JPH01247076A - Method and apparatus for fusing cell - Google Patents

Abstract

PURPOSE:To fuse heterogeneous cells in good yield, by preparing laminate of heterogeneous cells on a diaphragm by applying alternating voltage to the diaphragm, inserting electrodes so as to generate uniform electric field at the both sides of the diaphragm and applying alternating voltage and direct current pulse voltage to the electrodes. CONSTITUTION:Heterogeneous cells are separately charged at a definite time apart into a cell fusion chamber 1 installing a diaphragm 17 between a pair of electrodes 15 and 16 generating heterogeneous electric field and alternating voltage is applied whenever cells are charged to prepare a laminate of heterogeneous cells on the diaphragm. Then an electrode 21 is inserted so as to generate uniform electric field at the both sides of the diaphragm and alternating voltage and direct current pulse voltage are applied to the electrodes 21 to fuse heterogeneous cells. As a result, yield of heterogeneous fused cell can be raised.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing a heterologous fused cell A-B by fusing two types of cells A with Bt, and an apparatus therefor.

P) Prior art As a method for fusing cells together, there is a cell electrofusion method that uses electrical stimulation.

One of these methods, the cell electrofusion method, uses two types of cells A
, B'i is first applied to a mixed suspension containing B'i, and when all the cells come into contact with each other, a chain of cells called a pearl chain is formed. A high voltage pulse is then applied to fuse the cells and the cells (Rioscien
ce Report S, 'L 335~342L
1984).

In other cell electrofusion methods, the cells are brought into contact with each other using an alternating electric field, and the cell density is raised by 1 m to keep the cells in contact, and only high voltage pulses are applied (Hro/rECHNOLOGY, 4.57-60
(1986) see No.

Problems to be Solved by the I/V Invention When cell fusion is caused in a cell suspension containing two types of cells A and R, the possibility of cell fusion is A-A, R-
Since there are four types, R, AB, and RA, the yield of heterokaryons in which cells A and B are fused is 50% in principle.

The entire object of the present invention is to provide a cell fusion device that can increase the yield of heterokaryons by artificially increasing the frequency of association of different cells A and B.

2.) Means for Solving All Problems The present invention provides a means for solving all the problems in which different cells are placed in a cell fusion chamber in which a diaphragm is installed between a pair of electrodes that generates a non-uniform electric field. After a certain period of time, the cells were placed separately, and each time a cell entered, an AC voltage was applied to create a total cell area on the diaphragm. After that, all the electrodes were inserted with the FII membrane sandwiched between them so that a uniform electric field was generated. Apply alternating current voltage and direct current pulse voltage to fuse different cells.

In addition, the device of the present invention includes a liquid feeding section that feeds different cells separately, a cell fusion chamber with a built-in electrode for stacking and fusing all of the cells that have been fed, and one of the cells in the fusion chamber. The upper lid has a pair of electrodes fixed to it,
It consists of a power supply unit that switches and applies a predetermined voltage 7J11 to the electrodes in the cell fusion chamber and/or the electrodes of the upper lid, and a control unit that controls cell liquid feeding and voltage application.

Here, the electrode built-in cell fusion chamber has a pair of electrodes that generate a non-uniform electric field sandwiching a diaphragm, and a recess into which the upper lid to which the electrode plate is fixed is installed.

The electrode fixed to the upper lid has a shape to generate a uniform electric field with one electrode arranged in the cell fusion chamber, and the other electrode has a shape to generate a uniform electric field with one electrode arranged in the cell fusion chamber. Two types of electrodes are prepared: one electrode is placed on the other side, and the other has a metal shape to generate a non-uniform electric field.

(E) Function The present invention sends only 1.A cells to the cell fusion chamber. Then, an alternating current voltage is applied to cause the A cells to adhere onto the diaphragm. After attaching Am cells.

B cells are fed and a full alternating current voltage is applied to attach the B cell layer to the A cell layer on the diaphragm, forming a stack of AR cells.

