JP2007020507A - Apparatus for culturing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for culturing cells designed to judge the timing of completion of subculturing and culturing. <P>SOLUTION: The apparatus for culturing the cells is equipped with a photographing means 15 for photographing the cells in an incubator 17 during the culturing of the cells and a means for measuring the interval of the adjacent cells on the basis of photographed image information about the cells. The apparatus is further provided with a means for detecting the formation of colonies by density information about the cells obtained during the culturing of the cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culturing apparatus for culturing adhesive cells and determining the timing of passage and ending of culture by image processing.

In order to determine the passage and the end of culture by image processing, it is necessary to detect cell proliferation during the culture. Conventionally, a method for detecting cell proliferation using the number of cells, cell concentration, and cell occupation area has been proposed (for example, Patent Document 1). Alternatively, a change in the proliferation rate is detected from the cell proliferation curve (growth curve), and when the proliferation rate slows down, the passage and the end of the culture are performed. If the colony is left standing for a long time, the cell growth is slowed or differentiated. Therefore, when the colony is formed, it is necessary to immediately take the steps of passage and culturing. Conventionally, for colony detection, a low-magnification objective lens is used, the incubator is photographed over a wide area, and formation of colonies is detected from the light and shade distribution of the image data.
Japanese Patent Laid-Open No. 2001-275659

  In order to determine the timing of passage and completion of culture, it is necessary to determine a threshold value using a parameter that correlates with cell growth. The number of cells, the cell concentration, and the cell occupation area are parameters that correlate with cell growth, but do not directly represent how close adjacent cells exist. Therefore, it is desirable to manage the cell density during culture by introducing a parameter that directly expresses cell dispersion rather than the number of cells, cell concentration, and cell occupation area. In order to obtain a growth curve, data must be accumulated at regular intervals, but when data loss or data loss occurs, the time at which the growth rate slows down cannot be recognized. For this reason, it is necessary to calculate the cell dispersion using an algorithm that does not depend on changes over time. Preparing a low-magnification objective lens to detect colony formation leads to an increase in the size of the device and an increase in the number of parts, so it is necessary to measure cell dispersion and detect colonies with a single objective lens. Become.

  In order to solve the above problems, the following configuration is provided. An imaging means for imaging cells in the incubator during cell culture, and an interval between adjacent cells is measured based on the captured cell image information. Several processes are required to calculate the cell spacing. First, cell extraction processing is performed on the photographed cell image. Next, binarization processing is performed in which only the cell portion is white and the portion other than the cell is black. The binarization process is performed to simplify subsequent image processing, and is not essential. Next, a thinning process is performed. This is a process of extracting only the line passing through the center of the cell part after binarization. Depending on the extraction algorithm, the cell part is extracted thicker or thinner than the actual one, so measuring the cell interval described later with a line passing through the center can reduce the error of the extraction algorithm and extract it. There is an advantage that the algorithm can be simplified. On the other hand, if the accuracy of the extraction algorithm is high, or if the cell size varies, the cell state is more accurately reflected by measuring the cell interval from the binarized image itself without performing thinning processing. It will be a thing. Finally, a cell interval measurement process is performed. In this method, a line having an arbitrary direction is set on the image, and the number of pixels of cells on the line and cells adjacent to the cell are measured. The line in an arbitrary direction is the X direction, the Y direction, or the normal direction with respect to the cell. The cell interval may be measured on one line, but it is preferable to set and measure a plurality of lines.

  In addition, it comprises means for detecting colony formation based on the cell density information acquired during the cell culture. When colonies are formed, the fact that cells are sparse and dense in one image is used. After performing the above-described cell extraction process, binarization process, and thinning process, the image data is divided into a plurality of regions. The above-described cell interval measurement process is executed for each region, and colony formation is detected by comparing the cell intervals in the respective regions. Colony detection is performed simultaneously with the measurement of cell density, which can contribute to shortening the processing time.

  It is possible to measure the interval between adjacent cells, and to determine the timing of passage and end of culture. Furthermore, it becomes possible to detect the formation of colonies with an objective lens that captures cell dispersion, and the influence on the cells due to colony formation can be suppressed.

