JPS6260062B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cell isolation device for selecting and isolating cells that produce secreted substances such as monoclonal antibodies.

[Conventional technology]

Conventionally, a method for selecting and isolating cells that produce secreted substances such as antibodies, called the limiting dilution method, is as follows. After culturing the fused cells placed in the wells, the culture supernatant in the wells in which the fused cells are found to be proliferating by colony observation is dispensed, and the culture supernatant is collected using an Elizer. Detect whether the target antibody is contained within. Next, a diluent is added to the cell solution producing the antibody of interest to the extent that the number of cells can be counted using a microscope, the cells are uniformly dispersed, and then the number of fused cells is counted. Measurement is performed by counting the number of cells by microscopic observation and calculating the total number of cells contained in the cell fluid.

Next, based on the number of cells obtained, 1 in 3 wells
Dilute the cell solution so that aliquots are obtained. This is because in order to obtain a single clone, it is necessary to increase the probability that two or more cells will not be dispensed into one well. After adding thymocytes that do not have the ability to produce antibodies so that the diluted and dispensed cells proliferate, repeating the dilution-dispensing-culture procedure increases the probability that they are monoclonal and increases monoclonal antibody production. Isolate the cells.

[Problem that the invention seeks to solve]

Even if the conventional limiting dilution method described above is repeated,
There is still a probability that it is not a monoclonal clone, and it is possible to increase the probability that it is a monoclonal clone by repeating the limiting dilution method.
That much effort and time are required.

[Means for solving problems]

The present invention has been made to solve the above-mentioned problems, and its gist is to provide a cell isolation system comprising a preparative dispensing section, a tray transport section, a cell counting section, an isolation dispensing section, and a controller. In the device, the preparative dispensing unit includes a manipulator that grips a pipette and a pump for sucking and discharging a fixed amount of liquid with the pipette, and the tray transport unit includes a tray stand on which a tray with a plurality of wells is placed. and a driving means for transporting the tray stand to each predetermined position in the sorting and dispensing section, the cell number counting section, and the isolation and dispensing section, and the cell number counting section transports the preparation to a position under the microscope. It consists of a drive motor that moves the cells and a control circuit that counts the number of cells observed.
The isolation and dispensing section consists of a dilution section consisting of a dilution container, a thin tube through which the cell liquid in the dilution section passes, and a detection section that detects cells passing through the thin tube, and the controller has a pipette tip mounted on a tray. means for transmitting a signal to the pipette manipulator for moving the solution into the well and onto the preparation; means for transmitting a signal to the pump for injecting and ejecting the solution into the pipette; A means for transmitting a signal to the drive motor of the preparation transport unit to move the specimen to the observation position, and a means for transmitting a signal to the drive motor of the preparation transport unit, and when the number of cells counted by the control circuit is equal to or higher than a certain value, the tip of the pipette is moved to the same cell solution as observed by the microscope. A means for transmitting a signal to the manipulator to move the solution into the well containing the solution and onto the dilution container, and a dilution section that calculates the required dilution amount based on the counted number of cells and sends a signal to add the calculated amount of diluent to the dilution container. means for transmitting a signal to the cell isolation unit for introducing the solution in the dilution container into the capillary, and a signal for moving the tray stand at well intervals in synchronization with the detection signal of cells passing through the capillary. The cell isolation apparatus is characterized in that it is a device comprising means for transmitting a signal to a driving means of a tray conveyance section, and is a device for controlling each section according to input operating conditions of each section.

[Function and Examples of the Invention]

The operation of the invention will be explained below with reference to the drawings showing embodiments.

