrode 4 and the second electrode 5 may be lower than that of the first surface 21 of the base material. In that case, as shown in FIGS. 7A to 7C, the first accommodating portion 28a and the second accommodating portion 28b of the device 1f according to the fourth embodiment are all covered with the base material 2 except for the opening portion on the outer peripheral surface. (Modification 1 of device 1f according to the fourth embodiment). Therefore, the ends of the fifth stretched portion 46 and the sixth stretched portion 56 of the first modification of the device 1f according to the fourth embodiment shown in FIGS. 7A to 7C relate to the fourth embodiment shown in FIG. From the device 1f, the risk of being caught and damaged by other parts during handling is further reduced. The example shown in FIGS. 7B and 7C may be formed by providing the step portion 23 on the device 1f shown in FIG.

Examples are given below to explain each embodiment in detail, but these examples are provided merely as a reference for the specific aspects. These examples do not limit or limit the scope of the invention.

<Example 1>
[Making a device]
The device was manufactured by the following procedure.
(1) Polymethylpentene is used as the material of the base material 2, and the solution accommodating portion 3, the first electrode insertion groove 6, the second electrode insertion groove 7, the fifth stretched portion through hole 27a, and the sixth stretched portion are processed by cutting. A through hole 27b, a first accommodating portion 28a, and a second accommodating portion 28b were prepared.
(2) Stainless steel was used as the material for the first electrode 4 and the second electrode 5, and the device was manufactured by inserting the first electrode 4 and the second electrode 5 into the machined base material 2. FIG. 8 is a photograph of the device produced in Example 1. The distance between the first electrode 4 and the second electrode 5 of the manufactured device was 1.87, 1.91, 1.90 mm in the order of the left end portion, the center, and the right end portion shown in FIG.

[High temperature pressurization]
<Example 2>
Using an autoclave, treatment at about 120 ° C., 2 atm, for 20 minutes was performed 100 times on the device prepared in Example 1. The distance between the first electrode 4 and the second electrode 5 after 100 times of execution was 1.87, 1.88, 1.89 mm in the order of the left end portion, the center, and the right end portion shown in FIG.

<Comparative example 1>
The high temperature pressurization treatment was repeated in the same procedure as in Example 2 except that the commercially available cell fusion electrode LF497P2 (manufactured by Becks Co., Ltd.) shown in FIG. 9 was used instead of the device of Example 1. However, in the cell fusion electrode LF497P2 used in Comparative Example 1, a pair of electrodes (first electrode 4 and second electrode 5) are fixed with silicon so as to be at equal intervals. Further, the connector for connecting to the power supply device and the pair of electrodes are fixed with solder and then further fixed with an adhesive. When the device of Comparative Example 1 was visually inspected after the high temperature pressurization treatment was performed 10 times, the adhesive was discolored due to deterioration. Further, when the device of Comparative Example 1 was touched immediately after being taken out from the eau de clave, it was confirmed that the distance between the pair of electrodes was easily changed by the movement of the electrodes.

Based on the above results, the device produced in Example 1 does not use an adhesive or silicon for fixing the electrodes, but fixes the electrodes by a structure such as a groove or an insertion hole formed in the base material. It was confirmed that the distance between the first electrode 4 and the second electrode 5 hardly changed even when the high temperature pressurization treatment was repeatedly carried out.

<Example 3>
[Cell fusion experiment]
Next, using the device prepared in Example 1, a cell fusion experiment was performed according to the following procedure.
<Cell preparation>
(1) After emulsifying "BAL6MAP", which is a peptide of Pseudomonas aeruginosa, with Freund's adjuvant, BALB / c mice were immunized. A second immunization was performed at intervals of two weeks or more, and the plasma antibody titer of the mouse was confirmed.
(2) Two weeks later, the mouse whose antibody titer was sufficiently elevated was immunized for the third time (30 weeks after the second immunization), and three days later, the spleen of the mouse was removed, cells were prepared, and cells were prepared. Was cryopreserved. The cryopreserved cells were thawed at a later date, and a cell fusion experiment was performed using spleen cells and P3X, which is a myeloma cell.

