JPH0545199Y2 - - Google Patents

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a chamber for a cell fusion device that uses electrical stimulation to fuse cells together, and the present invention relates to a chamber for a cell fusion device that fuses cells together using electrical stimulation. The present invention relates to a centrifugal cell fusion chamber capable of causing fusion.

(Prior art) In conventional cell fusion chambers, a cell suspension containing cells is placed between a pair of electrodes, and an alternating current voltage is applied to the pair of electrodes to electrophores the cells. The cells are brought into contact, and then a high-voltage DC pulse is applied from a pair of electrodes to electrically stimulate the cells, causing them to fuse.

However, when cells are suspended in a solvent with a high salt concentration, such as seawater, the electrical resistance of the cell suspension is too low to apply a predetermined voltage to the cell suspension. Therefore, cell fusion is performed by diluting the cell suspension to increase its electrical resistance and then applying voltage.

Some cells may die if the components of the cell suspension are changed. For example, the cells of seaweed and seaweed die when the concentration of seawater changes. Therefore, it has been difficult to fuse such cells with conventional devices.

Therefore, as a cell fusion chamber that allows electrical stimulation to be applied to cells without changing the concentration of the cell suspension, as shown in FIG. A centrifugal cell fusion chamber has been proposed in which the cells are precipitated on the lower electrode 52, the cells are brought into close contact with each other, and DC pulses are applied to fuse the cells.

In the cell fusion chamber of FIG. 3, a lower electrode 52 is provided on the inner bottom surface of a chamber body 50 housed in a centrifuge container, and an upper electrode is placed along the inner wall of the chamber body 50 at a short distance from the lower electrode 52. 54
is provided.

The inner diameter of the chamber body 50 is approximately 2 mm.

Put the cell suspension into the chamber body 50, and follow the arrow 1
When centrifugal force is applied in three directions, cells move toward the lower electrode 5.
2, the lower electrode 52 and the upper electrode 54
Cell fusion can be caused by applying a voltage between them.

The centrifugal cell fusion chamber 20 is rotated in a centrifuge in the direction of arrow 22 and a centrifugal force G is applied in direction 13, as shown in FIG.

(Problems that the invention attempts to solve) In the cell fusion chamber shown in Figure 3, the electrodes are small;
Furthermore, since the lines of electric force 58 are concentrated at the center of the lower electrode 52 and parallel lines of electric force cannot be obtained, there is a problem that the amount of cells to be treated is small.

The object of the present invention is to provide a centrifugal cell fusion chamber that can process a large amount of cells and has high fusion efficiency.

(Means for solving the problem) To explain with reference to FIG. 1 showing an example,
In the centrifugal cell fusion chamber of the present invention, a lower electrode 2 is provided at the inner bottom of the container 1, and a screen-shaped upper electrode 5 is provided with holes of uniform size evenly arranged in a metal plate through which cells can pass. is provided in parallel with the lower electrode 2 with a distance therebetween, and is adapted to be attached to a centrifuge.

(Function) When the cell suspension 12 is put into this cell fusion chamber, attached to a centrifuge, and the centrifuge is operated, centrifugal force acts on the cell fusion chamber in the direction of arrow 13, and the cell suspension 12 Cells in 12 are on the upper electrode 5
It passes through the hole 6 and is laminated widely and uniformly on the lower electrode 2. After the cells are stacked on the lower electrode 2, the upper terminal 9
A high voltage is applied between the lower electrode 2 and the lower electrode 2 to cause cell fusion.

Although the upper electrode 5 has a screen shape, it functions satisfactorily as one of the parallel plane electrodes.

(Example) FIG. 1 represents one embodiment.