FIG. 1 is a perspective view showing an embodiment of the present invention.
A portion of the cell solution is taken out from a target well on the cell culture tray 1 in which cells are proliferating by a pipette 3 moved by a pipette manipulator 2 and dropped onto a preparation 6. The pipette manipulator 2 includes a horizontal articulated arm,
Orthogonal arms etc. can be used. The pump 4 controls the discharge amount based on the pressure value and pressurization time.
The preparation 6 onto which the cell solution has been dropped is moved to a position below the microscope 8 by the drive motor 5 of the preparation transport section 7. The image observed by the microscope 8 is sent to the control circuit 24 through the TV camera 9, and the number of cells is counted by digital processing. Here, the cell number counting section is composed of, for example, a microscope 8, a TV camera 9, a control circuit 24, an input device 23 which also functions as a monitor, a slide 6, and a slide transport section 7. If the number of cells exceeds a certain value, dispense the original cell solution into the dilution container 10 using the pipette 3 and pipette manipulator 2, and dispense the diluted solution in an amount determined based on the number of cells. Add and dilute using the diluting section 11.

Cell concentration is calculated by the following method. That is, the amount of cell solution in the well and the amount of sampling on the preparation 6 are kept constant, and the amount is calculated by back calculating the volume ratio from the number of cells on the preparation. The diluting unit 10 includes a diluent tank and a pump, and controls the discharge amount of the diluent by controlling the time of the pump. The diluted cell solution is inhaled by the cell isolation and dispensing section 12. The isolation/dispensing unit has a configuration as shown in Fig. 2, for example, in which a diluted electrolyte flows in a thin tube 29, and the resistance value of the electrolyte flowing between the electrodes changes depending on whether there are cells or not. Cells are detected by changes in the measured voltage value, which differs depending on the case. The isolation and dispensing unit 12 is composed of a suction pump 30 and the above-mentioned cell detection units 26 and 27, and when cells are detected, it transfers the culture tray 21 to the tray transport unit 19 in synchronization with this cell detection signal. The cells are then moved, and each cell is dispensed into each well in the culture tray 21 that is not used for culture.

This is the liquid feed pipe 3 from the cell detection unit to the well.
1 is set to a value smaller than the volume of the well, and once a cell detection signal is obtained, the suction pump of the isolation/dispensing section 12 is temporarily stopped. Then, after driving the tray transport unit 19 and moving the well to which cells are to be dispensed next to directly below the liquid feeding tube, the suction pump is restarted and the cells are dispensed into the well. In this case, there are two
If more than one cell exists, one cell cannot be dispensed into one well, so to prevent such a situation from occurring, the dilution container 10 is used to dilute to a necessary and sufficient value. This value is determined experimentally in advance. The drive means for the tray conveyance unit 19 is composed of stepping motors 13, 14, feed screws (ball screws) 15, 16, and guides 17, 18, and the tray 21 is positioned by the number of input pulses to the stepping motors 13, 14. I do. The driving means may also be air driven. The tray chuck 20 is installed on a tray stand 22 as shown in FIG. 3, and is driven by a motor to open and close in the θ direction,
Directional movement is possible. In addition, in Figure 3,
29 is a motor, and 30 is a peen rack.
In addition to the fractional dispensing section shown in FIG. 2, a rotating body such as a fraction collector can also be used. The controller is a device consisting of, for example, an input device 23 and a control circuit 24,
Each part is controlled according to the above-mentioned parts and operating conditions between the parts inputted from the input device 23.

A block diagram and an operational flow diagram showing the signal system in the present invention are shown in FIGS. 4 and 5, respectively. The control signal _S1 activates the manipulator 2 to move the pipette 3 held by the manipulator to one of the plurality of wells on the culture tray, and the control signal _S2 activates the pump to remove the cells in the well. Aliquot the solution. Thereafter, the control signal _S1 operates the manipulator 2 to move the pipette 3 to a position above the preparation 6, and the control signal _S2 moves the cell solution in the pipette 3 onto the preparation 6.
Drip onto the.

