JPS6174570A - Cell-selection apparatus - Google Patents

Abstract

PURPOSE:To enable the efficient selection of hybridoma cells, by automating the selection process with a controller. CONSTITUTION:The container 1 is transferred to the position of the pipette manipulator 4, and the cell solution is pipetted to the well of the culture tray 18, and cultured for a definite period. The supernatant liquid of the cell solution in the well containing a colony detected by the colony detection device is assayed by the assaying device 9. When the production of the objective antibody is certified, the cell in the well is diluted to one cell per one well to obtain a monocloned cell. The dilution procedure is repeated twice to increase the probability to form a monoclone.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a cell sorting device that can efficiently select target cells from a group of cells or a group of microorganisms containing several types of cells or microorganisms. It is related to.

[Conventional technology]

Monoclonal antibodies are used in the study of molecular structures, genes, and their functions in antibody chemistry and antigen chemistry, in the study of hormones and neurotransmitter receptors in pharmacology, and in other fields such as virology, parasitic history, and bacteriology. It is something that is used. In addition, in diagnosis, it is used in applications such as organ transplantation (histocompatibility), passive immunization (injection of antibodies), and treatment of malignant tumors.

As mentioned above, monoclonal antibodies have a wide range of applications.

Secretory products such as monoclonal antibodies are obtained from the cells that produce them, and conventional methods for selecting and producing monoclonal antibody-producing cells include, for example, the following method.

Tile cells (2,5X 10 cells), @cancer cells (2,5X
107 pieces) etc. and polyethylene glycol 11” M
Cell fusion is performed as a synthesis promoter, and after centrifugation, the fusion solution is discarded and HA
T culture solution (Hypoxanthine Am1nopf
A medium solution containing erin and Tbymidine f) is added and dispersed into the cells. After that, the cell solution obtained by dispersion is placed in a tray with a plurality of shells, for example, 0.2
Dispense in ml; liter portions, and then culture in a CO2 incubator for 2 weeks. This culture kills unfused cells and proliferates fused cells.
.

Whether or not fused cells are proliferating is observed by the presence or absence of colonies, and wells tζ in which fused cells are found to be proliferating are determined using ELISA (EIJSA: Enzy
me 1inked irrmuno 5orbent
assay) Place the culture supernatant on the detection tray.
Separate and dispense. Thereafter, whether or not the objective antibody is contained in the collected culture supernatant is detected by ELISA.

At this time, the number of wells into which the cell solution was dispensed is, for example, 48.
Those with 0 antibodies are now producing the desired antibodies.
Usually about 5 hours? :,Become.

Add a diluent to the cell solution in the well containing the antibody of interest to the extent that the number of cells can be counted using a microscope to uniformly disperse the cells (initial dilution).

Next, a portion of the cell solution to be initially diluted is counted on a counting board and observed under a microscope (I7iI number), which is then calculated as the total number of cells contained in the cell solution.

Based on the calculated number of cells, dilute the IGA so that when 0.2 ml is dispensed into each well, IGA is dispensed into 3 wells (cell solution dilution). This cell solution dilution is to increase the probability of dispensing more than one cell into one well in order to obtain a single clone.

Thereafter, 770 thymocytes with no antibody-producing ability are obtained because the cells need to be in a certain number in order to proliferate.

The above operations (HA T #! i Add the nutrient solution, disperse the cells, dispense the cell solution into the wells, then add the thymocytes, this series of operations i 1J Mitsting.
The procedure is repeated twice (referred to as dillsion) to increase the probability of monoclonality, and the resulting monoclonal antibody-producing cells are cultured in large quantities to produce monoclonal antibodies in large quantities.

[Problem that the invention seeks to solve]

Conventionally, as mentioned above, monoclonal antibody-producing cells have been manually selected from a group of cells that have undergone cell fusion, but the probability of obtaining the desired monoclonal antibody-producing cells is low, and However, there are drawbacks such as the low stability of the collected cells (that is, they are likely to die) and the risk of contamination with foreign matter during the selection process, making it difficult to produce a large amount of monoclonal antibodies with high activity. When trying to select cells, it is necessary to select from a huge number of fused cells.

