JPS63160575A - Cell fusion apparatus - Google Patents

Abstract

PURPOSE:To obtain the titled apparatus capable of receiving a cell into a receiver in a desired state, by providing a cell-detecting means to a feeding path of a cell suspension liquid, a means to transfer a receiver according to the signal transmitted from the detecting means and a liquid-sucking nozzle at the reverse-side of the cell receiver. CONSTITUTION:A plate 8 having a number of small holes (micro cell) 9 and a cell-trapping filter at the bottom is supported in a vessel 7 filled with a liquid and a liquid suction nozzle 10 is attached to the reverse side of the small hole 9. A feeding path 6 to flow down a cell suspension liquid is placed above the small hole 9. The feeding path 6 is provided with cell-detecting means 15, 14, 16 and the microcell for receiving the cell is displaced by moving the plate 8 according to the signal transmitted from the detecting means. The feeding channel 6 is connected with feeding tanks (microsyringe) 1, 2 of different kinds of cells via a three-way valve 4.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a cell fusion device, 4? The present invention relates to a cell fusion device for plant breeding that automatically and reliably fuses a large amount of different fine N& cells.

[Conventional technology]

In cell fusion, a hybrid f@AB is created from two genetically different types of cells, A and B. Polyethylene glycol (PEG) is often used as a fusion agent in plant breeding. However, in this case, not only AB is produced as a hybrid cell, but also fusions of AA and BH between homogeneous cells and multiple fusions such as ABH and BBBB are also produced. The selective medium method is used to select only hybrid cells from among these cells.

There are morphological classification methods, direct selection methods, etc. However, the selective culture medium method is limited to the plants to which it can be applied, and is not versatile.

Although the morphological classification method and the direct selection method are versatile, they are very inefficient because they are performed manually.

In addition to the method of using a fusion agent, an electric fusion device that uses electric pulses to perform fusion has also been proposed, but the rate at which hybrid cells can be created is around 10%. Another problem is that the application of electric pulses requires the judgment of a skilled person.

The above is an example of the current status and problems of cell fusion.

For Mori: ° Electrofusion and Selection”, Cell Engineering, Vol.

3. Nn6, 497-505.1984.

[Problem that the invention seeks to solve]

To summarize the problems of the above-mentioned conventional techniques, they cannot be applied to all plants and cannot efficiently select miscellaneous AI cells. An object of the present invention is to solve these problems of the prior art.

[Means for solving problems]

The above object is achieved by reliably fusing only the pair of cells A and B. To achieve this, a pair of cells is injected into a small container (microcell), and a fusion agent is applied thereto.

In particular, the tip of a first conduit for supplying cells and the tip of a second conduit for suctioning a solution are placed opposite each other in a container that holds the solution, and a plate is prepared in which various types of microcells having filters at the bottom are arranged. It is characterized by having a configuration in which desired cells are injected into desired microcells by placing the cells between the two tips and supplying the cells while manipulating the position of the plate.

[Effect]

That is, the solution containing cells is stably transported from the first pipe line to the second pipe line, and the cells are trapped in the microcell having the filter that is fed during this time. Therefore, it is the first type that should be fused.

The pair of cells of the second type can be injected into the microcell sequentially and accurately at high speed. The fusion agent is added after a pair of cells have been injected. Microcells are prepared in a number equal to the number of fused cells required.

Cells of the same species interact with each other and never fuse.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. A.M.
Cells and B cells are accumulated in supply tanks 1 and 2, respectively.

The #I cells used here are in the state of thin protoplasts without walls, and the solution is usually around ICC, so a microsyringe or the like may be used as the supply tank. Thin I
@ passes through the conduit 3 and selects either A or B cell by the three-way cut valve 4. The cells pass through channels 5 and 6,
Enter the solution filled in container 7. A plate of microcells 8 is set horizontally just below the tip of the conduit 6. Only one of the flowing cells enters the microcell. There is a filter 9 between the cells.

Cells are supplemented with this filter. In order to form a flow layer of fluid from the pipes 5 and 6 to the microcell 8, the nozzle 10 is placed opposite to the pipe 6, and a suction device 11 is used to apply a negative pressure to the nozzle 0.

