JP4907146B2 - Automatic culture method and cell culture apparatus - Google Patents

Description

  The present invention relates to an automatic culture method and a cell culture apparatus.

  In order to culture cells efficiently in large quantities, it is necessary to perform culture operations such as medium replacement treatment for nutrient supply and removal of waste products, and subculture treatment for reseeding cells in accordance with cell growth. The cell growth rate varies greatly depending on the cell tumor and the individual, and even if the cell culture is performed according to a preset culture schedule, the culture schedule must be changed as needed according to the cell growth. Changing the culture schedule according to cell growth requires the experience of skilled workers.

  Therefore, Patent Document 1 proposes to determine the timing of passage or medium replacement using the number of adherent cells, the adherent cell concentration, the adherent cell occupation area, the adherent cell occupancy, and the rate of increase thereof. . According to this, although the proliferation of cells is grasped using images taken during culture, complicated processing is required because the number of adherent cells is obtained by image processing such as pattern matching. There is a problem that it takes.

  On the other hand, it is known that the medium exchange treatment is performed by detecting the pH by utilizing the fact that the pH changes because the cells discharge waste products during the growth process. However, if a pH meter is directly immersed in the culture medium, a contamination problem occurs. For this reason, although there exists a proposal which samples a small amount of culture media and detects pH using a pH meter, processing operations, such as sampling, increase.

JP 2001-275659 A

  An object of the present invention is to support management such as a change of a culture schedule that allows cells to be efficiently cultured by simple image processing based on an image taken in an incubator.

The above problem can be solved by the means described below. How automatic culture of the present invention, the cells in an image obtained by photographing the inside incubator for culturing cells treated thinning into a single line in the longitudinal direction of the cells, one cell thin line ized cells The apparent cell number is calculated as follows, the region of the image is divided into a plurality of regions, the region having the largest apparent cell number is selected, and the apparent cell number in the region is compared with the set cell number, and the culture schedule It is characterized by managing.

  That is, since the cells in the image are white or black images, the cells can be easily extracted by extracting the white or black images. In addition, the number of cells can be easily calculated by a simple process of converting a plurality of cells in the extracted image into a single line by a well-known thinning process. Here, the number of thinned cells (apparent cell number) is different from the actual cell number, but is correlated with the actual cell number. In addition, the set values relating to the change of the culture schedule include a set value for performing the passage process and a set value for determining the end of the culture.

  In addition, the cells in the image of the incubator in which the cells are cultured are thinned, the direction of the thinned cells is obtained, the density of the cells is obtained based on the direction of the cells, and the density of the cells is When the set value is exceeded, execution of the passaging process can be determined. That is, cells proliferate in various directions when proliferating, but proliferate in the same direction as the distance between adjacent cells becomes shorter. That is, as the cell density increases, the direction of the cells is aligned. Thus, the cell density can be grasped by obtaining the degree of alignment in the cell direction. In this case, it is possible to calculate the angle of the direction of the cells and the number of appearances of the thinned cells at each angle, and determine the execution of the passage process when the number of appearances in the set angle range exceeds the set value. it can.

  By the way, when the apparent number of cells is calculated over the entire captured image, even if a colony having a high cell density exists in a part of the image, the colony may not be detected. Therefore, it is possible to divide the image area into a plurality of areas, calculate the number of apparent cells for each area, select the area with the largest number of apparent cells, and calculate the angle and the number of appearances for the selected image area. preferable.

  Moreover, execution of the medium replacement process can be determined by detecting a change in the pH indicator of the medium in an image obtained by photographing the inside of the incubator for culturing cells.

  ADVANTAGE OF THE INVENTION According to this invention, management of the change of the culture schedule etc. which can culture | cultivate a cell efficiently by simple image processing based on the image image | photographed inside the incubator can be supported.

  Hereinafter, an embodiment of the present invention will be described with reference to FIGS.

  FIG. 1 is a diagram showing a configuration of a cell culture device 1 of the present embodiment. As shown in the figure, the cell culture device 1 includes an incubator 2 for culturing cells and a thermostat 3 for accommodating the incubator 2. Inside the thermostat 3 are a color camera 4 for photographing the inside of the incubator 2, a camera driving device 5 for driving the color camera 4, an incubator driving device 6 for driving the incubator 2, and each of the driving devices. A motor controller 7 that controls driving, an operation terminal 8, and a converter 9 are provided.

  As shown in FIG. 2, the incubator 2 is installed in the thermostatic chamber 3, and is maintained at a temperature and CO2 concentration optimum for cell culture. The incubator 2 is provided so as to move linearly along the rail by the incubator driving device 6. A color camera 4 having a high-magnification objective lens for photographing cells is installed above the incubator 2 so as to photograph the inside of the incubator 2 from above. The color camera 4 is provided so as to move linearly by a camera driving device 5 on a rail installed at right angles to the rail of the camera. By moving the color camera 4 and the incubator 2, the operator can easily move the color camera 4 to the desired shooting position. Therefore, the color camera 4 can be accurately moved to the same position as the previous shooting position, and images with different shooting times can be obtained. Can be effectively compared.