  Embodiments of the present invention will be described below with reference to the drawings. As schematically shown in FIG. 1, the cell culture device 11 includes a thermostat 10 that provides an optimal temperature and CO2 concentration for cell culture, an incubator 17 for culturing cells, and an objective lens 16 for photographing cells. A camera 15 for digitizing the data of the objective lens 16, a converter 18 for transferring image data obtained from the camera 15 to the operation terminal 19, a camera driving device 13 for moving the camera 15, and an incubator 17 The incubator drive device 14 for moving the camera, the camera drive device 13 and the motor controller 12 for controlling the incubator drive device 14. By issuing a movement command from the operation terminal 19 to the motor controller 12, the camera driving device 13 and the incubator driving device 14 operate in the X and Y directions, respectively, and the camera 15 captures a cell at an arbitrary position in the incubator. It is possible.

  FIG. 2 shows the control terminal 19 in detail. The control terminal 19 communicates with the CPU 28 that performs arithmetic processing via the data bus 21, the main memory 23 that the CPU 28 uses as a temporary storage area, the external storage device 24 that stores image data and position information, and the motor controller 12. A communication port 27 for performing the operation, a monitor 22 for displaying an operation screen, a keyboard 26 for accepting user input, and a mouse 25 are provided.

  FIG. 3 is a perspective view of the thermostatic chamber 10 shown in FIG. FIG. 4 is a flowchart of the cell interval measurement process. The process will be described in detail with reference to FIG. The cell image processing start 61 starts processing for cell interval measurement. In the image data capturing 62, the image data at the photographing position determined by the camera driving device 13 and the incubator driving device 14 is transferred to the main memory 23 via the camera 15, the converter 18 and the data bus 21. In the cell extraction process 63, the CPU 28 performs threshold processing, difference processing, and the like to extract cell portions and store them in the main memory 23. The extraction of the cell portion may be any algorithm as long as the cells can be extracted. Next, binarization processing 64 is performed. This is to set the pixel value of a pixel recognized as a cell part in the cell extraction process 63 to 1 and set the other pixel values to 0. The binarization process 64 is not essential, but it is desirable to execute it because the process described later can be simplified. The thinning process 65 is a process of deleting a pixel of one cell portion from the left and right of the cell to form a line of one pixel passing through the center of the cell. The thinning process 65 may or may not be executed depending on the variation in cell size and the accuracy of the cell extraction process 63. Next, an area division process 66 is executed. In this case, one image is divided into m × n regions, and FIG. 6 is a diagram when the region is divided into 2 × 2. Each region does not need to have the same area, and may not be divided if colony formation is not detected. A cell interval measurement process 67 is executed for each divided area.

  In the cell interval measurement process 67, as shown in FIG. 5, a measurement line 52 is set on the image, and the interval between adjacent cells and the cell interval 51 are measured along the measurement line 52. Although a measurement line in the X direction is used in FIG. 5, a straight line or a curve having an arbitrary inclination may be used as the measurement line, and it is desirable to use a plurality of measurement lines for improving accuracy. Further, it is desirable that the cell image processing 61 is executed at a plurality of points on the incubator, and passage and collection are performed when the cell interval falls below a specified value at a plurality of points.

  Fig. 6 shows an image when a colony is formed.The cell spacing in the upper left, upper right, lower left, and lower right of the divided areas is 49, 21, 42, and 13 pixels, respectively, and the right half of the image, especially the lower right area, is shown. It shows that a colony is formed.

  Fig. 7 is a graph of changes in cell spacing during culture. Cell growth correlates with cell spacing, and even if data is not collected over time as in a growth curve, subculture and culture are performed. It indicates that the end can be determined.

The schematic diagram which shows the cell culture apparatus of this invention. The block diagram of the operation terminal of this invention. The perspective view which shows the thermostat of this invention. The process flowchart of this invention. The cell interval measurement figure of this invention. The image of colony formation of this invention. The graph of the culture | cultivation days of this invention and a cell space | interval.

Explanation of symbols

  11 Cell culture device, 12 Motor controller, 13 Camera drive device, 14 Incubator drive device, 15 Camera, 16 Objective lens, 17 Incubator

Claims (2)

  A cell culture apparatus comprising imaging means for imaging cells in an incubator during cell culture, and means for measuring an interval between adjacent cells based on the taken cell image information.   The cell culture device according to claim 1, further comprising means for detecting colony formation based on the cell density information acquired during the cell culture.

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