JP2006014693A - Cell culture system and method for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To properly and simply culture cells collected from a living body in a cell culture system. <P>SOLUTION: The cell culture system 1 is equipped with the following a counting apparatus 20 for counting the number of cells in a cell suspension prepared by placing the cell to be inoculated in a culture medium, a culture medium feeding apparatus 100 for feeding a fresh culture medium to the cell suspension to be inoculated, a controller 101 for controlling the amount of the fed culture medium from the culture medium feeding apparatus 100 to the cell suspension so that the cells are statistically at a properly culturable density for the counted number of the cells, a manipulator 102 for transporting the cell suspension in which the cells are regulated to a properly culturable density and inoculating the cells into the cell culture apparatus 103 and a cell culture apparatus 103 for culturing the inoculated cells. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell culture system and a cell culture method, and is particularly suitable for a cell culture system and a cell culture method in which cells are seeded and cultured automatically.

  In the conventional cell culture method, cells collected from a living body are seeded and cultured in a cell culture device, a part of the cells in the culture are collected, and the number of cells is measured to confirm the culture state. When the desired culture state can be confirmed by repeating the above measurement, the cell culture is terminated.

  As a conventional method for counting the number of cells, a method of counting the number of cells by taking in image data of corneal endothelial cells in culture and calculating the area of the proliferated cells (for example, Japanese Patent Laid-Open No. 5-184541 (Patent) Document 1)), a method for counting the number of cell groups immobilized on a substrate (for example, Japanese Patent Application Laid-Open No. 11-9297 (Patent Document 2)), and the amount of time variation in the amount of oxygen in a container during culture. There is a method of counting the number of cells by calculating (for example, JP-A-6-121667 (Patent Document 3)).

JP-A-5-184541 Japanese Patent Laid-Open No. 11-9297 JP-A-6-121667

  However, in the conventional cell culture method, since it is necessary to repeatedly measure the number of cells in culture, cell culture is extremely troublesome.

  In addition, in the method of counting the number of cells in Patent Documents 1 and 2, in the case of micro cells or cut cells directly collected from a living body, in order to obtain cell image data, the cells are uniformly arranged so as not to overlap the dish or the substrate. There is a problem that it is necessary to perform the work of fixing to the head, which takes time.

  In addition, in the method for counting the number of cells in Patent Document 3, since the number of cells is counted by a method of measuring the amount of a substance such as oxygen, the cells collected from a living body are placed in an automatic cell culture apparatus, and then are subjected to certain measurement conditions. It was necessary to count the number of cells after waiting until the condition became stable.

  An object of the present invention is to provide a cell culture system and a cell culture method capable of appropriately and simply culturing cells collected from a living body.

  In order to achieve the above object, the cell culture system of the present invention comprises a measuring device for measuring the number of cells in a cell suspension in which cells to be seeded are placed in a medium, and a fresh cell suspension to be seeded. A medium supply device that supplies a medium, and a medium supply amount from the medium supply device to the cell suspension so that the density of the cells can be statistically appropriately cultured with respect to the measured number of cells. And a manipulator that transports the cell suspension having a density at which cells can be appropriately cultured and seeds the cell suspension, and the cell culture device that cultures the seeded cells. It is in.

A more preferable specific configuration of the present invention is as follows.
(1) The measurement device includes an image imaging device that images the cell suspension in which cells to be seeded are placed in a medium, and the number of cells based on the image of the cell suspension captured by the image imaging device. And an image processing device for calculating
(2) A cell separation device for precipitating the cells in a container containing the cell suspension containing cells to be seeded in a medium and separating the cells from a liquid such as a medium, and the imaging device is precipitated. The image is picked up by the cell, and the image processing device detects the precipitation depth from the image of the imaged cell after precipitation, and calculates the number of cells from the correlation between the precipitation depth specific to each container and the number of cells. Be.
(3) A pipette manipulator that suspends the cells in a container containing the cell suspension in which cells to be seeded are placed in a medium and a predetermined amount of the medium is provided, and the imaging device is disposed in the container. An image of a state in which cells float apart in a predetermined amount of medium contained therein, and the image processing device counts the number of cells reflected in the image.
(4) A discharge device that discharges the cultured cells to the outside from the culture chamber in which the cell culture device is arranged is provided.

  In order to achieve the above object, the cell culturing method of the present invention measures the number of cells in a cell suspension in which cells to be seeded are placed in a culture medium, and measures the number of cells measured. The control unit controls the amount of medium supplied from the medium supply device to the cell suspension so that the cells can be statistically appropriately cultured, and the cell suspension is adjusted to the appropriate density. Is transferred to a cell culture device by a manipulator and seeded, and the cells of the seeded cell suspension are cultured by the cell culture device.

