JP6937982B2 - Cell sorting method - Google Patents

Description

The present invention relates to the field of production technology for cells for manufacturing pharmaceutical products.

In the biopharmacy manufacturing process, the cell bank system of the cells that produce the biopharmacy is important, among which the dispensing solution from a single cell line was prepared in a certain manner from the selected clonal cell line. Building a master cell bank is very important. On the other hand, in order to ensure the quality and safety of biopharmacy, it is important to record and store the history and records when constructing a master cell bank in detail.

In order to ensure the unity of the master cell bank, single cell cloning has been conventionally performed. This method is a method in which cells are seeded on a microplate using a limiting dilution method or a cell sorter system, and colony growth and monoclonal colony selection are performed by microscopic observation (Non-Patent Documents 1 and 2). ). Recently, a method of measuring the monoclonality of cells seeded on a microplate with an imaging device has been performed (Non-Patent Document 3). In the technique of injecting a gene into a cell and the technique of producing a fertilized egg, a method of using a manipulation system under a microscope has been developed (Non-Patent Document 4). Further, Patent Documents 1 to 3 describe a manipulation system developed for collecting floating cells by a capillary and performing microinjection on the collected cells.

"Single cell cloning", CeLLaviSta (Ceravista), Application Note (application note) March 8, 2017 Search Internet <URL: http: // www. primetech. co. jp / Portals / 0 / db / product / SynenTech / Cellavista_App_Single_Cell_Cloning_J_rev00_201330. pdf> Nobuo Ryomoto, Jie Xu, Masaya Kurokawa, Akihiko Kondo, Ikuo Fujii, Shunichi Kuroda, "Development of Fully Automatic 1-Cell Isolation and Analysis Device", 2011, Journal of Biotechnology, Vol. 89, No. 2, p. 72 ~ Page 78 "The Cell Metric CLD System", published by Solentim in 2014 Nobuaki Tanaka "Development of Manipulation System for Fine Operations", 2016, NSK TECHNICAL JOURNAL (NSK Technical Journal), No.688, p35-p44

Japanese Unexamined Patent Publication No. 2008-152044 Japanese Unexamined Patent Publication No. 2008-22423 Japanese Unexamined Patent Publication No. 2009-21102

Single-cell cloning using the limiting dilution method or the cell sorter system is very time-consuming and has the problem of poor reliability of unity. Therefore, although measures are taken to guarantee unity by a method using imaging, it takes a lot of time and effort to take an image of a single cell seeded at random, and an image in confirming unity is taken. The accuracy was poor.

As a result of diligent studies, the inventors secured unity by seeding cells one by one in the wells of a microplate using a manipulator and recording them with a video recording device mounted on a microscope. The idea was that the cell bank could be easily obtained and that the creation record of the master cell bank could be recorded and saved reliably and accurately.

The present invention
[1] In a method of separating cells using a manipulator equipped with a microscope, a video imaging device, an electric stage, and a collection pipette.
(1) A step of mounting a storage container and a culture container containing a cell solution on an electric stage mounted on a manipulator.
(2) A process of moving the storage container under the cell collection part of the manipulator by the horizontal movement of the electric stage.
(3) A step of lowering the upper part of the manipulator equipped with the collection pipette and inserting the collection pipette into the cell solution.
(4) A step of collecting one cell from a cell solution using a collection pipette attached to a manipulator.
(5) A step of raising the upper part of the manipulator equipped with the collection pipette to separate the collection pipette from the cell solution.
(6) A step of removing the storage container from under the cell collection part of the manipulator by moving the storage container horizontally.
(7) A step of moving one of the empty wells of the culture vessel under the cell collection part of the manipulator by the horizontal movement of the electric stage.
(8) A step of discharging one cell collected in an empty well of a culture vessel from a cell collection unit,
(9) The step of removing the well containing the cells discharged from under the cell collection section of the manipulator by the horizontal movement of the electric stage and moving the storage container under the cell collection section of the manipulator, and the above (2). )-(9) is repeated to separate cells one by one into individual wells.

