JPS6384481A - Cell-fusion apparatus - Google Patents

Abstract

PURPOSE:To enable a processing such as fusion of large quantity of cells in unit time, by connecting plural cell fusion chambers in the form of a ring, transferring the chambers one by one and carrying out the supply of cell suspension liquid, the application of electric potential at the position of microscopic observation, etc. CONSTITUTION:Plural cell-fusion chambers 2-1-2-n are connected in the form of a ring and the ring 2 of the connected cell-fusion chambers is rotatably supported. The cell-fusion chambers are transferred one by one by rotating the ring with a driving mechanism 28. The ring 2 is placed in a manner to pass above a stage 22 of a microscope. Feeding mechanisms 36, 38 to feed a cell suspension liquid to the cell-fusion chamber are placed along the rotational direction of the ring 2. The electrode pair of the cell-fusion chamber are made to contact with a pair of sliding electrodes 32, 34 at the position of the microscope stage 22 and recovery mechanisms 40, 42, 44 for cell suspension liquid are provided at the final stage of the ring 2. The treatment such as cell fusion or introduction of gene can be carried out at large quantities in a unit time by this process.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention is a cell fusion device for fusing cells together using electrical stimulation or for introducing macromolecules such as genes into cells. It is related to.

(Prior art) It has a pair of opposing electrodes, contains a cell suspension in which cells are suspended in the space between the electrodes, and electrically stimulates the cells by applying a voltage to the electrodes. Cell fusion chambers are used to fuse cells and introduce genes into cells.

Up until now, cell fusion has only been carried out on a small scale in the laboratory, and it is also necessary to supply cell suspensions, apply voltage, and cell fusion. Each operation, such as collecting the I suspension, is performed discontinuously.

(Problems to be Solved by the Invention) Until now, there has been no cell fusion device that attempts to perform operations such as cell fusion or gene introduction continuously and in large quantities.

An object of the present invention is to provide a cell fusion device that can continuously perform processes such as cell fusion and gene transfer, as well as mass processing, and that can automate these operations. .

(Means for Solving the Problems) Referring to FIG. 1 showing an embodiment, the cell fusion device of the present invention includes a plurality of cell fusion chambers (2-1 to 2-1).
2-n) are connected in a ring shape, and the connected cell fusion chamber ring (2) is rotatably supported, and the cell fusion chambers (2-1 to 2-n) of the ring (2) are connected in a ring shape.
A feeder* (26, 28) is provided to feed and rotate the ring (2) one by one, and the ring (2) is attached so that it passes over the stage of the microscope, and the position of the stage of the microscope is adjusted along the direction of rotation of the ring (2). Supply mechanism (3) that supplies cells @ suspension to the cell fusion chamber (2-1) in front of (30)
6, 38), sliding electrodes (32° 34) respectively abutting the pair of electrodes of the cell fusion chamber (2-2) at the stage position (30) of the microscope;
A cell suspension collection mechanism (40°42.44.46) was provided at the final stage position (2-n) of 2).

(Example) FIG. 1 is a schematic plan view showing one embodiment of the present invention.

FIG. 2 is a plan view showing a part of the ring of the cell fusion chamber in the same embodiment, and FIG. 3 is a sectional view taken along the line AB in FIG. 1.

2 is a ring of cell fusion chamber 2-1.2-2. ...2-n are connected in a ring shape.

Cell fusion chamber 2-1.2-2.・・・・・・2-n
As shown in FIGS. 2 and 3, each cell fusion chamber is separated by an insulating separator 4. Cell fusion chamber 2-1.2-2 separated by separator 4. Each of 2-n has a chamber body 6 made of an insulating member inside the ring, and a chamber body 8 made of an insulating member outside the ring, and the chamber bodies 6 and 8 are made of an insulating material. A ring-shaped bottom hole 10 made of transparent glass is fitted in between and bonded.

Each cell fusion chamber 2-1.2-2.・・・・・・
...2-n, the outer electrode 12 and the inner electrode 14 are provided parallel to each other, and the opposing parts of these electrodes 12 and 14 have a vertical surface at the bottom and a tapered top. It has spread. A space 16 formed by the opposing vertical surfaces of both electrodes 12 and 14 is a space for accommodating a cell suspension and applying electrical stimulation to the cells.

As shown in FIG. 1, the ring 2 of the cell fusion chamber is supported on the inside by four rollers 20 provided on the table 18 and can rotate on the table 18. The stand 18 is fixed to the stage 22 of the microscope at the microscope position 30, as shown in FIG.
2-2. . . . 2-n sequentially passes over the stage 22 of the microscope.

In FIG. 3, 24 is an objective lens of the microscope.

On the outside of the cell fusion chamber ring 2, one claw 26 is provided for each cell fusion chamber, and the platform 18 is provided with one claw 26 for each cell fusion chamber.
An air cylinder 28 is provided above, and when the air cylinder 28 pushes the claw 26, the ring 2 of the cell fusion chamber rotates regulated by the roller 2°. The ring 2 of cell fusion chambers is rotated at the pitch of the claws 26, that is, at the pitch of one cell fusion chamber at a time.

