JPH0661267B2 - Cell fusion method and cell fusion device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for fusing two types of cells by using electric pulses to prepare a new heterogeneous cell (hereinafter referred to as heterokaryon).

[0002]

2. Description of the Related Art As a technique for producing a heterokaryon, an electric fusion method and a microelectrode method are known. In the electrofusion method, a cell suspension in which two types of cells are suspended is placed between a pair of parallel electrodes, and an AC voltage is applied between the pair of parallel electrodes to arrange cells in a line. (Hereinafter referred to as pearl chain) is created, and then a direct current pulse is applied to fuse the cells in contact with each other. In the microelectrode method, a thin rod-shaped or needle-shaped electrode is inserted into a micro glass tube to expose the tip of the electrode and to enclose the glass tube wall in a portion other than the tip to enclose the micro electrode. An alternating voltage is applied to this microelectrode by using the above to attract cells in the cell suspension by the induction electrophoretic phenomenon to attach them to the tip of the microelectrode, and an electric pulse is applied to create a heterokaryon.

[0003]

In the above electrofusion method, it is difficult to fuse desired cells to obtain a desired heterokaryon. That is, it is impossible to specifically create new heterogeneous cells A and B by fusing two types of cells A and B having genetically different cell nuclei. Moreover, there is a possibility that three or more cells will be fused, and it is not possible to control that only two cells are fused.

The above-mentioned microelectrode method requires an operation of separating the created heterokaryons, and requires a high degree of skill and a great mental load for the operation of separating the heterokaryons from the microelectrodes. In addition, even a skilled person is liable to damage the fused heterokaryon.

The present invention has been made in view of the above-mentioned circumstances, and is an improvement of the above-mentioned microelectrode method. It is possible to attach only one arbitrary cell to the tip of the electrode and visually confirm the attached state, b. For one cell attached as described above,
Further attaching any one cell, and 2
The adhered state of individual cells can be visually confirmed, and c. Safely and securely holding and transferring one or a plurality of attached cells, and fusing a plurality of held cells together, d. Cell fusion that allows cells to be detached from the electrode without mechanically exerting an operating force on the retained cells (thus, without damaging the cells), and visually confirms the state. It is an object of the present invention to provide a method and a cell fusion device suitable for carrying out the above fusion method.

[0006]

The basic principle of the present invention created to achieve the above object will be briefly described as follows with reference to FIG. 1 corresponding to an embodiment.
That is, the needle electrode 1 is made to face the plate electrode 2 at a substantially right angle. As a result, when a voltage is applied to both electrodes, a non-uniform electric field (an electric field having a difference in density) is formed. The needle-shaped electrode 1 is covered with a tubular insulating cover 3 to form a minute gap G therebetween. In the vicinity of the tip of the needle-shaped electrode 1 where the electric field density locally increases, the inner diameter of the tubular insulating cover is reduced to form the fine holes 3
b is formed. When the electrode (FIG. 1) configured as described above is immersed in a cell suspension and an AC voltage is applied, the suspended individual cells a are attracted to the one having a larger electric field density by the induction electrophoresis phenomenon, It migrates as indicated by the additional arrow and adheres to the vicinity of the hole 3b of the insulating cover 3. If the hole 3a is formed in a size and shape that the cell a cannot pass through, the cell a does not come into contact with the needle electrode 1 and the insulating cover 3
Is attached and held on the outer peripheral surface of the. Since the cells a do not come into contact with the needle-shaped electrode 1, they are not directly charged with electric charge from the needle-shaped electrode 1 and are electrostatically attached and held in the vicinity of the hole 3b. Therefore, when the voltage value of the AC voltage is changed, the holding force changes, and by adjusting this holding force, it is possible to hold only one cell or two cells, and Retaining cells can also be released. It is convenient to perform the above operation within the visual field of the microscope, because the behavior of cells and the progress of the operation can be visually confirmed.