The present invention also
[2] The method for separating cells according to [1], wherein the cells are separated while taking a moving image.
[3] The method for separating cells according to [1] to [2], wherein one cell is collected and discharged by a collection pipette using a micropump.
[4] The method for separating cells according to [1] to [3], wherein the step of collecting one cell from the cell solution into a collection pipette is performed by using an automated function by the naked eye or image processing.
[5] The method for separating cells according to [1] to [4], which comprises a second moving image photographing device for photographing cells in a collecting pipette from a horizontal direction.
[6] The cell sorting method according to [1] to [5], wherein the cell sorting operation is provided for recording the entire cell sorting operation.
[7] The method for separating cells according to [1] to [6], wherein the storage container is a 6-well plate.
[8] The method for separating cells according to [1] to [7], wherein the culture vessel is a 96-well plate.
[9] The cell sorting method according to [1] to [8] and the cell sorting method according to [10] [2] to [9], wherein the electric stage is an XY table, are used. It relates to a method for inspecting monochromeity, which is characterized by the fact that.

The present invention further comprises [11] a manipulation system for operating a collecting pipette within the field of a microscope, and is a cell sorting device that separates one cell into one well using the collecting pipette. A first moving image capturing device that takes an image through the microscope to observe the pipette from above, a second moving image taking device that takes an image through a microscope that observes the collecting pipette from a horizontal direction, and the entire device. A third moving image shooting device that captures a bird's-eye view of an image, and a recording device that records images taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device in correspondence with time information. A cell sorting device, characterized by having
[12] The cell fraction according to [11], wherein the time information is the shooting time of each image taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device. [13] The device, and [13] a display device for simultaneously displaying each image taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device. Regarding the cell sorting device.

The manipulator equipped with the microscope, the moving image capturing device, the electric stage, and the collection pipette in the present invention is Z above the electric stage (Ss) on which the storage container and the culture container can be placed and automatically moved in the XY directions. Any manipulator that has a microscope (M1) that is coupled with an actuator (Sz) that can move in the axial direction and is equipped with a camera (C1) of a video recording device so that a collection pipette can be operated below this microscope. Anything may be used, but specifically, it is preferable to use an apparatus whose cross section is shown in FIG.

According to the present invention, a monoclone master cell bank composed of a single cell can be quickly and reliably constructed, and the unity of the cell bank can be completely preserved as an image record.

Manipulation system

The microscope used in the present invention may be any stereomicroscope to which a camera can be attached, but preferably a pipette operation performed under the microscope and a microscope that can automatically focus on cells in the pipette. preferable. Further, in the present invention, it is preferable to provide not only a microscope for observing the pipette operation from above as shown in M1 of FIG. 1 but also a second microscope as shown in M2 of FIG. 1 in which a camera is attached and the pipette operation is observed from the side. ..

The camera used in the present invention may be any camera as long as it can be attached to a stereomicroscope and can capture moving images, but the camera is not limited to the one attached to the microscope as shown in C1 of FIG. It is preferable to use a camera mounted on the second microscope M2 like C2 for photographing cells from the lateral direction, and a camera mounted at a position where the entire pipette operation can be overlooked like C3 in combination with C1.

The electric stage used in the present invention may be any XY table (stage) device for positioning driven by a motor, but it is controlled by a program and the wells in the storage container and the culture container are alternately measured by a microscope. Use a well that can be moved to a predetermined position below and can be sequentially selected as an empty well without cells and moved to a predetermined position below the microscope. Is preferable. As for the method of selecting an empty well, the selection order may be determined in advance according to the arrangement of the wells of the plate to be mounted, or an empty well without cells is searched by a sensor or the like and moved to a predetermined position under a microscope. It may be.

The collection pipette in the present invention may be any shape as long as it can collect cells and can be operated by a manipulator.