The control signal S ₅ is a signal for starting and stopping the drive motor in the preparation transport section. This control signal S ₅ moves the preparation 6 below the microscope 8, and the cell counting section counts the number of cells in the cell solution. Count. The measurement signal T ₁ contains the number of cells, and its value is stored in the controller as
Calculate the cell concentration in the cell solution. When the cell concentration is above a certain value, the control signal _S1 moves the pipette of the manipulator 3 to the well containing the cell solution at that cell concentration, and the control signal _S2 activates the pump to transfer the cell solution to the pipette. Separate. Next, the pipette that dispensed the cell solution is moved to the dilution container by the control signal _S1 , and the cell solution is poured into the dilution container by the control signal _S2 .
In step _S6 , the diluent is added to the dilution container to dilute it to a certain concentration or less, and the cell concentration in the cell solution is adjusted to an appropriate concentration for cell isolation and dispensing. The diluted cell solution is sucked into the cell isolation/separation unit 12, and when the cells pass through the thin tube 64, a measurement signal _T2 is transmitted.
The measurement signal T ₂ is a signal indicating that cells in the diluted cell solution have passed through the detection position of the cell solution passage of the cell isolation and dispensing unit. In synchronization with this cell detection measurement signal, the control signal S ₃ and S ₄ , the tray conveying section is operated to move the distance between the wells on the tray at the outlet 28 of the liquid feeding pipe 31.

Through the above-described process using the cell sorting device of the present invention, individual cells can be isolated from a group of cells.

[Effects of the invention] By using this device, it is guaranteed that the obtained antibody is monoclonal, and unlike selecting monoclonal antibody-producing cells by limiting dilution, there is no Since there is no need to repeat proliferation, monoclonal antibody cells can be selected in a short time. In addition, since these operations can be performed in a device with a sterile environment, there is no risk of contamination with bacteria, and advanced techniques are no longer required, making it possible to easily isolate monoclonal antibody-producing cells. .

From the above, it is clear that this invention will greatly contribute to the application of monoclonal antibodies.

[Brief explanation of the drawing]

Fig. 1 is a perspective view of the cell isolation device of the present invention, Fig. 2 is a perspective view of the cell isolation device of the present invention;
The figure is a schematic diagram of the cell detection unit in the present invention, and Figure 3A is a plan view of the tray conveyance unit in the present invention.
Figure b is its side view. FIG. 4 is a block diagram of the present invention, and FIG. 5 is an operational flow diagram of the present invention. 1...Cell culture tray, 2...Pipette manipulator, 3...Pipette, 4...Pump, 5...
... Drive motor, 6 ... Preparation, 7 ... Preparation transport section, 8 ... Microscope, 9 ... TV camera, 10 ... Dilution container, 11 ... Dilution section, 12 ...
...Isolation and dispensing section, 19...Tray transport section, 20...
Tray chuck, 21... Tray, 22... Tray stand, 23... Input device, 24... Control circuit, 2
5... Cell, 26... Electrode.

Claims (1)

[Claims] 1 A cell isolation device consisting of a preparative dispensing section, a tray transport section, a cell counting section, an isolation dispensing section, and a controller, wherein the preparative dispensing section includes a manipulator that grips a pipette, and a manipulator that holds a pipette, The tray transport section includes a tray stand on which a tray with a plurality of wells is placed, and the tray stand is connected to the preparative dispensing section,
It consists of a cell counting section and a driving means for transporting the specimen to each predetermined position in the isolation/dispensing section.The cell counting section counts a microscope, a drive motor that moves the preparation to a position under the microscope, and the number of observed cells. The isolation and dispensing section consists of a dilution section consisting of a dilution container, a capillary through which the cell liquid in the dilution section passes, and a detection section that detects the cells passing through the tubule. means for transmitting a signal to the pipette manipulator to move the tip into a well provided in the tray and onto the preparation; means for transmitting a signal to the pump for injecting and ejecting a solution into the pipette; means for transmitting a signal to the drive motor of the preparation transporting section to move the counting section to an observation position under the microscope; and means for observing the tip of the pipette under the microscope when the number of cells counted by the control circuit is above a certain value. A means for transmitting a signal to a manipulator to move the cell solution into a well containing the same solution and onto a dilution container, and calculating the required dilution amount based on the counted number of cells, and transferring the calculated amount of dilution solution to the dilution container. a means for transmitting a signal to the dilution section to add a solution to the dilution container; a means for transmitting a signal to the cell isolation section for introducing the solution in the dilution container into the tubule; A cell isolation device comprising a means for transmitting a signal for moving at well intervals to a driving means of a tray conveying section, and controlling each section according to input operating conditions of each section. .