In order to achieve the above-mentioned object (holding, fusing and releasing without damaging arbitrary cells) based on the above-mentioned principle, the cell fusion method according to the present invention is configured to generate a non-uniform electric field. One of the pair of electrodes thus formed is covered with an insulating cover made of an electrically insulating material, and minute holes are provided at a location where the distribution density of the non-uniform electric field is locally increased. A small gap is formed between the electrodes, the pair of electrodes is immersed in a cell suspension, and an AC voltage is applied to cause cells to adhere to the vicinity of the holes of the insulating cover by an induction electrophoretic phenomenon. After the treatment and the cell fusion treatment are finished, the application of the alternating voltage is canceled, and the fusion-treated cells adhering to the vicinity of the hole are detached from the insulating cover.

The cell fusion device according to the present invention, which was created to carry out the above method, has a structure in which a pair of electrodes configured to generate a non-uniform electric field and the pair of electrodes are An insulating cover made of an electrically insulating material for covering one of the electrodes, a minute hole provided in the insulating cover at a location where the distribution density of the nonuniform electric field is locally increased, and the one electrode A minute gap formed between the pair of electrodes and the insulating cover, a liquid tank for storing a cell suspension in which the pair of electrodes are dipped, and an AC power supply for applying an AC voltage to the pair of electrodes. And a DC power supply for applying a DC pulse voltage to the pair of electrodes.

[0009]

According to the above cell fusion method, one cell can be selectively attached to the insulating cover, and a holding force suitable for attaching two cells by increasing the AC applied voltage is further provided. As a state of having, it is possible to attach another cell and hold the above two cells in contact with each other. As described above, when cells of interest are attracted and adsorbed one by one so as to obtain the desired heterokaryon, and a direct current pulse is applied to break the cell membrane, they are in contact with each other.
Individual cells fuse. After that, when the application of the AC voltage is canceled, the fused cells that have adhered are detached from the insulating cover. In order to perform the above-mentioned withdrawal action only by voltage control without applying mechanical force and therefore without damaging cells, a. As shown in FIG. 1, the cell a cannot directly contact the needle-shaped electrode 1, and b. As shown in FIG. 1, it is necessary that a minute gap G is formed between the needle-shaped electrode 1 and the insulating cover 3 and that the above-mentioned configurations a and b are used. If the tip of the insulating cover 3 has a hemispherical surface 3a,
The cells a and the insulating cover 3 come into contact with each other in a state close to point contact,
Since it does not come into contact with a wide surface, the detaching action is further facilitated. The reason why the presence of the microscopic gap G in the above item (b) facilitates cell detachment has not yet been theoretically elucidated, but empirical facts confirmed by the present inventors by carefully repeating experiments. In addition, a substance having a high dielectric constant (insulating cover 3) is not brought into close contact with the needle-shaped electrode 1 and a high substance is provided through a thin layer of a substance having a low dielectric constant and conductivity (cell suspension). It is presumed that the electric field distribution for obtaining preferable results is formed by facing the material having the dielectric constant (insulating cover). When carrying out the present invention, it is necessary that the liquid such as the cell suspension or the culture solution flows into the minute gap G so that the liquid is filled in the minute gap G. .

[0010]

