JPS6232874A - Cell fusion apparatus - Google Patents

Abstract

PURPOSE:To enable the 1-to-1 contact of protoplastoid cells surely independent to the size of the cell, by using a cell-holding plate having plural cavities to perform the cell fusion and a means to transfer a cell from a storage tank to the cavity. CONSTITUTION:The objective cell-fusion apparatus for fusing protoplastoid cells with each other is furnished with a cell-holding plate 1 having plural cavities 11 to perform the cell fusion and a means to transfer the cells 12, 13 from the storage tank 4 to the cavity 11. The transfer means is composed of liquid- feeding pumps 5, 7, a detector 9 and a controller 10, etc. The cell in the storage tank 4 is transported together with the fluid, and when the cell is detected by the detector 9, the flow of the fluid is controlled by the controller 10 to release the cell through the preliminarily positioned opening 8 into the target cavity 11. The apparatus enables the 1-to-1 contact of two kinds of protoplastoid cells surely and automatically independent to the size of the cell.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to a cell fusion device for fusing protoplast-like cells, and particularly to a cell fusion device suitable for fusing two types of cells on a one-to-one basis. It is.

[Background of the invention]

Recently, using naked single cells (protoplasts) whose cell walls have been removed to expose the plasma membrane, hybrid cells have been created by fusion of cells without using reproductive cells, making use of the characteristics of naked single cells (protoplasts) in which the cell wall has been removed to expose the plasma membrane. There is a lot of research going on to improve varieties.

To obtain such hybrid cells, the cells of higher organisms such as plants are broken down into cells using enzymes, the cell walls are dissolved with enzymes to form protoplasts, the two types of cells in this state are brought into close proximity, and then chemicals such as polyethylene glycol are applied to the cells. However, in the process of bringing two types of cells close to each other, various types of contact occur, such as between cells of different types, between cells of the same type, and between cells composed of multiple cells, so that the desired It has been difficult to efficiently produce hybrid cells by fusing cells one-to-one.

As a method to facilitate the sieving of the fused cells mentioned above, two porous metal plates such as those described in ``A new method for cell fusion'' published in the Nikkei Sangyo Shimbun, October 6, 1980, were used.
There is a method of using two metal plates, leaving one cell of a different type in each hole in the upper and lower metal plates, and fusing them, but this still requires a lot of manual labor.

[Purpose of the invention]

The purpose of the present invention is to solve such conventional problems, and when fusing two types of protoplast-like cells in a one-to-one ratio, it is an object of the present invention to provide a method for achieving the one-to-one contact configuration regardless of the size of the cell diameter. An object of the present invention is to provide a cell fusion device that can perform cell fusion automatically and reliably.

[Summary of the invention]

In order to achieve the above object, the cell fusion device of the present invention is a cell fusion device that fuses cells.

A cell holding plate (1) having a plurality of recesses (11) for performing the fusion process, and means for transporting the cells from the storage tank (4) to the recesses (11) (liquid pump 5.7, etc.) ) is characterized by how many.

[Embodiments of the invention]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of a cell fusion device showing one embodiment of the present invention. In the figure, 1 is a cell holding plate having a large number of recesses 11 for holding cells 12 and cells I3, 2 is a container for storing the cell holding plate I together with a liquid 15, and 3 is a buffer solution 14 and cells 12.13. It is a flow path for conveying the liquid, and has an opening 8 in the middle to connect the liquid feeding pumps 5 and 7. 4 is a cell storage tank for storing cells 12 or 13; 5
7 is a liquid pump that sends the cells 12 and 13 in the cell storage tank 4 to the channel 3; 7 is a liquid pump that collects the buffer solution 14 in the channel 3 and flows it into the waste reservoir 6; and 8 is a pump in the channel 3. Cell 12.1
An opening that releases 3 to the outside.

9 is a detector that detects cells 12 and 13 in the channel 3 and notifies the controller 10; 16 is an opening 8 that
This is a moving mechanism that moves the recess 11 onto an arbitrary recess 11.

Cell retention plate 1-1 has a concave part that prevents two types of protoplast-like cells, which are placed close to each other one by one in a pair, from separating or moving freely, in order to ensure one-to-one cell fusion. 11 are provided in large numbers. Therefore, the diameter of the recess 11 is set to a value slightly larger than twice the maximum cell diameter in the cell population to be applied in this plate 1, so that the protoplast-like cell pairs placed close to each other are stably retained. (20-200 μm).

If the lengths of the cells you want to fuse are large and small when paired, there is a large difference in length.

Several plates 1 with different diameters of recesses 11 are prepared.

The control unit 10 drives the liquid feeding pump 5 so that the cells 12 and 13 in the cell storage tank 4 are transported through the channel 3 together with the buffer solution 14, and the liquid feeding pump 7 drives the buffer solution in the channel 3. The moving mechanism 16 is controlled so that the opening 8 is located above each of the recesses 11 by driving the liquid 14 to be collected together with the conveyed amount. By performing each of these operations, as shown in FIG. The cells 12 are transported through the opening 8, and one cell is transferred to each of the four parts 11 one after another.
Place them one by one. Note that the liquid 15 in the container 2 at this time is a buffer similar to the buffer 14 flowing through the channel 3.