In order to make the cell flow n one-dimensional in the conduit 6, the supply tank 12
The sheath source flows into the conduit 13. The effect of the sheath liquid is the same as that of the sheath flow used in blood cell counters and cell sorters. The passage of cells through the conduit 6 is detected by irradiating light from a laser light source 14 onto the conduit, guiding light scattered by the cells to a photomultiplier tube 15 via an optical system such as a mirror or lens, and detecting the state of the scattering. be done. The photomultiplier tube signal is analyzed by a control circuit 16 to record the passage of cells. Since the distance from the cell detection point to the microcell is constant and the cell flow rate is kept constant, the time to reach the microcell is uniquely determined.

In this way, by driving the microcell coupled to the container 7 with the feeding mechanism 17 according to the judgment of the control circuit 16, the W
Cells flowing from channel 6 are supplemented one after another with microcells. Therefore, it becomes possible to set one pair of Ai cells, H, and iQ in each microcell. The conduit 6 and the nozzle IO are in opposing positions and fixed, and the plate having the microcells moves in two dimensions.

A pair of thin IL! into a microcell plate, tube 1
8, a polyethylene glycol solution is injected into the container 7, and after a certain period of time, a calcium ion solution is injected into the container 7 through a conduit 19. As a result, these solutions diffuse into the microcell and are injected, but the conduit 20 may be opened to facilitate the injection.

A method for removing the microcell plate from this overall apparatus as a means for taking out the fused side @ generated in the microcell, and a method for removing the buffer solution from the three-way switching valve 225 through the pipe 2.
There is a method in which the liquid is injected back into the microcell from the nozzle 10 via the pipe 23 and then taken out from the conduit 23. In the former method, after removing the plate.

Remove the fused cells by shaking manually in fluid or applying ultrasonic vibration.

Note that before extracting A and B cells, the conduit 21 and the three-way switching valve 22. Spray the cleaning solution onto the microcell plate from the nozzle 10 to remove the clogging of the filter.

and supplied sheath fluid. However, the solution in the container 7 may be used as the sheath liquid without preparing the sheath liquid for transfer.

What is the configuration in this case? Shown in Figure 42. By making the diameter of the suction nozzle 10 as narrow as the cell diameter, a narrow channel is formed from the conduit 6 to the nozzle 10, and the cells form a one-dimensional line near the n1 microcell 8. Detection of cells from light source from outside of container 7] From 4? This is achieved by irradiating light onto a@. Other configurations are the same as in Figure 1. Figures 1 and 2 In the embodiment shown, there is only one conduit for supplying cells, and a three-way switching valve is used to switch between two types of cells. Ta. However, dedicated conduits may be provided for each.

Furthermore, although the microcell 8 is moved together with the container 7 in FIGS. 1 and 2, the microcell alone may be moved by an arm 24 that straddles the container 7, as shown in FIG.

〔Effect of the invention〕

According to the present invention, it is possible to reliably fuse cells of A. 82 species with different genetic properties one pair at a time, thereby eliminating the need for means for selecting hybrid cells and efficiently performing cell fusion of all kinds of plants. It can be carried out by threading.

[Brief explanation of the drawing]

FIG. 1 is a system diagram showing an embodiment of the present invention. The shaded area shows the longitudinal section. FIG. 2 is a longitudinal sectional view showing an embodiment different from FIG. 1, and FIG. 3 is a longitudinal sectional view showing an embodiment different from FIGS. 1 and 2. 1... A cell supply tank, 2... B cell supply tank, 8...
・My small cell% 10...nozzle, 12...sheath flow supply. ]4... Laser light source, 15... Photomultiplier tube, 17
...Feeding mechanism.

Claims (1)

[Claims] 1. A conduit for supplying cells, a solution suction device installed at a position facing the tip of the conduit, and a filter installed in the gap between them to allow passage of the solution, but with a filter at the bottom to trap cells. It is equipped with a plate having a large number of microvessels in the cell, a means for detecting the presence of cells flowing through a conduit, a feeding mechanism for moving the plate, and a means for adding a fusion agent necessary for cell fusion, and for operating a solution suction device. Pour the cells into the tube,
A cell fusion device that controls a plate feeding mechanism based on a cell presence detection signal, injects two types of cells one pair at a time into microvessels on a plate, and then adds a fusion agent.