In the present invention, the manipulator may be any manipulator as long as it can move the attached pipette to perform fine cell collection operations, but specifically, the manipulator is used as a driving target and has a threaded portion on the outer peripheral side. A screw shaft, a motor that freely supports the rotation of the rotary shaft in the axial direction and rotationally drives the rotary shaft, and a screw shaft fixed to the rotary shaft and screwed to the screw shaft, and the screw shaft is screwed to the shaft. It has a screw element that freely supports movement in a direction, and a fine movement mechanism that finely drives the rotating shaft along the axial direction according to a voltage applied to the piezoelectric element, and is accompanied by the fine movement mechanism in a three-dimensional space. A pair of manipulators having a three-dimensional axis moving table for moving and controlling the position of the pipette, a microscope means for observing cells collected by the manipulator, a control means for controlling the drive of the manipulator, and the above. Anything may be provided as long as it includes an operating means for driving the manipulator via a control means. The control means may be automated by a program or may be manual. A controller for instructing the motor of the manipulator to drive the manipulator may be provided, and the controller may instruct the piezoelectric element to drive the pipette after instructing the motor to drive the manipulator.

The following elements can be added when configuring the manipulator system.
(1) An element in which the controller is connected to a changeover switch and commands drive to the motor or the piezoelectric element according to a changeover operation of the changeover switch.
(2) The controller is connected to a magnification recognition device that recognizes the magnification of the microscope, and is an element that commands the motor or the piezoelectric element to drive according to the recognition result of the magnification recognition device.
(3) The controller is an element connected to the joystick and instructing the motor or the piezoelectric element to drive according to the operation of the joystick.
(4) The controller is connected to a personal computer display that displays a microscope image, and is an element that commands drive to the motor or the piezoelectric element according to the operation of a mouse that operates the microscope image.

In the present invention, the step of mounting the storage container and the culture container containing the cell solution on the electric stage mounted on the manipulator of (1) is a storage container and an empty container containing the cell solution at a designated position of the electric stage. The process of attaching the culture container of the above is shown, and the storage container may be any container such as a petri dish, a well for beaker cell culture, etc. Is preferable. An empty culture vessel refers to a vessel having a well containing no cells, and if it is a cell culture well plate, it is a 6-well plate, a 12-hole plate, a 24-hole plate, a 48-hole plate, a 96-hole plate, or a 384-hole plate. Anything may be used, but it is preferable to use a 48-hole plate, a 96-hole plate, or the like. It is preferable to add a medium to each well of the cell culture vessel in advance in order to culture the cells to be distributed one by one. The cell culture medium used in the present invention is any cell such as iPS cell, ES cell, A549 cell, CTC cell, hybridoma, CHO cell, lymphocyte, leukocyte, yeast, etc. that requires preparation of a monoclonal strain. Although cells may be used, it is particularly preferable to use CHO cells, iPS cells and the like. The medium used for the culture medium may be any medium as long as it is suitable for the growth of the exemplified cells (CD CHO Medium, CD OptiCHO Medium, etc.). The concentration of the cell solution is 1 X 10 ⁴ Cells / ^{mL to 1 X 10 8} Cells / mL, preferably 1 X 10 ⁵ Cells / ^{mL to 1 X 10 7} Cells / mL, particularly preferably 5 X 10 ⁶ Cells / mL. ~ 5 X 10 ⁷ Cells / mL.

In the present invention, the step of moving the storage container (2) under the cell collection unit of the manipulator by the horizontal movement of the electric stage is a drive signal of the electric stage having a drive mechanism in the X direction (horizontal direction). The step of moving the storage container under the cell collection part according to the above is shown.

In the present invention, the step of lowering the upper part of the manipulator equipped with the collection pipette (3) and inserting the collection pipette into the cell solution until the tip of the collection pipette can be inserted into the cell solution in the storage container is By lowering the upper part of the manipulator that can automatically ascend and descend in the Y direction (vertical), the collection pipette attached to the upper part of the manipulator also descends, and the tip portion thereof is in the cell solution in the storage container. Indicates the operation to insert in.