EXAMPLES Next, using the cell fusion device according to the present invention,
An example of carrying out the cell fusion method according to the present invention will be described. FIG. 1 is a sectional view of a main part in one embodiment of a cell fusion device according to the present invention, and 2 is a sectional view taken along line II-II thereof.
Reference numeral 1 is a needle-shaped electrode, the tip of which is sharply polished. The needle electrode is made to face the plate electrode 2 at a substantially right angle.
In carrying out the present invention, the plate-shaped electrode 2 does not necessarily have to be a flat plate, and a conductive member having an electrode surface significantly wider than the tip of the needle-shaped electrode 1 is sufficient. Reference numeral 3 denotes an insulating cover made of a glass tube, which is fixed to each other so as to be concentric with the needle electrode 1, and when the needle electrode moves, this insulating cover also moves together. It has become. The tip of the glass-made tubular insulating cover (specifically, the edge on the same side as the sharp tip of the needle-shaped electrode 1) is polished to a hemispherical surface 3a. Good results are obtained by setting the diameter of the spherical surface between 20 and 100 microns. If the diameter is larger than this, the effect of facilitating the detachment of cells becomes insufficient, and if the diameter is smaller than this, the setting relationship with the hole 3b described below becomes difficult. Insulation cover 3 made of glass
A fine hole 3b is provided at the tip of the concentric with the center line.
This hole 3b is inevitably located at a position facing the tip of the needle-shaped electrode 1, and the electric field density is extremely large in the non-uniform electric field formed by the pair of electrodes (1, 2). It will be located in the place. The diameter of this hole 3b is made smaller than the diameter of the cell to be handled, and if the diameter of the cell to be handled is unequal, it is made smaller than the smallest of them. In short, the fine holes 3b are provided so that the cells a do not pass through. If the type or property of the cell to be handled cannot be specified, setting the diameter of the hole 3b to 5 to 30 microns provides versatility except in special cases. Most of the instruments used for cell fusion require a sterilization operation, but if the insulating cover is made of a glass material as in the present embodiment, there is no risk of softening and deformation even when subjected to high temperature sterilization. Moreover, even if it is subjected to ultraviolet sterilization, there is no possibility of deterioration such as synthetic resin. In addition, stains are less likely to adhere, and when stains are present, the degree of stains can be easily identified visually, and the stains can be easily cleaned. Moreover, the glass material is convenient because the hemispherical surface 3a and the fine holes 3b can be processed by applying a so-called glassworking technique. However, the practice of the invention does not prohibit the use of electrically insulating materials (eg, ceramics) other than glass. The needle electrode 1 is
A tip of a wire having a diameter of 0.1 mm to 1.0 mm is ground to be sharpened and inserted into the insulating cover 3. The size of the illustrated minute gap G is 1 to 50 microns, preferably 20 microns.

FIG. 3 is a schematic explanatory view showing a mechanism for applying a voltage to the pair of electrodes 1, 2. 4 in the figure
Reference numeral a is a DC power supply, which has a function of generating a DC pulse voltage. Also, 4b in the figure is an AC power source,
It has a function of generating an alternating voltage of 0.1 megahertz to 1.0 megahertz and a mechanism for adjusting the voltage value. The DC pulse generated from the DC power supply 4a and the AC voltage generated from the AC power supply 4b are generated by the switch 5
The opening / closing and switching are controlled by the
It is applied to the pair of electrodes 1, 2. The electrical operating state is monitored and recorded by the measuring unit 6. The pair of electrodes 1 and 2 has the structure described with reference to FIG.
It is immersed in the cell suspension 9 in the liquid tank 8. More specifically, the electrodes 1 and 2 are supported by manipulators (small robots) not shown in the figure, the distance between the electrodes can be adjusted, and both electrodes are kept in a pair. The structure is such that the liquid tank 8 can be moved together with it. The liquid tank 8 of this example is made of transparent glass. When the present invention is carried out, at least the bottom surface (correctly, the bottom wall) of this liquid tank is made of a transparent member, and is installed on the stage 11 of the inverted microscope 10. Although not shown, a means for illuminating the vicinity of the electrodes 1 and 2 is provided so that the area sandwiched between the electrodes 1 and 2 can be enlarged and observed.
It is desirable that the magnification of the microscope can be changed according to the type of plant to be fused, and the magnification is 100 to 40 times.
A range of 0 times is commonly used. The above-mentioned liquid tank 8 is provided with partition ribs 8a, 8 having a partition lower than the liquid surface of the cell suspension therein.
It is divided into three sections A ', B', and C'by b. This is a compartment A ′ for storing the isolated cell a of the cell A and a compartment B ′ for storing the isolated cell b of the cell B.
And a compartment C'for releasing the heterokaryons a and b in which the cells A and B are fused into the culture solution, and the number of compartments when the present invention is carried out is not necessarily 3 compartments. Although not limited thereto, it is desirable to divide the 3 combs into more. The movement of the electrodes 1 and 2 by the manipulator (not shown) described above has a structure capable of moving between these compartments below the liquid surface. In this case, all of the electrodes 1 and 2 do not necessarily have to be submerged below the liquid surface, but at least the tip of the needle electrode 1 and the vicinity of the hole 3b of the insulating cover can move below the liquid surface. There must be. This is because the isolated cells attached to the tip of the insulating cover 3 are kept in the liquid without being exposed to the air.