Next, from the cell storage tank 4 in which the protoplast-like cells 13 were prepared, the flow path 3. The cells 13 are transported through the opening 8 and placed one after another in each recess 11 in which a cell 12 already exists.

Thereafter, the liquid 15 in the container 2 is replaced with a fusion treatment liquid such as polyethylene glycol to cause cell fusion and simultaneously produce a large number of desired hybrid cells.

Note that the cell holding plate 1 may be transferred to another container that already contains the fusion treatment liquid without replacing the liquid 15.

In order to release the transported cells 12 and 13 from the opening 8 into the recess 11 in a timely manner, the control unit 10 controls the cell 1
When it is determined from the output of the detector 9 that the cells 2 and 13 have approached the opening 8, the liquid pumps 5 and 7 are controlled to stop the flow of the buffer solution 14 in the flow 13, and the cells are removed from the entrance 8. 12.
13 is released and falls into the recess 11. Thereafter, while maintaining this state, the moving mechanism 16 is controlled so that the opening 8 that has finished discharging is positioned above the next empty recess 11. By automatically repeating the above operations, the recess 1
One cell 12 and one cell 13 are placed for each cell.

Note that the moving mechanism 16 uses a known servo mechanism that is capable of fine horizontal movement.

The control unit 10 controls the intense buffer solution 14.15 near the opening 8.
In the case where liquid flows in and out, adjustment is made by synchronizing the operations of liquid pumps 5 and 7.

Furthermore, the opening 8 may be replaced with an opening/closing mechanism that opens and closes the outlet using a control signal from the control section 10. ■When the cells 12 and 13 reach the opening 8, the liquid pump 5.7 may be driven to slightly increase the fluid pressure in the channel 3 to forcefully release the cells 12 and 13. . (2) It is also possible to provide a vibrator in the opening 8 to speed up the release of the cells 12 and 13.

(2) A detector similar to the detector 9 may be provided near the exit of the opening 8 to detect the release of the cells 12, 13 and determine the timing of movement of the opening 8 by the moving mechanism 16.

The detector 9 is equipped with a light emitting diode and a light receiving element, and uses an optical detection method to detect cells 12.13 from changes in electrical conductivity.
This can be achieved by methods such as detecting In addition, the control unit 10
By adding a function to identify the output waveform from the detector 9 and changing the standard value for identification, it is possible to distinguish between cells 12.13 and foreign substances such as unnecessary dust, and to increase the number of hybrid cells produced. .

In this way, each cell 12.13 is placed in channel 3. Since one cell is placed in each recess 11 of the cell holding plate 1 through the opening 8, cell fusion can be performed in which two different types of cells 12, 13 are brought into reliable one-to-one contact. Cell fusion according to the present invention does not require special operations to remove cells of the same type and unnecessary fusion products of three or more.

In addition, by appropriately selecting the diameters of the four parts 11 of the cell holding plate 1, it is possible to ensure a one-on-one relationship even if the cell diameters vary.
fusion is possible, and there is no restriction on the cell diameter in the cell population to be fused.

In the cell fusion of the present invention, the aperture 8 is positioned by mechanical technology that allows transportation and release of cells 12, 13 and precise position control using microelectronic technology, allowing for extensive automation in the cell fusion process. It is.

〔Effect of the invention〕

As explained above, according to the present invention, protoplast-like cells 12 are first conveyed together with the buffer solution 14 through the channel 3 to each recess 11 of the cell holding plate l, and then released from the opening 8 to form a cell. After each cell is placed, another type of cell 13 is similarly placed one by one, so the two types of cells to be fused are surely brought into one-to-one contact without restricting variations in cell diameter.

In addition, the cell fusion process is greatly simplified.

[Brief explanation of drawings]

FIG. 1 is a block diagram of a cell fusion device showing one embodiment of the present invention, and FIG. 2 is a diagram for explaining the operation of FIG. 1. 1: Cell holding plate, 2: Container, 3: Channel, 4: Cell storage tank, 5, 7: Liquid pump, 8: Opening, 9: Detector, 10: Control section, 11: Recess, 12 .13: Cell, 14
: buffer solution, 15: buffer solution or fusion treatment solution, 16: transfer mechanism. Figure 1

Claims (2)

[Claims] (1) A cell fusion device for fusing cells, characterized by comprising a cell holding plate having a plurality of recesses and performing the fusion process, and means for transporting the cells from the storage tank to the recesses. Cell fusion device. (2) The conveying means conveys the cells in the storage tank together with the fluid, controls the flow of the fluid when the cells are detected, and releases the cells from a pre-positioned opening into the target recess. The cell fusion device according to claim 1, characterized in that the cell fusion device is arranged.