In the present invention, the step of collecting one cell from the cell solution of (4) using a collection pipette mounted on a manipulator is a step of collecting one cell in the cell solution by suction force of a micropump in the collection pipette. The process of collecting is shown. One cell may be collected from the cell solution with the naked eye and manually, or an automated function by image processing may be used. The automation function by image processing indicates a function of moving the tip of a collection pipette relative to the vicinity of cells by an inhalation control means and inhaling cells by a micropump. The control means includes a recognition means for recognizing cells in a solution based on an image signal output from an imaging means, and a position calculation means for calculating the position of the cells recognized by the recognition means. Based on the position information of the cells output from the calculation means, the tip of the collection pipette is relatively moved to the vicinity of a single cell in the solution. Further, the inhalation means of the present invention can hold the inhaled cells at an appropriate position in the collection pipette by inhaling gas from the tip of the collection pipette after inhaling one cell in the liquid, and the cells can be inhaled. You can maintain unity.
The unity of the collected cells is the image of three imaging devices: the first moving image shooting device in the upward direction, the second moving image shooting device in the horizontal direction, and the third moving image shooting device in a position that allows a bird's-eye view diagonally upward. It can be confirmed by the record.

In the present invention, the step (5) of raising the upper part of the manipulator to which the collecting pipette is attached and separating the collecting pipette from the cell solution indicates an operation in which the tip portion of the pipette is separated from the cell solution in the storage container. ..

In the present invention, the step of separating the storage container (6) from under the cell collection part of the manipulator by the horizontal movement of the electric stage is a driving mechanism in the X direction (horizontal direction) according to a predetermined program. The process of moving the storage container from under the cell collection unit to another position in response to a drive signal is shown.

In the present invention, the step of moving one of the empty wells of the culture vessel (7) under the cell collection part of the manipulator by the horizontal movement of the electric stage is the X direction (7) according to a predetermined program. The process in which an electric stage having a drive mechanism in the horizontal direction (horizontal direction) moves an empty well in which cells in the culture vessel are not inserted is moved under the cell collection section in response to a drive signal.

In the present invention, the step of discharging one cell collected in the empty well of the culture vessel (8) from the cell collection unit is the upper part of the manipulator capable of automatically ascending and descending in the Y direction (vertical). As the descending, the collection pipette that binds to the upper part of the manipulator and holds only one cell in it also descends, and its tip rests above an empty well in the culture vessel. The process in which cells are discharged from the collection pipette into the empty well by the discharge force of the micropump is shown. In addition, the unity of the discharged cells is the three imaging devices: the first moving image shooting device in the upward direction, the second moving image shooting device in the lateral direction, and the third moving image shooting device in a position where a bird's-eye view can be seen diagonally upward. It can be confirmed by the image recording of.

In the present invention, the step (9) of moving the storage container under the cell collection section of the manipulator by removing the well containing the discharged cells from under the cell collection section of the manipulator by the horizontal movement of the electric stage. The electric stage having a drive mechanism in the X direction (horizontal direction) separates the well part of the culture container containing a single cell from under the cell collection part in response to the drive signal, and the storage container is a cell. The process of moving it under the sampling part is shown. When a container having a plurality of wells such as a 6-well plate is used as a storage container, for example, a liquid medium may be placed in one well and used as a washing container. As a result, after the single cell is transferred to the well of the culture vessel once, air can be blown out by a micropump in the liquid medium of the washing container to wash the collection pipette. ..

In the present invention, the method characterized in that cells are separated into individual wells one by one by repeating the operations (2) to (9) is described in the above (2) to (9). This means that the series of operations performed is automatically repeated, and by this repeated operation, the cells in the storage solution are collected one by one as a single cell by a collection pipette bound to the manipulator, and one as a single cell. Each is discharged into a separate well on the culture vessel.

By using this method, a single cell clone can be automatically created, and a series of repetitive operations can be performed with the first moving image shooting device in the upward direction, the second moving image shooting device in the horizontal direction, and a bird's-eye view diagonally upward. It can be saved by recording images of the three imaging devices of the third moving image capturing device at a position where it can be, and the unity of the master cell bank can be proved.