<Cell fusion>
(I) Equipment used, reagents, equipment used: Electric cell fusion device CFB16-HB (manufactured by Becks Co., Ltd.)
-Electrodes used: Electrodes of Example 1 after 100 autoclaves-Cell culture medium: RPMI1640 10% FBS
-Cell fusion buffer: 0.3 M mannitol, 0.1 mM MgCl ₂ , 0.1 mM CaCl ₂ , HAT supplement (added at 2%), briclone (added at 5%)

(Ii) Cell fusion procedure (1) The medium and cell fusion buffer were warmed in a constant temperature bath at 37 ° C.
(2) Spleen cells were cell-counted.
(3) Myeloma cells were collected and cell-counted.
(4) Spleen cells: Myeloma cells were mixed at a ratio of 2: 1 and centrifuged at 1500 rpm for 5 min at 4 ° C. (Spleen cells 6x10 ⁶ P3X cells 3x10 ⁶ )
(5) After removing the supernatant, tapping was performed, the cells were suspended in 10 ml of cell fusion buffer, and centrifuged at 1500 rpm, 5 min, and 4 ° C. (Washing)
(6) The above (5) was repeated.
(7) After removing the supernatant, tapping was performed and the cells were suspended in a cell fusion buffer so as to have a concentration of ^{1 × 10 7 / ml.} (0.9mL)
(8) The first electrode 4 and the second electrode 5 of the device prepared in Example 1 were washed twice with a cell fusion buffer, and 0.8 mL of a cell suspension was placed between the electrodes.
(9) The cable of the cell fusion device was connected to the first electrode 4 and the second electrode 5, and the resistance was measured.
(10) Cell fusion was performed under the conditions of ACV: 26V, AC Time: 20s, DCV: 300V, On Time: 30us Off Time: 500ms, DC cycle: 3, Post Time: 7s, Fade: On. The resistance value at this time was 0.904 kΩ.
(11) The device after cell fusion was placed in a sterilized petri dish for incubation and incubated in a _{CO 2 incubator for 10 minutes.}
(12) Cells were collected from the device, co-washed with a cell fusion buffer, and then centrifuged at 1500 rpm for 5 min.
(13) The supernatant was removed, and after tapping, 10 mL of 2% HAT supplement and a medium containing 5% briclon were added, and the mixture was allowed to stand at 37 ° C. for 15 minutes.
(14) The 96-well plate was seeded at 100 μL / well.
(15) HAT supplement and medium containing briclon were added at 100 μL / well. After 8 days, the number of hybridomas formed was measured and the medium was exchanged.

(Iii) As a result of cell fusion, the formation of hybridomas with an average of 3.16 cells per well and a total of 303 cells was confirmed.

From the above results, it was confirmed that the device prepared in Example 1 can be used for cell fusion even if the high temperature pressurization treatment is repeated.

The device disclosed in this application can be processed at high temperature and high pressure. Therefore, it is useful in the fields of cell fusion and electroporation.

1, 1a-1f ... device,
2 ... Base material, 21 ... Base material first surface, 22 ... Base material second surface, 23 ... Step portion, 23a ... First step portion, 23b ... Second step portion, 24 ... Electrode insertion hole, 25a ... First Stretched part insertion hole, 25b ... 2nd stretched part insertion hole, 26a ... 3rd stretched part through hole, 26b ... 4th stretched part through hole, 27a ... 5th stretched part through hole, 27b ... 6th stretched part through hole, 28a ... 1st accommodating part, 28b ... 2nd accommodating part,
3 ... Solution accommodating part, 31 ... Opening, 32 ... First wall surface, 33 ... Second wall surface, 34 ... Bottom surface, 35 ... Spacer,
4 ... 1st electrode, 41 ... Electrode 1st surface, 42 ... Electrode 2nd surface, 43 ... 1st protruding part, 44 ... 1st stretched part, 45 ... 3rd stretched part, 46 ... 5th stretched part,
5 ... 2nd electrode, 51 ... Electrode 1st surface, 52 ... Electrode 2nd surface, 53 ... 2nd protruding part, 54 ... 2nd stretched part, 55 ... 4th stretched part, 56 ... 6th stretched part,
6 ... 1st electrode insertion groove, 61 ... Insertion groove 1st surface, 62 ... Insertion groove 2nd surface,
7 ... 2nd electrode insertion groove, 71 ... Insertion groove 1st surface, 72 ... Insertion groove 2nd surface

