JPH0191767A - Cell fusion chamber - Google Patents

Abstract

PURPOSE:To simplify the take-out operation of fused cell and to reduce the size of a cell fusion chamber, by forming a cell fusion space between a pair of parallel electrodes placed on a flat transparent plate and forming a cell- holding space at a part separated from the cell fusion space by a prescribed distance in a specific direction. CONSTITUTION:A cell fusion space 3 is formed between a pair of parallelly disposed electrodes 2a, 2b positioned on a flat transparent plate 1. Cells to be fused are put into the cell fusion space. A high-frequency electric potential superposed with DC pulse is applied to the electrodes 2a, 2b to effect the fusion of the cells. The flat transparent plate is transferred to position the objective cell at the center of the field of a microscope. The cell is sucked with a micropipette, transferred by a prescribed distance and discharged into a cell holding space 4.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a cell fusion chamber for performing electrical cell fusion, and in particular for use in conjunction with a micromanipulator capable of simple automatic control, for example. Regarding the fusion chamber.

<Conventional technology> In general, electric cell fusion involves applying a high-frequency voltage to cells in a culture medium to bring the cells into contact with each other, and then applying a direct current pulse to fuse the cells in contact. Conventionally, this has been carried out using a chamber that has a space between electrodes that face each other at a constant interval and accommodate cells together with a culture medium.

<Problems to be solved by the invention> In the electric cell fusion performed using the conventional chamber as described above, a pipette or the like is used to remove the fused cells.
It is necessary to transfer it to a separately prepared container such as a petri dish. This operation is extremely difficult because it requires selecting and removing the fused cells and transferring them one by one to another container while there are also unfused cells.

<Means for Solving the Problems> The present invention has been made to simplify the work of removing cells after fusion, and in particular, by combining them with a micromanipulator that can be easily and automatically controlled, fusion can be easily and quickly performed. The purpose of this invention is to provide a cell fusion chamber from which cells can be sorted and removed.

The structure for achieving this object will be explained with reference to FIG. , a cell fusion space 3 formed between 2b and accommodating cells to be fused;
The cells are formed at a predetermined distance Δ in the direction perpendicular to the direction in which they face each other, and are characterized by having a cell storage space 4 for accommodating the cells after fusion.

<Operation> Cells in the cell fusion space 3 can be visually recognized through the transparent plate 1 using an inverted microscope. As shown in Figure 2, fusion/non-fusion cell sorting can be performed by shifting the transparent plate 1 so that the target cells are in the center of the microscope field of view.At this time, the cell storage space 4 is also For example, by attaching a cell suction device 23 to a micromanipulator 20 that has the function of moving in parallel by a certain distance according to a command, the selected cells can be aspirated, then moved a certain distance and then ejected. All of the selected fused cells can be accommodated in the cell storage space 4.

<Example> Embodiments of the present invention will be described below based on the drawings.

FIG. 1 is a diagram showing the configuration of an embodiment of the present invention, in which (a) is a plan view, (b) is a left side view thereof, and (C) is a sectional view taken along line cc.

A transparent flat plate 1 made of glass such as a slide glass is used as a substrate,
A pair of cell fusion electrodes 2a and 2b are embedded and fixed in parallel to each other on the upper surface thereof. This electrode 2
Between a and 2b, an electrode 2a from the upper surface of the transparent flat plate 1,
A rectangular recess with a depth equal to the amount of filling 2b is formed, and this recess forms the cell fusion space 3.

A rectangular recess is also formed on the transparent flat plate 1, adjacent to the cell fusion space 3 and separated by a distance Δ in the longitudinal direction, and this recess is connected to the cell storage space 4.
is formed.

The length and width B of the cell storage space 4 are set to be equal to or greater than the length l and width of the cell fusion space 3, respectively. Moreover, the center lines of the cell fusion space 3 and the cell storage space 4 in the width direction are substantially aligned.

Next, how to use it will be explained.

FIG. 2 is an explanatory view of how to use the embodiment of the present invention, and is a view showing the entire device from vertically above.

The transparent flat plate 1 is placed on the stage of an inverted microscope (not shown), and the electrodes 2a and 2b are connected to the cell fusion device 1, respectively.
Connected to 0.