Specifically, the images taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device are recorded in the recording device in correspondence with the time information, that is, the shooting time, and have the same shooting time. Each image taken by the moving image shooting device, the second moving image shooting device, and the third moving image shooting device can be confirmed at any time by the display device having a function of displaying a plurality of images at the same time. Whether or not the cells have been injected can be confirmed not only at the time of cell dispensing but also after long-term record storage, so if the cells dispensed for each cell proliferate and become master cells, it is GMP compliant. It is an inspection method for monochromeity. The display device is attached to the manipulator of the present invention by recording the images taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device corresponding to the time information on some information recording medium. without as hereinbefore, node on preparative by personal computers and the like of the mobile display device or display device in the manipulator and elsewhere in the present invention, the first moving image shooting apparatus, the second video with the same photographing time Each image taken by the photographing device and the third moving image photographing device is reproduced, and the unity of the cells can be confirmed.

In the cell sorting device of the present invention whose configuration is described in FIG. 1 using a microscope, an imaging device, an electric stage, and a manipulator equipped with a collection pipette, a storage container and a culture container are placed and automatically operated in the XY directions. A collection pipette connected to an electric stage (Ss) and a microscope (Mp) having a function of holding the collection pipette on desired wells of a storage container and a culture container was attached by moving the pipette. The first microscope (M1), which is coupled with an electric XYZ manipulator and an actuator (Sz) that can move in the Z-axis direction and is equipped with the camera (C1) of the first moving image imaging device, and a pipette for collecting cells from the side. The cells of the second microscope (M2) and the vertical C3 holder (A2), which are horizontally attached to the vertical M2 holder (A1) for observation and equipped with the camera (C2) of the second video imaging device. A cell sorting device (hereinafter referred to as the present device) characterized by including a camera (C3) of a third moving image capturing device mounted at a position where a bird's-eye view of the picking operation can be performed will be described.

Since the base plate (B1) of the apparatus is a precision apparatus, it is installed on a table, a laboratory table, etc. via a vibration isolator (G1). The electric XYZ manipulator is installed at the lower part of the first microscope and its surroundings at a position where the collection pipette can be operated, and the operation is performed while looking at the screen of the image device projected by the first or second moving image capturing device or the field of view of the first microscope. I can do it. Configure the device. The operation can be performed using a joystick connected to a personal computer. It should be noted that all of the moving image shooting devices have a shot video recording function, and all the shot images of the cell sorting operation can be used as proof of monoclone. In order to confirm whether or not the collected cells are single, the first moving image photographing device photographs the cells from the upper Z-axis direction, and the second moving image photographing device photographs the cells from the lateral X or Y axis direction. Since only those confirmed to be single cells by one image are recognized as single cells, a backlight (L) that brightens the part where the collection pipette is held is also required for the configuration of the present device. be. The electric drive unit of the device is connected to the electrical panel, and the personal computer is used by connecting the electrical panel and the joystick. Further, the images taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device are recorded and displayed on the recording device (R) having a function of recording in correspondence with the time information. The reproduced image can be confirmed by the device (D).

Examples of the present invention will be described below. In order to carry out the present invention, the system shown in FIG. 1 was constructed mainly by combining devices manufactured by NSK Ltd.

1. 1. Preparation of cell solution (1) ^{CHO cells incorporating an antibody-expressing gene at a cell density of 1 × 10 6} Cells / mL in a liquid medium (CD CHO Medium, Thermo) in a centrifuge tube (100 / mL, manufactured by AGC Technograss). Suspended in Fisher Scientific (manufactured by Fisher Scientific).
(2) The cell suspension was divided into one well of a 6-well plate (Falcon ^TM 6-well transparent flat bottom untreated multi-well cell culture plate, manufactured by Corning Inc.) using the cell suspension as a cell storage container. One well of the 6-well plate (Falcon ^TM 6-well transparent flat-bottomed untreated multi-well cell culture plate, manufactured by Corning) contains only a liquid medium (CD CHO Medium, Thermo Fisher Scientific) for use as a washing container. rice field.
(3) The cell storage container was centrifuged at 1000 rpm for 5 minutes, and the cells were submerged in the bottom surface of the dish. Since the single cell isolation and transport steps were _{performed at room temperature outside the CO 2} incubator, the cell isolation step was promptly started after adjusting the cell suspension.
(4) In advance, about 100 μL of the medium was dispensed into a 96-well plate used as a culture vessel.