1 is a cylindrical container made of polycarbonate resin. A lower electrode 2 having a flat surface is screwed and fixed to the bottom of the container 1. 3 is an O-ring that seals between the lower electrode 2 and the container 1. An upper electrode 5 is placed on the lower electrode 2 with an insulating spacer 4 interposed therebetween. The upper electrode 5 is made of a stainless steel plate with a thickness of about 0.1 mm, and the diameter is made by photolithography and etching as shown in Fig. 2.
It is a metal screen in which holes 6 of about 100 μm are evenly drilled over the entire surface at intervals of about 150 μm. Upper electrode 5
is fixed to the distal end surface of a substantially cylindrical metal support member 7 by welding. The upper end surface of the support member 7 protrudes beyond the upper end of the container 1 and is in contact with an upper terminal 9 attached to a lid 8 screwed into the upper end of the container 1. The upper terminal 9 is glued to the lid 8. 10 is an O-ring that seals between the support member 7 and the container 1. The lid 8 is designed to be screwed into a screw provided at the upper end of the container 1, and when the lid 8 is screwed in, the upper terminal 9 presses against the support member 7, and the spacer 4
The upper electrode 5 is pressed against the lower electrode 2 via. The distance between the lower electrode 2 and the upper electrode 5 set by the spacer 4 is about 1 to 2 mm. Further, the diameter of the flat surface of the lower electrode 2 surrounded by the spacer 4 is about 10 mm.

A step is provided at an intermediate position of the support member 7,
A backflow prevention member 11 that engages with the step is provided. The backflow prevention member 11 is made of an insulating material such as polycarbonate resin, and has conical holes opening upward and downward, and both holes are narrowed in the center and connected to allow liquid to pass through. ing. 12 is the injected cell suspension.

Next, a case will be described in which cell fusion is caused using the cell fusion chamber of this example.

Attach the lower electrode 2 from the bottom of the container 1,
The upper electrode 5 is fitted from above together with the support member 7 via the spacer 4, and the backflow prevention member 11 is inserted. As shown in the figure, the cell suspension 12 is injected from the upper opening of the container in a vertically installed state, and is introduced into the lower side through the hole of the backflow prevention member 11. Thereafter, a lid 8 is screwed onto the top of the container 1, the support member 7 and the upper terminal 9 are brought into contact with each other, and the upper electrode 5 is pressed against the lower electrode 3 via the spacer 4 to set a predetermined interval.

Next, this cell fusion chamber is attached to a centrifuge. At this time, the cell fusion chamber is mounted laterally. The cell suspension 12 is prevented from flowing back to the top of the container 1 by the backflow prevention member 11 .
Upper terminal 9 and lower electrode 2 when attached to the centrifuge
Attach a lead to each. The lead is
For example, the centrifugal separator can be connected to a power source by providing a terminal on its rotating shaft and bringing the terminal into contact with a brush connected to the power source.

When the centrifuge is operated, centrifugal force acts on the cell fusion chamber in the direction shown by the arrow 13, and the cell suspension 12 is biased toward the lower electrode 2, causing the cell suspension 1
The cells in 2 pass through the holes 6 of the upper electrode 5 and are stacked on the lower electrode 2. At this time, since the holes 6 are evenly provided over the entire surface of the upper electrode 5, the cells are also evenly stacked on the lower electrode 2. When the cells are stacked on the lower electrode 2, a high voltage is applied between the upper terminal 9 and the lower electrode 2 to cause electrical fusion of the cells.

In the example, voltage is applied while applying centrifugal force, but after cells are stacked on the lower electrode 2 by centrifugal force, voltage is applied after removing the cell fusion chamber from the centrifuge. You can do it like this.

(Effects of the invention) In this invention, the screen-shaped upper electrode, which has holes of uniform size evenly arranged in the metal plate through which cells can pass, is provided parallel to the lower electrode with a distance between them. It is easy to increase the area and therefore a large amount of cell suspension can be processed at once.

Since the cells are widely and uniformly stacked on the lower electrode through the evenly provided holes of the upper electrode, cell fusion can be carried out smoothly.

Furthermore, by providing a lead for power supply to the centrifuge, cell fusion can be caused while applying centrifugal force.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal sectional view showing one embodiment, FIG. 2 is a partial plan view showing the upper electrode in the same embodiment, FIG. 3 is a longitudinal sectional view showing a conventional centrifugal cell fusion chamber, and FIG. 4 is a longitudinal sectional view showing an example. It is a schematic diagram showing a state in which a cell fusion chamber is attached to a centrifuge and is being rotated. 1... Container, 2... Lower electrode, 4... Spacer,
5... Upper electrode, 7... Support member, 6... Hole.

Claims (1)

[Scope of utility model registration request] A lower electrode is provided at the inner bottom of the container, and a screen-shaped upper electrode, in which holes of uniform size are evenly arranged and bored in a metal plate through which cells can pass, is provided parallel to and spaced from the lower electrode. A centrifugal cell fusion chamber that is attached to a centrifuge.