Claims

A device used for cell fusion or electroporation
The device is
With the base material
Solution container and
With the 1st and 2nd electrodes,
The first electrode insertion groove and the second electrode insertion groove,
Including
The base material is
Formed of resin,
It has a first surface of the base material and a second surface of the base material which is a surface opposite to the first surface of the base material.
The solution container is
Formed from the first surface of the base material toward the second surface of the base material,
The first wall surface and
A second wall surface formed so as to be substantially equal to the first wall surface,
On the bottom and
Have,
The first electrode insertion groove is formed from the bottom surface toward the second surface of the base material so as to be substantially parallel to the first wall surface.
The second electrode insertion groove is formed from the bottom surface toward the second surface of the base material so as to be substantially parallel to the second wall surface.
The first electrode and the second electrode are
It has a first surface of the electrode and a second surface of the electrode which is a surface opposite to the first surface of the electrode.
The first electrode is
The first surface of the electrode faces the first wall surface and the first surface of the insertion groove of the first electrode insertion groove.
So that the second surface of the electrode cannot move in the direction of the second wall surface.
Inserted and held in the first electrode insertion groove,
The second electrode is
The first surface of the electrode faces the second wall surface and the first surface of the insertion groove of the second electrode insertion groove.
So that the second surface of the electrode cannot move in the direction of the first wall surface.
A device that is inserted and held in the second electrode insertion groove.
(1) One end of the first electrode has a first stretched portion that extends from the solution accommodating portion to the inside of the base material.
The base material includes a first stretched portion insertion hole for inserting and holding the first stretched portion.
The first stretched portion is inserted and held in the first stretched portion insertion hole so as to be immovable in a substantially vertical direction with respect to the first surface of the electrode and the second surface of the electrode.
And / or
(2) One end of the second electrode has a second stretched portion that extends from the solution accommodating portion to the inside of the base material.
The base material includes a second stretched portion insertion hole for inserting and holding the second stretched portion.
The first aspect of claim 1, wherein the second stretched portion is inserted and held in the insertion hole of the second stretched portion so as to be immovable in a substantially vertical direction with respect to the first surface of the electrode and the second surface of the electrode. Device.
(1) The other end of the first electrode has a third stretched portion that extends from the solution accommodating portion to the outside of the base material.
The base material includes a third stretched portion through hole for penetrating and holding the third stretched portion.
The third stretched portion is penetrated and held in the through hole of the third stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode.
And / or
(2) The other end of the second electrode has a fourth stretched portion that extends from the solution accommodating portion to the outside of the base material.
The base material includes a fourth stretched portion through hole for penetrating and holding the fourth stretched portion.
The first or second aspect of the present invention, wherein the fourth stretched portion is penetrated and held through the through hole of the fourth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode. Device.
(1) The other end of the first electrode has a fifth stretched portion extending from the solution accommodating portion to the base material.
The base material is
A through hole for the fifth stretched portion for penetrating and holding the fifth stretched portion,
The first accommodating portion accommodating the end portion of the fifth extending portion and
Including
The fifth stretched portion is penetrated and held in the through hole of the fifth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode.
And / or
(2) The other end of the second electrode has a sixth stretched portion extending from the solution accommodating portion to the base material.
The base material is
A through hole for the sixth stretched portion for penetrating and holding the sixth stretched portion,
A second accommodating portion accommodating the end portion of the sixth extending portion and
Including
The first or second aspect of the present invention, wherein the sixth stretched portion is penetrated and held through the through hole of the sixth stretched portion so as to be immovable in the substantially vertical direction of the first surface of the electrode and the second surface of the electrode. Device.
The device according to claim 1 or 2, wherein a protrusion protruding from the first surface of the base material is formed on the first electrode and / or the second electrode.
A step portion is formed on the outer peripheral portion of the first surface of the base material, and a step portion is formed.
The device according to any one of claims 1 to 5, wherein the step portion is formed so as to have a step in a direction opposite to the second surface of the base material.