JPS62179380A - Apparatus for cell fusion - Google Patents

Abstract

PURPOSE:To provide a cell fusion apparatus composed of a specific centrifugal tube holding tool, a centrifugal tube heating device, a pipette driving device, a liquid feed controlling device, a solution tube heating device and a controller and capable of realizing accurate and reproducible stirring, dilution, etc., in cell fusion process. CONSTITUTION:Solutions of two kinds of cells to be fused are mixed together in a centrifugal tube 1, which is treated with a centrifugal separator. After removing supernatant liquid, the centrifugal tube 1 is fixed to a holding tool 2. The solution is dripped from the solution tubes 5-7 into the centrifugal tube with a pipette 3 according to the procedure programmed by a controller 4 and the centrifugal tube is stirred by a pipette driving device. A centrifugal tube heating device 9 and a solution tube heating device 10 are furnished with a heat sensor and a heater. The pipette driving device is composed of a vertically driving means 81, a horizontally rotating means 82 and a radially driving means 83 and is operated according to the output of the controller 4.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a cell fusion device.

[Prior Art] The cell fusion method is used in various industries such as medicine, food, agriculture, biology, medicine, etc. as a means to newly create a characteristic proliferative hybrid cell line such as a monoclonal antibody-producing proliferative cell line. , is frequently used in basic pharmaceutical research.

As a cell fusion method, polyethylene glycol (PEG)
), methods using chemical cell fusion-promoting substances such as lysolecithin, glycerol, and oleic acid esters, methods using inactivated viruses with cell fusion-promoting ability such as Sendai virus, and electric cell fusion methods are known. . Among these methods, the method most frequently used at present is the method using PEG. For this method, Ba1b/c mouse tile cells and myeloma cells (e.g. Sp210-A
A typical example using g+4) will be described below.

The spleens were removed from several immunized mice (I3alb/c), and the visceral cells were squeezed out using forceps or the like in a 100 mm diameter Nyare filled with a Hank's strainer.

Pour the tile cell suspension into a centrifugal duplex with a volume of about 5 liters, centrifuge (1600 rpm, 5 minutes), discard the supernatant, add a new hemolytic solution 5-7!!Q, Add 5 to 7 mQ of Hank's solution, centrifuge, and discard the supernatant. Add 10 mQ of Hank's solution and discard the centrifuged supernatant. Repeat this operation. Repeat the procedure twice and suspend the precipitate in an appropriate amount of medium or Hank's solution. Count the number of living cells using a hemocytometer and use mouse bond cells of +09 ([! The cells that have been subcultured for several days before the test were washed 2 to 3 times with Hank's solution by the same centrifugation procedure as above, and the precipitate was suspended in an appropriate amount of culture solution or Hank's solution, and the cells were collected using a hemocytometer plate. Count the number of living cells and prepare 10'g of myeloma cells.

Prepared 2xl O” cells, 5xi myeloma cells
O'~IxlOJl were mixed in a plastic centrifugal duplex with a capacity of about 50 ml, and centrifuged (1600 rpm).

5 minutes) and discard the supernatant. Add 50% PIEG (molecular weight approximately 1500 to 4000) I5mM HEPES, DME to this cell precipitate while keeping it warm at 37°C.
While stirring with a pipette, gradually add 2 mQ of the solution (in 1 minute).After this, stir with a pipette for 1 minute and 30 seconds, then add 1 mQ in the first 4 minutes while stirring.
2Rρ every minute then I2mQ for 2 minutes, total 20J11
Add 2 of DME. Furthermore, 15mM HEPES,
DME solution 20! Add JQ and centrifuge (1600 rpm,
5 minutes) and completely remove the supernatant.

Add 20% FCS and DME solution to the cells while still in the precipitated state.
Layer xQ and leave to stand for about 30 minutes. After that, 20%FC
8. Add DME solution to make the total volume 40 times, and add 2 portions of this solution per well to two 96-well plates for agitation culture.
Dispense 00 μg each and start culturing.

An example of a cell fusion method using PEG was explained, and in this series of operations, from dispensing 50% PEG to HEPES, DM
The optimal method for dispensing up to 20 mQ of E solution has not been established, and the amount of solution dispensed, dispensing speed, stirring time, stirring method, etc., depend on the type of cells used for fusion and the amount of solution. It varies depending on the type and experimenter. These operations are performed manually, so stirring and dilution are always accurate and reproducible.
It was very difficult to do so, and there were cases where bacteria were introduced into the system.

[Object of the Invention] The object of the present invention is to provide an apparatus that can accurately and reproducibly perform such stirring and dilution during cell fusion, prevent contamination with various bacteria, and can respond to a variety of conditions.