After forming a stack of AH cells, a metal lid is placed on the top lid provided with an electrode shaped to generate a uniform electric field within the chamber, and a predetermined electrical stimulation is applied to perform cell fusion. After the fusion, the fused cells are collected by electrophoresis toward the collection port in the chamber, where all 9 upper lids are replaced.

(f) Example Embodiments of the present invention will be described based on the drawings.

FIG. 1 shows a complete overview of the apparatus according to the present invention.

1 is a cell fusion chamber, 2.2' is a syringe for cell delivery, 3 and 3' are fluid delivery channels, 2 and 3 are for A cells, and 2' and 3' are for B cells.

In the cell fusion chamber 1, a washing channel 6 and a recovery channel 7 are connected to a washing liquid syringe 41 and a recovery syringe 5, respectively. Also.

Electrode in cell fusion chamber 1 (described later)
It is connected to the power supply unit 9 via '13. The power supply section 9 is composed of a switch 12, an AC voltage generation section 10, and a DC pulse voltage generation source 11, and the switch 12 switches the electrodes in the chamber between the two power sources.

In addition, the operation of the cell liquid feeding syringe 2.2', the washing liquid syringe 41 and the collection syringe 5, and the switch 12 of the power supply section 9
Is the switching controlled by the control unit 14? I can cut B.

Next, the structure of the cell fusion chamber 1 will be explained.

FIG. 2 is a top view of the interior of the cell fusion chamber 1, and FIG. 3 is a side view of the interior of the cell fusion chamber 1.

The chamber 1 has a concave main body 25 and a pair of electrodes 15.
．． 16 are installed, and the opposing area of the electrode 16 is the same as the electrode 15 so that a non-uniform electric field is generated between the pair of electrodes.
The opposing area is smaller than 0.

Between the electrodes 15 and 16 is a diaphragm 1 formed of a mesh.
7 is arranged, and the pore size of the mesh is smaller than the cell pore size XV so that the cells are stacked on the mesh. Note that the diaphragm 17 is fixed with 23-1 and 23-2.

The body part 25 is formed with four position regulating parts 19 so that the upper lid can be fixed in a predetermined position.

Further, on both side walls of the main body 25, there are flow channels 3 for cell liquid feeding that communicate with the central opening 18. A cleaning flow path 6° and a recovery flow path 7 are provided.

With the above configuration, the operation of this embodiment is performed as follows.

(1) First, the syringe 2 is operated according to a command from the control unit 14 to deliver a predetermined amount of the suspension of cells A to the cell fusion chamber 1. At this time, the mv to be sent is the electrode 1 in the chamber.
The entire length of 5 is the entire distance between the a1 electrode 15 and the diaphragm 17,
If the cell diameter is d and the cell density in syringe 2 is ρ, then t
v = (a, /dJ, /ρ. The values of d and ρ are input to the control unit 14 in advance.

2) Next, according to a command from the control section 14, the switch 12 of the first power supply section 9 is connected to the AC voltage generating terminal 10, and an AC electric field is applied between the electrodes 15 and 16. At this time, the electrode 15,
A non-uniform electric field is generated between the electrodes 16, and the cell A migrates in the direction of dense electric lines of force, that is, toward the electrode 16. However, between the electrodes V
Cell C has a diaphragm 17, and since the cells are larger than the gap 1 of the diaphragm 17, a monolayer of cells A is formed at the diaphragm.

Transfer the cell B suspension to Yamabuki in the same manner to create a monolayer of cells B on the monolayer of cells A. (4) Above (υ~(3J)
Repeat approximately '/(dA+da) times, but d^
, dB is the diameter of cells A and B, respectively, and the fourth
Place the upper lid metal for the chamber electrode as shown in the diagram. The chamber electrode upper lid has a protrusion 24 that can engage with the recess 19 of the main body 25 and an electrode 21 (or 22). The opposing area is equal to the area of the electrode 15 so that a uniform electric field is generated between the electrode 21 and the electrode 15. Also, the pole 2
2 is configured so that a non-uniform electric field is generated between the electrode 15 and the sub-electrode 15 (so that the electric lines of force at the electrode i become dense), and the opposing area of the electrode 22 is larger than the entire opposing area of the electrode 15.