An example of performing a cell fusion operation using the cell fusion device configured as described above will be described below with reference to the accompanying drawings. For convenience of explanation, it is assumed that two different types of cells are cell A and cell B, and one of the isolated cells is represented by a and b. In the present embodiment, the cultured cells obtained from the seeds of grass (Zoysia japonica) and cells A, Zoysia tenuifolia (Zo
The cells collected from the tip of the runner of ysia matrella were designated as cell B. As the cultured cells A of Shiva, seeds were placed on a medium containing plant hormones, and the obtained callus was shaken in a liquid medium to obtain suspension cells. On the other hand, the cell B of Korushishiba cleaves the tip of the runner, and hypertonic solution (0.5M mannitol, calcium chloride
Shake with water salt) for 30 minutes to 1 hour. 3% cellulase RS, 2% each of these cells A and B
Macerozyme R-10, 0.1% pectolyase Y-2
Isolation was performed by treatment with an enzyme solution containing 3, 0.5 M mannitol (both are trade names) at 26 ° C. and 30 rpm for 3 to 6 hours to obtain naked cells (protoplasts). (Fig. 3
(Reference) A compartment A'in the liquid tank 8 is filled with a cell suspension containing a large number of isolated cells a of Shiva obtained as described above.
Since the isolated cells a have a larger specific gravity than the liquid, they do not cross the partition ribs 8a and stay in the compartment A '. Then, the cell suspension containing a large number of the isolated cells b of Korrushiba is placed in the compartment B ′. In this manner, the electrodes 1 and 2 are moved into the compartment A ', the plate-like electrode 2 is fixedly supported in the compartment A', and the needle electrode 1 is movable.
Place it in the field of view of 0 and adjust the illumination to enable observation of a magnified image. (See FIG. 4) When an alternating voltage is applied to the needle-shaped electrode 1 and the plate-shaped electrode 2, a non-uniform electric field is induced because the electrode having the sharp tip faces the electrode having the surface. For this reason, the isolated cell a is attracted toward a portion having a high electric field density (induced electrophoretic phenomenon), and adheres to the vicinity of the hole 3b as indicated by an additional arrow. Since this portion is polished into a spherical shape, the isolated cell a is not damaged. In this case, when a large number of isolated cells are attracted and adhered, they become tufts of grapes, which hinders the subsequent fusion operation. The voltage is controlled to be suitable for adsorbing and holding. This operation is performed in the transparent liquid tank 8 on the microscope stage 11 and observed by the microscope 10. In this way, when adjusting the AC voltage to control the number of isolated cells a to be attached,
It is also possible to adjust the electrode interval dimension of the pair of electrodes 1 and 2. However, in the present example, the adhesion force of the isolated cells was adjusted by adjusting the voltage value of the AC voltage, and the adjustment of the dimension between the electrodes was used when applying the DC pulse voltage described later with reference to FIG. 7. (See FIG. 3) The pair of electrodes 1 and 2 are positioned in the compartment A ′, and one isolated cell a is attached to the tip of the insulating cover 3 as described above (FIG. 4). A route is selected so that the detached cells a are not exposed on the liquid surface, that is, the pair of electrodes 1 and 2 is moved to the compartment B ′ (FIG. 3) while keeping the hole 3b of the insulating cover 3 below the liquid surface. , Isolated cell a
Are transferred into compartment B '. The isolated cells b are previously placed in this compartment B '. (See Figure 5) At this stage,
Since the voltage value of the applied AC voltage is a value suitable for attaching and holding one isolated cell, the voltage is appropriately raised to a level at which two isolated cells can be attached and held. Then, in addition to the already attached isolated cell a, another isolated cell b is attracted and attached. This operation state can also be observed by the microscope 10 described above.
FIG. 6 is a schematic view showing a state in which one isolated cell a and one isolated cell b are in contact with each other and attached near the tip of the insulating cover 3 (on the hemispherical surface 3a, near the hole 3b). In this state, the AC voltage is continuously applied.In the state of Fig. 6, the switch 5 (Fig. 3) is operated to apply a DC pulse to the pair of electrodes 1 and 2. A voltage is applied to fuse the isolated cells a and b. In this operation, the electrode gap size of the pair of electrodes 1 and 2 is adjusted so that the distribution gradient of the DC pulse voltage is the cell A, The value should be an appropriate value (for example, 0.1 to 10 kilovolts / centimeter) according to the type of B. After this fusion operation, the application of the alternating voltage is continued to show the fused heterokaryon. 7
It is attached to the tip of the insulating cover 3 as shown in FIG. In this state, let stand for 30 seconds to 100 seconds (preferably 30 seconds) and wait until the heterokaryon returns to a spherical shape.
Although the cell suspension 9 is stored in the liquid tank 8,
Compartment C'has no isolated cells.