  ADVANTAGE OF THE INVENTION According to this invention, the cell culture system and cell culture method which can culture | cultivate the cell extract | collected from the biological body appropriately and simply can be provided.

  Hereinafter, a plurality of embodiments of the present invention will be described with reference to the drawings. The same reference numerals in the drawings of the respective embodiments indicate the same or equivalent.

  First, a first embodiment of the present invention will be described with reference to FIGS. In this example, a cell culture system 1 and a cell culture method for culturing adhesive cells will be described.

  First, the overall configuration of the cell culture system of the present embodiment will be described with reference to FIG. FIG. 1 is a schematic configuration diagram of a cell culture system according to a first embodiment of the present invention.

  The cell culture system 1 includes a cell input device 108, a cell separation device 104, a measurement device 20, a manipulator 102, a medium supply device 100, a cell culture device 103, a control device 101, and a cell discharge device 107.

  The collected cell loading device 108 is a device that loads a container containing a cell suspension in which cells for seeding are placed in a medium into the cell separation device 104. After introducing the cells into the container, the medium supplied from the medium supply device 100 is added to a certain amount under the control of the liquid transport control device 101.

  The cell separation device 104 is a device for precipitating cells in the container 2 containing a cell suspension containing cells to be seeded in a medium and separating the cells from a liquid such as a medium. Adhesive cells including stem cells collected from a living body are separated by adding trypsin by a human hand, and are put into the container 2 of the cell separation device 104. This container 2 is a container in which the correlation between the depth of cells precipitated in the container and the number of cells is known in advance.

  The cell separation device 104 is constituted by a centrifuge provided with a rotating disk 104a on which the container 2 is installed and a motor 104b that rotationally drives the rotating disk 104a. The centrifuge precipitates cells of a cell preservation solution composed of dissociated corneal cells and a medium, etc., separates excess substances as a supernatant, and exchanges this supernatant with a fresh medium under the control of the control device 101. Repeat to remove excess material.

  The measuring device 20 is a device that measures the number of cells in a cell suspension in which cells for seeding are placed in a medium. The measuring device 20 includes an image imaging device 105 that images a cell suspension (cell suspension in the container 2) in which cells to be seeded are placed in a medium, and a cell suspension imaged by the image imaging device. And an image processing device 106 that calculates the number of cells based on the image.

  Specifically, the image capturing device 105 is a device that captures an image of a container containing cells separated and precipitated by the cell separating device 104 using a CCD camera or the like.

  Further, the image processing device 106 specifically detects the precipitation depth from the imaged cell image after precipitation, and calculates the cell number from the correlation between the precipitation depth specific to each container and the cell number. . The image processing apparatus 106 includes a bottom position detecting unit 110 that detects the bottom position of the container 2, a top position detecting unit 111 that detects the top position of the precipitated cell, and a depth of the precipitated cell by obtaining a difference between them. Stored in the microcomputer is a depth calculation means 112 for calculating the number of precipitated cells, and a cell number calculation means 113 for calculating the number of cells based on the correlation between the depth of the cells precipitated in the container 2 and the number of cells. Have soft.

  The image processing device 106 includes a device main body 106a having a microcomputer, a display device 106b for displaying an image, a keyboard 106c and a mouse 106d as input devices.

  The manipulator 102 is a device that transports the container 2 containing the cell suspension in which the number of cells has been measured and seeds it in a culture container such as a dish or a cartridge inside the cell culture apparatus 102.

  The medium supply apparatus 100 is an apparatus that supplies a fresh medium to a cell suspension seeded in the cell culture apparatus 103, has a refrigeration function capable of storing the medium, and from there to a place that can be controlled by the control apparatus 101. This is a device for supplying a culture medium by a pump 100b.

  The medium supply device 100 is for supplying the medium so that the density of the cells can be statistically optimally cultured with respect to the number of cells counted by the image processing device 106. The culture medium supply apparatus 100 includes a container 100a that stores a fresh culture medium, and a pump 100b that supplies the culture medium in the container 100a to the cell culture apparatus 103. The container 100a and the cell culture device 103 are connected via a pipe 100c and a pump 100b.

  The cell culture device 103 is a device for culturing cells of a cell suspension seeded in a culture container such as a dish or a cartridge to a transplantable state. That is, the cell culture device 106 is a device in which a dish or cartridge for culturing can be placed inside, and manages the ratio of carbon dioxide gas and air inside the device, the temperature and humidity inside the device to a constant state suitable for culture. Device.

  The control device 101 is a device that controls the manipulator 102, the medium supply device 100, and the cell separation device 104. The control device 101 controls the manipulator 102 to transport the container containing the cell suspension, which has been separated by the cell separation device 104, to the cell culture device 103. In addition, the control device 101 supplies a medium supply amount from the medium supply device 100 to the cell suspension of the cell culture device 103 so that the density of the cells can be statistically appropriately cultured with respect to the measured number of cells. To control.