It also includes a drive section 21 that incorporates a pulse motor or the like as an actuator, and a control section 22 that can output a signal for driving the drive section 21 by a preset distance in at least one direction (X direction). A micromanipulator 20 is prepared, and its drive unit 21 is equipped with a micropipette 23 that can aspirate and discharge cells.

First, cells to be fused are housed in the cell fusion space 3 along with a culture medium. Next, the cell fusion device 10 is operated to apply a high frequency voltage between the electrodes 2a and 2b to bring the cells in the cell fusion space 3 into contact, and then a DC pulse is applied to fuse them. This process can be observed using an inverted microscope through the transparent flat plate 1.

After completing the above operations, only the fused cells in the cell fusion space 3 are picked up, but at this time, the transparent plate 1 must be moved appropriately relative to the optical axis of the microscope by moving the microscope stage, etc. This brings the cells to be sorted to the center of the microscope field of view. If fused cells are found by visual inspection through a microscope, the micromanipulator 20 is operated to remove the micropipette 2.
3 is displaced to near the center of the microscope field of view with its central axis parallel to the electrodes 2a and 2b, and the cells are aspirated. Note that at this time, as shown in FIG. 3, it is desirable to aspirate the cells W into the micropipette 23 together with the culture solution CL.

Next, give a command to the micromanipulator 20 to
direction, and is automatically driven a certain distance toward the cell storage space 4 side. This fixed distance is set in advance to the following distance.

D=1+Δ+α However, 0<α<l, -f Due to this drive, no matter where the cells are aspirated in the cell fusion space 3, the tip of the micropipette 23 is always placed above the cell storage space 4. Therefore, if the cells are discharged after this drive, the sorted cells will be transferred to the cell storage space 4.
contained within. By repeating the above operations, only the fused cells are collected in the cell storage space 4. After completing all the desired sorting, move to cell storage space 4.
Transfer the cells inside to the medium using a pipette, etc., and complete the entire treatment.

Note that the length and width B of the cell storage space 4 do not necessarily have to be larger than the length l and width B of the cell fusion space 3, but suction can be carried out at any position within the cell fusion space 3. Thin.

When cells are automatically moved into the cell storage space 4 by the micromanipulator 20, somewhat complicated movement control is required because the movement distance cannot be made constant.

Further, in the above embodiment, an example was shown in which the cell storage space 4 was formed by providing a recess on the surface of the transparent flat plate 1, but the present invention is not limited to this, and the cell storage space 4 is formed by a low wall on the surface of the transparent flat plate 1. It is also possible to provide a wall and use the interior surrounded by this wall as a cell storage space.

<Effects of the Invention> As explained above, according to the present invention, there is a cell fusion space sandwiched between a pair of electrodes facing each other on a single transparent plate, and an electrode for this cell fusion space. Since we have provided a cell storage space formed at a predetermined distance in a direction orthogonal to the facing direction, the fused cells in the cell fusion space can be sorted at the center of the field of view of an inverted microscope, and the cell storage space can be easily accessed using a micromanipulator, etc. can be housed within. Furthermore, there is no need for another container such as a petri dish, and both spaces can be made compact.

Furthermore, if the length and width dimensions of the cell storage space are equal to or greater than the length and width dimensions of the cell fusion space, the micromanipulator can be moved a certain distance when transferring the sorted cells to the cell storage space. It is extremely easy to control the movement.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the configuration of an embodiment of the present invention, in which (a) is a plan view, (bl is a left side view thereof, and (C) is a sectional view taken along line CC thereof. FIGS. 2 and 3) 1 is an explanatory diagram of how to use the embodiment of the present invention. 1... Transparent flat plates 2a, 2b... Electrodes 3... Cell fusion space 4... Cell storage space 10... Cell fusion device 20. ...Micromanipulator 21...Drive section 22, controller, control section 23...Micropipette patent applicant Agent, Shimadzu Corporation
Patent Attorney Nishi 1) Arata

Claims (2)

[Claims] (1) A cell fusion space formed between a pair of parallel opposing electrodes provided on a transparent plate to accommodate cells to be fused; A cell fusion chamber, which is formed at a predetermined distance in a direction perpendicular to the direction in which the electrodes face each other, and includes a cell storage space for accommodating cells after fusion. (2) The cell storage space has a rectangular shape on the transparent flat plate, and its length and width are equal to or greater than the length and width of the cell fusion space,
A cell fusion chamber according to claim 1.