2. Single cell isolation step (1) A collection pipette was attached to an actuator (Sz: Z-axis motor).
(2) The storage container of the 6-well plate containing the cells and the culture container of the 96-well plate containing the cells were attached to the electric stage (Ss: electric sample stage).
(3) The electric stage automatically moves in the horizontal direction, and a well containing the cell solution of the 6-well plate of the storage container is placed directly under the collection pipette, and the actuator automatically lowers to the tip of the collection pipette. Was placed on the bottom of the saucer in the storage container.
(4) The joystick (JS: operation interface, JS: operation interface,) under the observation of the image projected by the first microscope (M1: stereomicroscope SZX16, manufactured by Olympus Corporation) and the first video recording device (C1: first camera, manufactured by Basler). A single cell was aspirated into a pipette by operating a Saitek (manufactured by Saitek).
(5) After the suction of a single cell, the actuator (Sz) and the electric stage (Ss) automatically moved, and the pipette was moved to the observation position of the second microscope (M2) equipped with the second video imaging device. ..
(6) In order to confirm that the single cell image taken by the first imaging device is correct, the first moving image photographing device (C2: second camera, manufactured by Basler) is used. It was confirmed that only a single cell was present in the pipette even at an angle of 90 degrees from C1), and it was recorded as an image. In addition, the depth of focus of the second microscope (M2: second microscope, manufactured by Edmund Optics) was automatically changed, and an image for denying foreign matter from adhering to the outer wall of the pipette was also recorded.
(7) After taking the image in the above step (6), the electric stage (Ss) automatically moves the storage container, and the 96-well plate of the culture container is placed under the collection pipette, and the actuator (Sz) moves. It descended automatically and ejected cells to the bottom of any well on a 96-well plate. Then the collection pipette was raised.
(8) During the steps (5) to (7) above, C1 recorded the isolation process of a single cell as a moving image while focusing on the cells in the pipette.
(9) After the cells are discharged in the above step (7), the electric stage (Ss) automatically moves the culture container and collects a well containing only the medium of the 6-well plate of the storage container used as a washing container. It was placed below the pipette.
(10) The actuator (Sz) was automatically lowered, the collecting pipette was placed in the liquid medium of the washing container, and air was blown out by a micropump (MP: electric micropump) to wash the inside of the pipette.
(11) By repeating the operations from the steps (3) to (10), a single cell clone was prepared on each well of the 96-well plate.
(12) In the above operations (1) to (11), a bird's-eye view image was taken by the third moving image shooting device, but the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device having the same shooting time. The image taken by the above was recorded on the recording device (R) and reproduced on the same screen of the display device (D), so that the monochrome nature of the master cell was verified.

M1: First microscope M2: Second microscope C1: First camera C2: Second camera C3: Third camera & lens Sz: Z-axis motor Ss: Electric sample stage M p: Electric micropump St: Manipulator A1: M2 holder A2: C3 holder N: Dedicated needle L: Backlight B1: Base plate G1: Anti-vibration member R: Recording device D: Display device

Claims (14)