  The control device 101 includes a device main body 101a having a microcomputer, a display device 101b for displaying an image, a keyboard 101c and a mouse 101d as input devices.

  The cell discharge device 107 carries out the cells that have been cultured in the cell culture device 103 to the outside of the cell 10. In 10, a cell culture device 103, a cell separation device 104, an image capturing device 105, and a cell discharge device 107 are arranged.

  Next, a cell culture method using the cell culture system of the present embodiment will be described with reference to FIG. FIG. 2 is a configuration flowchart of the cell culture system of FIG.

  Cells containing stem cells collected from a living body are separated by adding trypsin, and the cells are put into the container 2 of the cell separator 104 to obtain a cell preservation solution. By driving the cell separation device 104 in which the container 2 is installed, the cells in the container 2 are precipitated, and excess substances are separated as a supernatant. Under the control of the control device 101, the supernatant is sucked out, a fresh medium is injected into the container 2 from the medium supply device 100, the cell separation device 104 is driven again, and thereafter, the target cell and the medium are repeated. Remove excess material except.

  Thereafter, the state in which the target cells and the medium are contained in the container 2 is captured by the image capturing device 105, and the captured image is sent to the image processing device 106. The image processing device 106 calculates the number of cells based on the captured image. Specifically, the bottom position detection means 110 detects the edge of the bottom position of the container 2, the top position detection means 111 detects the top edge position of the precipitated cell, and the precipitation cell depth calculation means 112 detects the edge. The depth of the precipitated cells is calculated by obtaining the difference, and the cell number is calculated by the cell number calculating means 113 from the correlation between the cell depth and the cell number precipitated in the container.

  The control apparatus 101 controls the culture medium supply apparatus 100, and an amount of fresh culture medium that is considered to be statistically optimal for the calculated number of cells to grow is added from the container 100a of the culture medium supply apparatus 100 to the container 2. To do. The control device 101 controls the manipulator 102 so that the container 2 with the cells and medium optimized for growth is conveyed by the manipulator 102 and seeded in a dish or cartridge in the cell culture device 103.

  The cells seeded on the dish or cartridge are cultured in the cell culture device 103. When this culture is completed, the dish or cartridge is discharged from the measuring device 20 to the outside from the cell discharging device 107.

  According to the cell culture system of the present embodiment, a measuring device 20 that measures the number of cells in a cell suspension in which cells to be seeded are placed in a medium, and a medium that supplies a fresh medium to the cell suspension to be seeded. A control apparatus 101 that controls the supply amount of the medium from the culture medium supply apparatus 100 to the cell suspension so that the density of the cells can be statistically appropriately cultured with respect to the measured number of cells; A manipulator 102 that conveys a cell suspension having a density at which cells can be appropriately cultured and seeds the cell suspension 103, and a cell culture device 103 that cultures the cells of the seeded cell suspension. Therefore, the cells collected from the living body can be cultured appropriately and easily.

  In addition, the cell separation device 104 for precipitating the cells in the container 2 containing the cell suspension containing the cells to be seeded in the medium and separating them from the liquid such as the medium, and the image capturing device for imaging the precipitated cells 105 and an image processing device 106 that detects the precipitation depth from the image of the imaged cell after precipitation and calculates the cell number from the correlation between the precipitation depth inherent to each container and the cell number. It is possible to easily count the number of cells even in a short period of time.

  Next, a second embodiment of the present invention will be described with reference to FIGS. FIG. 3 is a schematic configuration diagram of the cell culture system according to the second embodiment of the present invention, and FIG. 4 is a configuration flowchart of the cell culture system of FIG.

  In the second embodiment, the manipulator 102 is constituted by a pipette manipulator and does not include the cell separation device 104 of the first embodiment. The manipulator 102 pipettes and suspends the cells and the medium or other liquid for storing the cells in the container 2 containing the cells input from the cell input means and the medium or other liquid for storing the cells. It is a device to do. The manipulator 102 also serves as a device for transporting the container 2 containing the cell suspension in which the number of cells has been measured and seeding it in a culture container such as a dish or a cartridge inside the cell culture apparatus 102.

  The collected cell input device 108 is configured to input a container containing a cell suspension in which cells for seeding are placed in a medium to the manipulator 102.

  In addition, the image capturing apparatus 105 irradiates the container 2 containing the cells and the medium or other liquid for storing the cells with scattered light, captures some of the images with a CCD camera or the like, and performs image processing on the captured images. The data is transmitted to the device 106.