When the isolated cells a and b are contacted and DC pulses are applied (fused) in the section B'of the liquid tank 8 shown in FIG. , The pair of electrodes 1 and 2 are moved again, and the fused cells (heterokaryon) a and b (FIG. 7) attached to the insulating cover 3 are partitioned beyond the partition ridges 8b so as not to be released into the air. When it is transferred to C'and the application of the AC voltage is canceled, the heterokaryons a and b are released from the insulating cover 3 and released into the culture solution as shown in FIG. Since the portion where the isolated cells or the heterokaryons adhere to the insulating cover 3 is ground to the smooth hemispherical surface 3a, there is no risk of the adhered cells or the heterokaryons being damaged. Further, since the heterokaryon is in contact with the hemispherical surface 3a while being attached to the insulating cover 3, the contact area is extremely small. Therefore, when the application of the AC voltage is canceled, the heterocaryon can be easily detached, and it is not necessary to mechanically assist the detachment, and the heterokaryon can be detached without damaging the heterokaryon.

The action and effect clarified by the above-described embodiment is obtained as shown in FIG.
The needle-shaped electrode 1 is covered with an insulating cover 3 via a minute gap G, (b) a fine hole 3b is provided at the tip of the insulating cover 3, and (c) a voltage is applied to the needle-shaped electrode 1. Is applied to induce a non-uniform electric field, and the present invention creates an auxiliary structure that promotes the effects of the configurations of (a), (b), and (c). At the same time, by utilizing the unique effects obtained by the configurations of (a), (b), and (c), an operation method that further enhances its practical value is created. Above (a), (b),
By applying the configuration of (c), it becomes possible to retain any one isolated cell by utilizing the induction electrophoretic phenomenon (a), as in the above-mentioned example,
(B) It becomes possible to attach and retain any one other isolated cell to the retained one isolated cell, and (c) fuse the two retained isolated cells. And (d) it becomes possible to retain and transfer any isolated cells or fused cells, and
(E) The transferred isolated cells or the fused cells can be released without applying mechanical force, and (f) the needle-shaped electrode is used for holding the isolated cells and fusing for fusion. It can also serve three roles of applying voltage and holding fused cells. In this way, one component having many functions can reduce the number of components of the entire apparatus, simplify the structure, and reduce the manufacturing cost.