Furthermore, this sorting work requires advanced technology,
Acquiring this technique usually requires 1 to 2 years of education, so there are very few engineers who can select monoclonal antibody-producing cells.

For this reason, the number of cells that can be treated in a series of experiments is limited, and the probability of obtaining the desired monoclonal antibody-producing cells is low, and even if they are obtained, the result is only a low antibody-producing ability, and monoclonal This is an inhibiting factor in antibody application Q.

[Kth method to solve all problems]

The present invention has been made to solve all of the above-mentioned problems, and its gist consists of a cell sorting and dispensing section, a tray transport section, a culture section, an assay section, an isolation and dispensing section, and a control section. In the apparatus, the preparative dispensing section includes a pipette, a manipulator for gripping the pipette, and a pump for suctioning and discharging a certain amount of liquid from the pipette, and the tray conveying section includes a tray stand on which the tray is placed, and a manipulator for gripping the pipette. It consists of a tray stand and a driving means for transporting the tray to a predetermined position in the preparative dispensing section, the culture section, the assay section, and the isolation dispensing section,
The culture unit includes a tray stock that stores one of the trays, and the controller receives input information according to the operating conditions of each unit. The present invention resides in a cell sorting device that is characterized by being a controlled device.

[Action of the invention]

The operation of the device of the present invention will be explained below.

The solution containing the fused cell group is dispensed in fixed amounts into the wells (l\\liquid reservoir) on the culture π tray by the dispensing unit, and the tray for the number of litters is placed on this culture tray by the tray transport unit. Contains a retractable tray stock and transports it to the Bamboo Co2 culture section. The culture tray that has been maintained in the CO2 culture section for a certain period of time under conditions that allow the growth of target cells is returned to the tray transport section, and if it is necessary to detect proliferation by colony observation, a colony observation section is provided.
This detects whether colonies are growing in each well. A fixed amount of the supernatant liquid of the well in which colony growth has been detected is dispensed into a detection tray by an automatic sorting/dispensing section.

The detection tray filled with the supernatant liquid is carried into the assay section by the tray conveyor groove, and it is detected whether or not the supernatant liquid contains antibodies. As a result, if the culture tray wells are confirmed to contain antibodies in the supernatant, for example, if the cell concentration in the solution is unknown or inconsistent, some of the wells may be further automatically removed. The cell number is transferred to the cell counting section Cζ by the preparative separation section, and the cell concentration in the solution is measured. If the cell concentration is higher than that at which it is possible to separate and dispense each cell into 1-11 cells, dilute it to the desired concentration using a grading mechanism, and then use the diluted liquid separation and dispensing section to separate the cells into new cells. Dispense the cells so that each well contains 1 - cells in a culture tray. This culture tray (with one cell dispensed into each well) is stored in the CO2 culture section after adding pond cells if necessary for proliferation, and from there, individual cells are dispensed. Monoclonal antibody-producing cells are obtained by repeating the above-described mechanism one more time in sequence until the point mentioned above. These operations are automatically controlled by the controller according to conditions input from the outside. In addition, among the above operating shallow grooves, in cases where it is not necessary to measure the degree of cell proliferation, or when the number of non-proliferating cells is suitable for the normal operation of the isolation and dispensing mechanism, It is not necessary to provide a colony observation section and a dilution mechanism.

〔Example〕

Embodiments of the present invention will be explained with reference to all the drawings and their functions. Is Fig. 1 the cell sorting device of the present invention? FIG.