  Further, the image processing device 106 distinguishes the image 120 captured by the image capturing device 105 into a black and white image, the device 121 that specifies the threshold value of the black and white image boundary, and the cell image and other images. Separately, means 122 for converting to a binary image, means 123 for removing extraneous images such as dust appearing smaller than the size of the cell, and means for filling a hole in an image of a cell that is poorly reflected and has a hole 124 and means 125 for counting the number of remaining points are included in the software stored in the microcomputer.

  In the cell culturing method of the second embodiment, the medium and target cells in the container 2 are suspended by the pipette manipulator 102, and the liquid after suspension is imaged by the image capturing device 114. The captured image is converted into a black and white image by the image processing device 115, a threshold value is specified to distinguish the cell image from the other images, and then converted into a binary image that appears smaller than the cell size. After removing unnecessary images such as dust, filling the holes in the image of cells that are poorly reflected and having holes, the number of cells is calculated by counting the number of remaining points.

  Thereafter, an amount of medium that is considered to be statistically optimal for the calculated number of cells to grow is added by controlling the pipette manipulator 102 with the control device 101, and then added to the dish or cartridge in the cell culture device 106. Sowing. After completion of the culture, the dish or cartridge is discharged from the cell discharge device 107.

  According to the second embodiment, the manipulator 103 for suspending the cells in the container 2 containing the cell suspension containing the cells to be seeded in the medium and a predetermined amount of the medium, and the container 2 An image capturing device 105 that captures a state in which cells are floating in a predetermined amount of medium, and an image processing device 106 that counts the number of cells reflected in the image. It is possible to easily count the number of cells even in a short period of time.

1 is a schematic configuration diagram of a cell culture system according to a first embodiment of the present invention. 2 is a configuration flowchart of the cell culture system of FIG. 1. It is a block schematic diagram of the cell culture system of 2nd Example of this invention. FIG. 4 is a configuration flowchart of the cell culture system of FIG. 3.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Cell culture system, 2 ... Container, 10 ... Culture room, 20 ... Measuring device, 100 ... Medium supply device, 101 ... Control device, 102 ... Manipulator, 103 ... Cell culture device, 104 ... Cell separation device, 105 ... Image Imaging device 106 ... Image processing device 107 ... Cell discharge device 108 ... Cell input device 110 ... Bottom side position detection means of container 111 111Upper side position detection means of precipitated cells 112 ... Precipitation cell depth calculation means, 113: Cell number calculating means, 120 ... Converting to black and white image, 121 ... Means for specifying threshold value of black and white boundary, 122 ... Means for converting to binary image, 123 ... Means for removing excess image, 124 ... Cell Means for filling holes in the image, 125... Means for counting the number of points on the image.

Claims (6)

A measuring device for measuring the number of cells in a cell suspension containing cells for seeding in a medium;
A medium supply device for supplying a fresh medium to the cell suspension to be seeded;
A control device for controlling the amount of medium supplied from the medium supply device to the cell suspension so that the density of the cells can be statistically appropriately cultured with respect to the measured number of cells;
A manipulator that conveys the cell suspension having a density at which cells can be appropriately cultured and seeds it in a cell culture device;
A cell culture system comprising the cell culture device for culturing seeded cells.   2. The cell culture system according to claim 1, wherein the measurement device includes an image imaging device that images the cell suspension in which cells to be seeded are placed in a culture medium, and the cell suspension imaged by the image imaging device. A cell culture system comprising: an image processing device that calculates the number of cells based on an image of a suspension.   3. A cell separation apparatus according to claim 2, wherein the cell separating apparatus for precipitating the cells in a container containing the cell suspension in which cells to be seeded are placed in a medium and separating the cells from a liquid such as a medium. The imaging device is for imaging the precipitated cells, and the image processing device detects the precipitation depth from the image of the imaged cells after the precipitation, and determines the precipitation depth and the number of cells unique to each container. A cell culture system characterized in that the number of cells is calculated from the correlation.   The cell culture system according to claim 2, further comprising a pipette manipulator that suspends the cells in a container containing the cell suspension in which the cells to be seeded are placed in a medium and a predetermined amount of the medium. The image capturing device captures a state in which cells float apart in a predetermined amount of medium contained in the container, and the image processing device counts the number of cells reflected in the image. A cell culture system characterized by being.   5. The cell culture system according to claim 1, further comprising: a discharge device that discharges the cultured cells to the outside from the culture chamber in which the cell culture device is disposed. Count the number of cells in the cell suspension in which the cells to be seeded are placed in the culture medium,
Control the amount of medium supplied from the medium supply device to the cell suspension by the control device so that the cells can be statistically cultivated appropriately with respect to the measured number of cells,
The cell suspension having an appropriate density is conveyed to a cell culture device by a manipulator and seeded,
A cell culture method comprising culturing cells of the seeded cell suspension using the cell culture device.

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