[0015]

As described above, when the cell fusion method of the present invention is carried out using the cell fusion device of the present invention, a. It is possible to attach only one arbitrary cell to the tip of the electrode and visually confirm the attached state, b. For one cell attached as described above,
Further attaching any one cell, and 2
The adhered state of individual cells can be visually confirmed, and c. Safely and securely holding and transferring one or a plurality of attached cells, and fusing a plurality of held cells together, d. It is possible to detach the cells from the electrode without mechanically exerting an operation force on the retained cells (thus, without damaging the cells), and visually confirm the state. Has excellent practical effects.

[Brief description of drawings]

FIG. 1 is a vertical cross-sectional view illustrating the vicinity of a needle electrode in one example of a cell fusion device according to the present invention.

FIG. 2 is a transverse cross-sectional view showing the vicinity of a needle electrode in one example of the cell fusion device according to the present invention.

FIG. 3 shows one embodiment of the cell fusion device according to the present invention,
It is sectional drawing which added the electrical diagram.

FIG. 4 is a cross-sectional view of an essential part for explaining the operation of one embodiment of the cell fusion device according to the present invention.

FIG. 5 is a cross-sectional view of essential parts for explaining the operation of the embodiment of the cell fusion device according to the present invention.

FIG. 6 is a cross-sectional view of a main part for explaining the operation of one embodiment of the cell fusion device according to the present invention.

FIG. 7 is a cross-sectional view of an essential part for explaining the operation of one embodiment of the cell fusion device according to the present invention.

FIG. 8 is a cross-sectional view of an essential part for explaining the operation of one embodiment of the cell fusion device according to the present invention.

[Explanation of symbols]

1 ... Needle-shaped electrode, 2 ... Plate-shaped electrode, 3 ... Insulating cover, 3a ...
Hemispherical surface, 3b ... Fine holes, 4a ... DC power supply, 4b ... AC power supply, 5 ... Switch, 6 ... Measuring part, 7 ... Control part, 8 ... Liquid tank, 8a, 8b ... Partition ridge, 9 ... Cell suspension Suspension, 10 ... Microscope, 11 ... Microscope stage, a ... Isolated cell of cell A,
b ... Isolated cell of cell B, A ', B', C '... compartment, G
… A small gap.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 5/14 13/00

Claims (22)