Container 1, such as a test tube, containing fused cells and culture medium
The container 1i is grabbed by the container handler 2 and moved to the location of the pipette manipulator 4. At the tip of the container handler 2, a chuck operated by an air-driven mandrel or a motor is attached, and the chuck grips the container i6. The pipette 3 is inserted into the container 1, and the cell solution is aspirated using the pump 5. The pipette manipulator 4t is moved to the well position of the culture tray 18, and the pump 5 discharges a certain amount of cell solution into the well. Tray transport #Move the pipette 3 and another well by moving the pipette manipulator 4 and transfer a certain amount of cell lysed Wt in the same way.
Discharge into k-well. These operations are repeated to dispense the cell solution into each well on the culture tray 18. The pipette manipulator 4 can use a horizontal multi-joint arm or an orthogonal arm, and the operation of the tray conveying section M can be carried out by, for example, a motor drive as shown in FIG. 2 or an air drive as shown in FIG. Such driving means can be used all around. In addition, in Fig. 2, 39 is a feed screw (ball screw).
, 40 is a pulse motor, 41 in FIG. 3 is a near cylinder, 42 is an m-brake, 43 is a potentiometer, and 44 is an electromagnetic valve. The pond wire can be used for driving minutes. Culture medium tray 1 with solution dispensed
8 is a CO2 incubator 24 via a tray stand 19.
The cells are stored in a tray stock 20 inside the cell and maintained in a cell culture environment for a certain period of time. In order to store the culture tray 18t in the tray transport unit w2r)t, first, the tray transport unit 16 moves all of the culture trays 18 to a position where they are in contact with the tray stand 19, and then the tray chuck on the tray stand I9]7 By moving the culture tray 18 to the tray stand 1
9 and is stored in the tray stock 20. The tray chuck 17 can be opened and closed in the θ direction and moved in the x and z directions by being driven by a motor as shown in FIG. In FIG. 4, 15 is a motor, 4G is a pole screw, and 47 is a beon rack. Alternatively, these operations can also be performed by air drive. The culture tray 18 that has been kept in the cell culture environment in the CO2 incubator 24 for a certain period of time is moved to the tray transport section 16 by the reverse operation of inserting it into the tray stock 20, and the culture tray 18 is placed at the position of the colony observation section 26. Move each well above 18 and detect if colonies are growing. The colony detecting section 26 can detect colonies by processing an image taken by a television camera or by detecting the amount of light transmitted.

The pipette 3 moved by the pipette manipulator 4 is drawn into the supernatant of the cell solution from each well on the culture tray 18 in which colonies have been detected to be growing, and the supernatant of the cell solution is drawn into the well on the detection tray 9. Dispense a certain amount into each well. Furthermore, using the bed manipulator 4, a fixed amount of the assay sample 174 is added to the wells on the detection tray 9 containing the cell solution supernatant, and the assay sample 174 is transferred to the assay section by motor drive or air drive in the same way as the tray transport section 16. 8
f! to see if the target substance is contained in the cell solution supernatant placed in each well of the detection tray 9.
:To detect. Assay parts include liquid limousine and ELISA (ELISA).
An immunosorbent assay) analyzer or the like can be used. Detection that the target substance is contained in the cell solution supernatant liquid means that the cells in the wells of the original culture tray 18 are producing the target substance. . A portion of the cell solution in the well of the culture tray 18 that is producing the target substance is dispensed onto the preparation of the preparation transport section 31 using the pipette manipulator 29, and then the cells on the preparation are counted by the total cell number counting section 25. Count the number of cells contained in the solution. As the cell number counting section 25, for example, a drawing processing system using a W camera with a microscope, a Coulter counter, or the like is used. The cell concentration of the cell solution in the original well on the culture tray 18 is calculated based on the number of cells counted by the cell number counting section 25, and the pipette manipulator 30 is used to dilute the cell solution to the desired concentration that allows isolated inoculation. The solution is dispensed using the dilution container 33t, and a predetermined amount of diluent is added to the dilution section 34& to achieve the desired concentration.

The undiluted cell solution is inhaled by the isolation/dispensing section 35.