In a method of separating cells using a microscope, a video recording device, an electric stage, and a manipulator equipped with a collection pipette.
(1) A step of mounting a storage container and a culture container containing a cell solution on an electric stage mounted on a manipulator.
(2) A process of moving the storage container under the cell collection part of the manipulator by the horizontal movement of the electric stage.
(3) A step of lowering the upper part of the manipulator equipped with the collection pipette and inserting the collection pipette into the cell solution.
One cell (4) cell solution was inhaled using a sampling pipette mounted on the manipulator, by inhaling the gas from collecting pipette tip, the unity inhalation cells in harvested pipette The three imaging devices, the first moving image shooting device in the upward direction, the second moving image shooting device in the lateral direction, and the third moving image shooting device in a position that allows a bird's-eye view diagonally upward, while holding and holding the cell unity. The process of recording on an image,
(5) A step of raising the upper part of the manipulator equipped with the collection pipette to separate the collection pipette from the cell solution.
(6) A step of removing the storage container from under the cell collection part of the manipulator by moving the storage container horizontally.
(7) A step of moving one of the empty wells of the culture vessel under the cell collection part of the manipulator by the horizontal movement of the electric stage.
(8) A step of discharging one cell collected in an empty well of a culture vessel from a cell collection unit ,
(9) A step of removing the well containing the discharged cells from under the cell collection section of the manipulator by the horizontal movement of the electric stage and moving the storage container under the cell collection section of the manipulator .
A method characterized by separating cells one by one into individual wells by repeating the above operations (2) to (9). Collection of one cell by sampling pipette, methods who claim 1 Symbol placement and performing by the still and discharge micropump. Method person according to claim 1 or 2 Symbol mounting, characterized in that a step of collecting the one cell to the harvesting pipette from the cell solution using automation gross or image processing. Method towards any one of claims 1-3, wherein the storage container is 6well plate. Method towards any one of claims 1-4, wherein the culture vessel is 96well plates. Method towards any one of claims 1-5, characterized in that the motorized stage is XY table. The method according to any one of claims 1 to 6, wherein the first moving image capturing device and the second moving image capturing device are mounted via a microscope. A method for testing monochromeity, which comprises using the method for sorting cells according to any one of claims 1 to 7. Any of claims 1 to 7, wherein the images taken by the first moving image shooting device, the second moving image shooting device, and the third moving image shooting device are recorded in the recording device in correspondence with the time information, that is, the shooting time. A method for demonstrating unity of a master cell bank using the cell sorting method described in item 1. A cell equipped with a microscope, a video imaging device, an electric stage, and a collecting pipette , has a manipulation system for operating the collecting pipette within the field of the microscope, and separates one cell into one well using the collecting pipette. A preparative device having a function of holding a single cell in a collection pipette, and taking an image through the microscope in order to observe the single cell in the collection pipette from above. A moving image photographing device, a second moving image taking device for taking an image through a microscope for observing a single cell in the collecting pipette from a horizontal direction, a third moving image taking device for taking a bird's-eye view of the entire device, and the above-mentioned A cell characterized by having a recording device that records an image taken by the first moving image photographing device, the second moving image taking device, and the third moving image taking device in correspondence with the time information of the photographed image of a single cell. Sorting device. The cell sorting device according to claim 10 , further comprising a first moving image capturing device, the second moving image capturing device, and a display device that simultaneously displays each image captured by the third moving image capturing device. It is provided with a recognition means for recognizing cells in a solution and a position calculation means for calculating the position of the cells recognized by the recognition means based on an image signal output from the photographing means, and is output from the position calculation means. The device according to claim 10 or 11, further comprising an inhalation control means for moving the tip of a collection pipette relative to the vicinity of a single cell in a solution based on the position information of the cells. The electric stage is a motor-driven positioning XY table (stage) device that can be programmed to move wells in storage and culture vessels alternately to a predetermined position below the microscope. The apparatus according to any one of claims 10 to 12, wherein the apparatus is characterized. The manipulator is fixed to the rotary shaft, a screw shaft having a threaded portion on the outer peripheral side, a motor that freely supports the movement of the rotary shaft in the axial direction and drives the rotary shaft to rotate, with the pipette as a drive target. The screw element is screwed to the screw shaft to freely support the movement of the screw shaft in the axial direction, and the rotating shaft is finely moved along the axial direction according to the voltage applied to the piezoelectric element. The device according to any one of claims 10 to 13, further comprising a fine movement mechanism for causing the movement .

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