[Claims] 1. A part of a pair of electrodes configured to generate a non-uniform electric field is covered with an insulating cover made of an electrically insulating material, and the distribution density of the non-uniform electric field is locally increased. A minute hole is provided, a minute gap is formed between the one electrode and the insulating cover, the pair of electrodes is immersed in a cell suspension, and an AC voltage is applied to induce cell migration by an induction electrophoresis phenomenon. Is attached near the hole of the insulating cover, cell fusion treatment is performed, and after the cell fusion treatment is finished, the application of the AC voltage is canceled, and the fusion treatment attached near the hole is completed. A method of cell fusion, characterized in that the cells of the above are detached from the insulating cover. 2. One of the pair of electrodes that generate the non-uniform electric field is a needle-shaped electrode, and an insulating cover that covers the needle-shaped electrode is a tubular member attached concentrically with the needle-shaped electrode. The opening of one end of the tubular insulating cover has a minute hole diameter, and the other of the pair of electrodes has an electrode surface substantially orthogonal to the needle electrode. The method for cell fusion according to claim 1, wherein 3. The cell fusion method according to claim 2, wherein the tubular insulating cover is made of glass. 4. The cell according to claim 1, wherein the inner diameter of the minute hole is set to be smaller than the outer diameter of the smallest of the plurality of types of isolated cells to be fused. Fusion method. 5. The cell fusion method according to claim 1, wherein the cell fusion treatment is performed according to the following procedure. a. Immersing a pair of electrodes in a suspension of cells A and applying an AC voltage to attach the isolated cells of cells A near the pores of the insulating cover, b. Without moving the pair of electrodes above the liquid surface, and moving them below the liquid surface into the cell B suspension, c. AC voltage is applied to the pair of electrodes to bring the isolated cells of the cell B into close contact with the isolated cells of the cell A, and d. A DC pulse voltage is applied to fuse the isolated cells of cells A and B described above. 6. The operation for releasing the fusion-treated cells by canceling the application of the alternating voltage is performed by moving the pair of electrodes into the culture medium without pulling them up to the liquid surface. The cell fusion method according to claim 5, wherein 7. The above-mentioned cell A-attachment operation, cell B-attachment operation, cell A-B fusion operation, and fused cell detachment operation are performed on a petri dish that is subdivided into at least three areas under the liquid surface. In one of the above compartments, the isolated cells of cell A are allowed to settle, in the other one the isolated cells of cell B are allowed to settle, and in the other one The cell fusion method according to claim 6, wherein the cell culture medium is filled with the culture medium. 8. The cell A attachment operation, the cell B attachment operation, the cell A and B fusion operation, and the fusion cell detachment operation are performed while observing from below the Petri dish with a microscope. The cell fusion method according to claim 7, wherein 9. The holding power of the isolated cells to be adhered is adjusted by changing the AC voltage, so that one A
The cell fusion method according to claim 6, which is controlled so that the cells and one B cell adhere to each other. 10. A cell A, B by applying a DC pulse voltage.
After fusing the isolated cells of, the application of an AC voltage is canceled, and the fused cells are released into the culture medium.
The cell fusion method according to claim 6. 11. A pair of electrodes configured to generate a non-uniform electric field, an insulating cover made of an electrically insulating material for covering one of the pair of electrodes, and the non-uniform electric field. Minute holes formed in the insulating cover so as to be located at a position where the distribution density of the electrode locally increases, a minute gap formed between the one electrode and the insulating cover, and the pair of electrodes. And a DC power supply for applying a DC pulse voltage to the pair of electrodes, a liquid tank for storing a cell suspension in which the cell is immersed, an AC power supply for applying an AC voltage to the pair of electrodes, and a DC power supply for applying a DC pulse voltage to the pair of electrodes. A cell fusion device characterized in that 12. The DC power supply has a switch means for controlling opening / closing of an AC voltage supply circuit to the pair of electrodes, and an adjusting means for changing an AC voltage value. The cell fusion device according to claim 11, wherein: 13. The one electrode is a needle-shaped electrode,
13. The insulating cover is a tubular member arranged concentrically with the needle electrode.
The cell fusion device according to 1. 14. The insulating cover is a glass tubular member, and the diameter of the opening on the electrode tip side of the glass tube is the smallest among the isolated cells of a plurality of types of cells to be handled. The cell fusion device according to claim 13, which is smaller than the outer diameter of the isolated cell. 15. The cell fusion device according to claim 14, wherein the diameter of the opening on the electrode tip side of the glass tube is 5 μm to 30 μm. 16. The glass tubular insulating cover has a diameter of 20 μm to 100 at its electrode tip end.
The cell fusion device according to claim 13, wherein the cell fusion device has a micron hemispherical shape. 17. The liquid tank for storing the cell suspension is characterized in that it is divided into at least three parts by a partition lower than the liquid surface.
The cell fusion device according to 1. 18. The liquid tank, at least the bottom wall of which is made of a transparent material, has a structure that can be mounted on a microscope stage facing a microscope objective lens held upward. The cell fusion device according to claim 17, wherein: 19. The pair of electrodes are provided between the at least three compartments without raising the tip of the needle electrode and the vicinity of the tip opening of the insulating cover above the liquid surface. The cell fusion device according to claim 17, which has a movable structure. 20. The pair of electrodes has a structure capable of adjusting a distance between the electrodes.
The cell fusion device according to 1. 21. The size of the minute gap formed between the one electrode and the insulating cover is 1 μm to 5 μm.
The cell fusion device according to claim 11, wherein the cell fusion device is 0 micron. 22. The cell fusion device according to claim 11, wherein the frequency of the AC power supply is 0.1 MHz to 1.0 MHz.