In the single-dispenser dispensing section, a diluted electrolyte flows in a thin tube, and the resistance of the electrolyte flowing between the electrodes is different depending on whether there are cells or not, and this is used to calculate the current value. Detect cells by changes. The mechanism of the isolation and dispensing section is, for example, as shown in FIG.

In addition, referring to FIG. 5, 48 is a cell, 49 is an electrode, and 50 is a cell.
is an ammeter, and 51 is a well.

A new culture tray 18 is moved by the tray transport unit 15 in synchronization with this cell detection signal, and the culture tray 18
Dispense one round of cells into each well. - The culture tray on which individual cells have been dispensed is again stored in the tray stock 20 in the CCh incubator 24 to serve as cell culture.

Further, in the same manner as described above, colony detection and target substance detection are performed, and each part is controlled according to the above-mentioned parts and the operating conditions between each part inputted by the input device. be.

〔Effect of the invention〕

Since the cell sorting device of the present invention has the above-described configuration, it has the following effects. It is something that you have.

Therefore, it can be easily carried out without special advanced technology and the labor force can be significantly reduced.

←) There is no risk of contamination with bacteria, and the amount of processing can be increased, so monoclonal antibody-producing cells can always be stably obtained and efficiency can be completely increased.

(c) Conventionally, it took two to three months to obtain one monoclonal antibody-producing cell, but with this device, it can be obtained in about one month, including the proliferation of a single clone antibody-producing cell. ,
During this time, several types of single clones can be processed simultaneously.

[Brief explanation of the drawing]

FIG. 1 is a perspective view of the cell sorting device of the present invention, and FIG. 2 is a schematic diagram of an example of a tray conveyance unit driven by a motor. The side view shown in Figure 3 is an example of a tray transport mechanism that is driven by air. The side view shown in FIG. 4 is an example of the drive of the tray stand, the tray chuck, and the tray chuck in the tray conveying section. In the diagram shown,
(A) is its plan view, (B) is its side view, and FIG. 5 is a schematic diagram of the isolation and dispensing section. 1. Container 2. Container handler 3.
Pipette 4 Pipette manipulator 5 Pump 6 Pipette exchange section 7 Assay reagent 8 Assay section 9 Detection tray 10 Drive motor 11 Rapid motor 12・Feed screw 13
...Feed screw 14.Guide 15..Guide 16..Tray transport section 17..Tray chuck 18..Culture tray 19..Tray stand 20..Tray stock 21..
Drive motor 22...Feed screw 23...Guide 24...Co2 incubator■ 25...Cell number counting unit 26...Colony detection =
Part 27 - Control part 28 Pump 29...
Drive motor 30... Pipette manipulator 31 Pipette 32... Preparation transport section 33... Dilution container 34... Dilution section 3
5... Isolation/dispensing section 36... Controller 3
7... Input device 38 - Control unit 39...
Feed screw 41...Air cylinder 40...
Pulse motor 43... Potentiometer 42...
・・Electromagnetic brake 45・・Motor 44・・Solenoid valve 47・・Peon rack 4+6・
...Ball screw 49...Electrode 48...Cell 51...Well 50...Ammeter size #2 #4 #5

Claims (1)

[Claims] (1) A cell sorting device consisting of a preparative dispensing section, a tray transport section, a culturing section, an assay section, an isolation dispensing section, and a controller, wherein the preparative dispensing section includes a pipette and a manipulator that grips the pipette. and a pump for aspirating and discharging a fixed amount of liquid with the pipette, and the tray transport section includes a tray stand on which the tray is placed, and the tray stand is connected to the preparative dispensing section, the culture section, the assay section, and the isolation/dispensing section. the culture unit comprises a drive means for transporting the trays to a predetermined position in the culture unit, and the culture unit includes a tray stock that stores a plurality of the trays;
A cell sorting device characterized in that the controller is a device that controls each of the units according to input operating conditions of each unit.