Assuming that the cell fusion chamber 2 - 2 is now located at the microscope position 30 , the cell fusion chamber at this microscope position 30 has a sliding terminal 32 pressed against the outer electrode 12 and a sliding terminal 34 pressed against the inner electrode 14 . is being forced. The sliding terminal 32 is connected to a negative power source, and the sliding terminal 34 is connected to a positive power source.

Cell fusion chamber 2-2 at microscope position 30 with respect to the rotation direction of ring 2 of the cell fusion chamber (counterclockwise in the figure)
At a position in front of the cell fusion chamber 2-1, there is provided a hopper 36 that supplies a cell suspension containing cells. The cells are supplied to the cell fusion chamber 2-1.

A recovery port 40 for recovering the cell suspension is provided in the last cell fusion chamber 2-n in the rotational direction of the ring 2 of the cell fusion chamber. The collection port 40 can be moved up and down by an air cylinder 42 to place the collection port into the cell fusion chamber. The collection port 40 is a collection box 44
After that, the cells are suctioned by a vacuum pump 46, and the fused cells are collected into a collection box 44.

Next, the operation of this embodiment will be explained.

A fixed amount of cell suspension is supplied from the hopper 36 to the cell fusion chamber 2-1 by the syringe piston 38. Next, by driving the air cylinder 28, the cell fusion chamber ring 2 is rotated by one cell fusion chamber. This makes the cell fusion chamber 2-1 supplied with the cell suspension visible? & The empty cell fusion chamber 2-n that is sent to the mirror position 30 and collected at the first collection port 40 is sent to the position of the hopper 36. Microscope position 30
Then, the predetermined voltage required for cell fusion is applied to the sliding terminal 32.34.
is applied, and the fusion state is observed under a microscope. By sequentially driving the air cylinders 28, the cell fusion chamber containing the electrically stimulated cell suspension at the microscope position 30 is sequentially transported to the collection port 40. At the final stage position, the cell suspension after the fusion operation is collected by the collection port 40.

The supply of the cell suspension from the hopper 36, the application of the fusion voltage at the microscope position 30, the fusion time after the voltage application is finished, and the collection of the cell suspension from the third collection port 40 are performed continuously, Moreover, these series of operations are sequentially performed for all cell fusion chambers 2-1, 2-2°, . . . , 2-n.

The rotational speed at which the air cylinder 28 feeds the cell fusion chamber is approximately 10 to 20 seconds per step. Then, the cell S suspension is supplied, the cells settle, and at the microscope position, a voltage is applied for electrophoresis to cause the cells to migrate from the sliding terminals 32 and 34, and a pulse is applied for fusion, and after a fusion time, The time until the final collection from the collection port 40 is about 5 to 15 minutes. Therefore, the processing time of each of these steps and the rotation speed of the ring 2 of the cell fusion chambers can be adjusted depending on the number of cell fusion chambers.

(Effect of Ryogetsu) In the cell fusion device of the present invention, a plurality of cell fusion chambers are connected in a ring shape, and the cell fusion chambers of this ring are fed one by one to supply a cell suspension and perform operations at the microscope position. Since the voltage application, waiting for the fusion time after the voltage application is completed, and collection of the cell suspension from the collection port are performed continuously, the following effects can be achieved.

(1) A large amount of cell fusion and gene transfer can be performed in a unit time.

(2) Automatic control can be performed without any manual effort.

(3) Miniaturization is possible, and sterile operation can be performed within a clean bench.

[Brief explanation of the drawing]

FIG. 1 is a schematic plan view showing one embodiment of the present invention, FIG. 2 is a plan view showing a part of the ring of the cell fusion chamber in the same embodiment, and FIG. 3 is the position of line A-B in FIG. 1. FIG. 2...Ring of cell fusion chamber, 2-1~2
-n...Cell fusion chamber, 20...
Roller, 22...Microscope stage, 26...Claw. 28.42...Air cylinder, 30...
...Microscope position. 32.34...Sliding electrode, 36...Hopper, 38...Syringe piston. 40... Collection port, 44... Collection box, 46... Vacuum pump.

Claims (1)

[Claims] (1) A plurality of cell fusion chambers are connected in a ring shape, and a ring of the connected cell fusion chambers is rotatably supported.
A feeding mechanism is provided to feed and rotate the ring one by one, and the ring is attached so as to pass over the stage of the microscope, and the cells are suspended in a cell fusion chamber located in front of the stage of the microscope along the direction of rotation of the ring. A supply mechanism for supplying the liquid is provided, a sliding electrode is provided that contacts each of the pair of electrodes of the cell fusion chamber at the stage of the microscope, and a cell suspension recovery mechanism is provided at the final stage of the ring. Cell fusion device installed.