[Means for Solving the Problems] The gist of the present invention is a cell fusion device that includes a centrifugal duplex holder, a centrifugal tube warming section, a pipette drive section, a liquid feeding control section, a solution duplex warming section, and a controller. , (a) the centrifugal tube holder consists of means for fixing the centrifugal duplex; (b) the centrifugal duplex heat-retaining section and the solution duplex heat-insulating section have a heat-insulating means for keeping the duplex warm and a thermal sensor for measuring the temperature thereof; C) Pipette drive unit:
It consists of a pipette and a driving means capable of driving the pipette vertically, horizontally, and rearward, and (d) the liquid feeding control section selects a pump for feeding liquid to the pipette and liquid feeding of two or more types of solutions to the pipette. The cell fusion device has a valve, and (e) the controller controls a pipette drive section, a liquid feeding control section, a centrifuge tube warming section, and a solution tube warming section according to operating conditions input in advance.

The invention will be explained below with reference to the drawings which show examples.

Note that the present invention is not limited to the following embodiments.

FIG. 1 is a schematic diagram showing a typical embodiment of the cell fusion device of the present invention. Mix the two types of cell solutions to be fused in a centrifuge tube (1), centrifuge the centrifuge duplex (1), remove the supernatant, and fix the centrifuge duplex (1) to the holder (2). . The centrifuge tube (1) used is a typical plastic conical tube (eg Falcon 2070) commercially available for cell processing. The retainer (2) is typically a Falcon 207
0 (30 mm in diameter), and the duplex is held at a position where the heat retaining effect of the centrifuge tube heat retaining part is efficiently maintained.

The pipette (3) drips the solution from the solution tube (586×7) into the centrifuge tube according to the procedure programmed by the controller (4), and performs a stirring operation using the pipette drive unit. Typically, centrifuge tubes (1
) while stirring the liquid part in the solution duplex (5) for 1 minute.
) of 50% PEG was pumped at 2 mQ/min for 1 minute, and then the DME in the solution duplex (6) was pumped at 2 mQ/min by switching the liquid feeding valve.
The liquid is pumped for 4 minutes at mQ 1 minute and then for 2 minutes at 6 iQ/minute. Finally, add l 5mM HEPE from the solution tube (7).
S, DME solution is dispensed for 10 seconds at 120 ml/min. The stirring method and liquid feeding method of the pipette (3) are set in advance in the memory in the controller (4).

Centrifuge tube heat retention part (9) and solution tube heat retention part (
10) has a thermal sensor (for example, a thermocouple) and a heater, and the container housing them should include a block bath or a constant temperature water bath.

The pipette drive unit includes a vertical drive unit (81), a horizontal rotation unit (
82) and a radial drive unit (83), which is supported by a controller (4) and operated by a Stesobinog motor, etc., to move the pipette tip evenly within the centrifugal duplex, resulting in a stirring effect by the pipette. Let me. Any drive system may be used as long as it can realize spatial movement of the pipette.

The controller (4) controls the driving direction, driving time, and driving speed of the pipette (3). Regarding the liquid feeding from the solution tube (5×6) (7), the type of liquid (valve selection), liquid feeding speed, and liquid feeding time are controlled. Regarding the heat retaining parts (9) and (10), the temperature is calculated from the electrical signal from the heat sensor, and the temperature of the heater is 0N1.
0FF control is performed to maintain a preset temperature (eg, typically 376C).

The liquid feeding control unit (11) has a pump for liquid feeding, such as a peristaltic pump, and a valve for switching liquid, such as a solenoid valve, and the control of the valve and the pump is performed by the controller (4).

[Effect of the invention] The advantages of the cell fusion device of the present invention are as follows.

(1) Stirring and dilution can be performed accurately and reproducibly according to the instructions of the controller, so the optimal fusion conditions for cell fusion can be easily determined, and effective cell fusion can be performed with high reproducibility.

(2) Prevent contamination with germs.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing a typical embodiment of the cell fusion device of the present invention. l...Centrifugal tube, 2...Centrifugal duplex holder,
3. Pipette, 4. Controller, 5.6.7.
- Solution tube, 81, 82, 83... Pipette drive unit, 9... Centrifugal tube heat retention unit, IO... Solution tube heat retention unit, 11... Liquid feeding control unit.

Claims (1)

[Scope of Claims] 1. A cell fusion device comprising a centrifuge tube holder, a centrifuge tube warming section, a pipette drive section, a liquid feeding control section, a solution tube warming section, and a controller, comprising: (a) a centrifuge tube holder; (b) The centrifuge tube heat retention unit and the solution tube heat retention unit include a heat retention unit that retains the temperature of the tube and a thermal sensor that measures the temperature thereof; (c) The pipette drive unit includes:
It consists of a pipette and a driving means that can drive the pipette in up, down, left, right, front and rear directions, and (d) the liquid feeding control section includes a pump that sends liquid to the pipette, and a valve that selects the feeding of two or more types of solutions to the pipette. (e) A cell fusion device characterized in that the controller controls a pipette driving section, a liquid feeding control section, a centrifugal tube warming section, and a solution tube warming section according to operating conditions inputted in advance.