In this process, in order to perform cell fusion, the chamber electrode upper lid 20-1, on which the electrode 21 is installed, is fully used.

The cross-sectional view when the upper lid is fixed to the chamber body is shown in FIG. 21 can be changed.

(5) Next, the control unit 140 commands V electric t! A full AC voltage is applied between f15 and electrode 21. Thereafter, a 12-karat gold switch is applied to apply a DC pulse voltage to fuse cells A and B. If you apply multiple pulses, repeat this. At this time, since the opposing areas of one electrode 15 and the electrode 21 are equal, the bottom of the opening 18 of the chamber 1 is two-tiered and the electrode 21 side is deeper, so that a uniform DC pulsed electric field is applied to the cells. Note that FIG. 5(a) shows a top view of the chamber 1 when pulses are applied.
Show VC.

(6) After the pulse mark U[l, move the upper member 20-1 to the electrode 22
Replace the 2O-2VC installed. and electrode 15
An alternating current voltage is applied between the electrode 22 and the electrode 22. Since the opposing area of the electrode 220 is V larger than that of the electrode 15, a non-uniform electric field is applied.

The fused cell AB is in the direction of dense electric lines of force, that is, the electrode 15
The fused cell A migrates toward the diaphragm 17 and is caught on the septum 17.
R is off. A top view of the chamber 1 at this time is shown in FIG. 5 (bJ).

(7) Create the syringe 5 according to the command from the control unit 14 and collect the fused cell suspension from the flow path 7. The cell suspension stored in the syringe 5 flows through the flow path 8 and 9.

The cleaning liquid passes through the channel 6 and settles in the chamber 1.

The cleaning contaminated liquid is collected by the syringe 5. The flow path of the syringe 5 is equipped with a cock, which is opened only when necessary.

The collection of the fused cells is not limited to the above-mentioned method, but may also be performed by removing the diaphragm 17 from the champer and sieving it into another container.

() Effect According to the present invention, the yield of hybrid cells of A and B is 100%.

In addition, recovery of the cell suspension after fusion becomes easy.

[Brief explanation of the drawing]

- Fig. 1 is an overall view of the apparatus for carrying out the method according to the present invention, and Fig. 2 is a top view of the cell fusion chamber. Figure 3 is a side view of the cell fusion chamber, Figure 4 (aJ is a side view showing the upper lid of the cell fusion chamber, Figure 4 (bJ is a side sectional view when the upper lid is fixed to the cell fusion chamber, Fig. 5 (ta) is a top view of the chamber when the upper lid 20-1 is fixed, and Fig. 5 (bJ is a top view of the chamber when the upper lid 20-2 is fixed. 1... Cell fusion Chamber 15.16.21.22・
・・Electrode 17−・・Diaphragm T+ Kuroshi 5 Ward

Claims (1)

[Claims] 1. Different cells are placed separately at a certain time into a cell fusion chamber in which a diaphragm is installed between a pair of electrodes that generate a non-uniform electric field, and each time a cell enters, an AC voltage is applied. is applied to create a stack of different cells on the diaphragm, then electrodes are inserted to sandwich the diaphragm to generate a uniform electric field, and AC voltage and DC pulse voltage are applied to fuse the different cells. Cell fusion method. 2. A liquid feeding unit that feeds different cells separately, a cell fusion chamber with a built-in electrode for stacking and fusing the fed cells, and an electrode that pairs with one electrode of the fusion chamber. A cell fusion device that is equipped with an upper lid to which a cell fusion is fixed, a power supply unit that switches and applies a predetermined voltage to the electrodes in the cell fusion chamber and/or the electrodes of the upper lid, and a control unit that controls cell liquid feeding and voltage application. Device.