  The operation terminal 8 controls the entire cell culture device 1, sends a command to the motor controller 7 to control the drive of each drive device when photographing the inside of the incubator 2, and photographs from the color camera 4. The obtained image signal is obtained via the converter 9. Specifically, as illustrated in FIG. 3, the operation terminal 8 includes a CPU 22 that performs arithmetic processing via a data bus 21, a main memory 23 that the CPU 22 temporarily uses as a storage area, and image data and position information. An external storage device 24 for storing given information, a communication port 25 for communicating with the motor controller 7, a keyboard 26 and a mouse 27 for inputting information by the user, and a monitor 28 for confirming culture information by the user. It is configured. A converter 9 that exchanges signals with the color camera 4 is connected to the data bus 21.

  The operation of the cell culture device 1 of the present embodiment configured as described above will be described with reference to FIGS. First, when cell culture is started by the cell culture apparatus 1, a target cell number at the end of the culture is set in advance. This setting can be set, for example, by searching a cell culture image corresponding to the target cell number from a group of images taken in the past using the setting screen shown in FIG. The cell culture image photographed in the past is stored in the operation terminal 8 in advance. The target cell number setting dialog 41 shown in FIG. 4 includes an image display area 42, an image advance button 43 and an image return button 44 for scrolling an image, a determination button 45 for determining a screen, a cancel button 46 for canceling, An image registration button 47 for registering an image is provided. In the image display area 42, a cell culture image photographed in the past is displayed, and by scrolling the image, the target cell number is set by selecting an image close to the target cell number. In addition, in the cell culture images taken in the past, an apparent cell number and cell profiling described later are held as numerical values. Since the target cell number is set based on a cell culture image photographed in advance, the target cell number and cell profiling (cell density) can be designated simply and intuitively.

  The image is selected by scrolling the image in the image display area 42 using the image advance button 43 and the image return button 44 to display the target cell state (cell number), determining the screen with the enter button 45, When canceling, the cancel button 46 is pushed. The target cell number is determined in the target cell number setting dialog 41 and culture is started.

  The process shown in FIG. 5 starts when a certain time has elapsed after the start of culture. FIG. 5 is a flowchart showing the procedure of the automatic culture method according to one embodiment of the present invention, which is executed in the operation terminal 8. First, when processing is started, cell image processing shown in the flowchart of FIG. 6 is performed. As shown in FIG. 6, the operation terminal 8 takes in image data from the color camera 4 photographed in the incubator 2 through the converter 9 (S61), and then performs cell extraction processing (S62).

  Here, image data obtained by photographing the inside of the incubator 2 with the color camera 4 will be described. The color camera 4 of the present embodiment takes an image of the inside of the incubator 2 with two focuses that are photographed by inverting the cell portion. The photographing field of view of the color camera 4 is set to, for example, 15 mm long and 20 mm wide. By the way, various sizes of the incubator 2 are used for the purpose of culturing, but since the cells are evenly seeded in the incubator 2 at the start of culture, the color camera 4 is not necessarily used for the entire area in the incubator 2. There is no need to shoot. Therefore, for example, when the incubator 2 having a radius of about 25 cm is used, as shown in FIG. 7, 16 measurement points each indicated by a black circle in the figure are arranged on the circumference of a circle with a radius of 2 cm and a circle with a radius of 4 cm. Set. Then, by taking an image of 15 mm in length and 20 mm in width with each measurement point as the center, it is possible to almost confirm the proliferation of cells in the entire incubator 2. Note that the number and position of the measurement points are not limited to the example of FIG. 7 as long as the state of the entire incubator 2 can be confirmed.

  The image data photographed in this way is transferred to the operation terminal 8 via the converter 9. The transferred image data is taken into the main memory 23 and also taken into the external storage device 24 at the same time.

  In the cell extraction process in step S62 of FIG. 6, a pixel (inverted portion) having a luminance equal to or higher than a threshold determined by the CPU 22 is extracted as a cell (S62). Thus, the cell extraction process does not require advanced processing, and any algorithm can be used as long as it can extract the feature points of the cell. When the cell part and the part other than the cell are recognized, the CPU 22 and the main memory 23 are used to perform binarization processing in which the pixel value of the part other than the cell is 0 and the pixel value of the cell is 1 (S63).

  Next, thinning processing is performed for drawing a single line passing through the center of the cell portion of the extracted cell (S64). In this thinning process, as shown in FIG. 8, one cell is gradually swollen or thinned from the counter electrode direction (FIG. 8A), and finally converted into a single line (FIG. 8). 8 (b)). Details of the thinning process are described in the document “Introduction to Image Processing in C Language (ISBN 4-7856-3124-4) published by Shosodo”. When the simple image processing called thinning processing is performed on the cells in the image as described above, the number of cells can be easily calculated.

  Here, if the number of apparent cells in the entire image is calculated based on the thinned image, the data is averaged if the cell density is biased, and colonies with particularly high cell density are detected. Can not be. Therefore, as shown in FIG. 9, the original image (FIG. 9A) is divided into a plurality of regions (4 in the illustrated example) (FIG. 9B) (S65). This number of area divisions can be appropriately set according to the culture state. For each region, a continuous line calculated by the thinning process in step S64 is calculated as one cell, and a cell counting process is performed to determine the apparent number of cells in each divided region (S66). ). The apparent cell number is different from the actual cell number, but is correlated with the actual cell number. At the time of this cell counting process, the start point coordinates and the end point coordinates of each line of the thinned cells are obtained and stored in the main memory 23.

  Next, an area with the largest number of apparent cells is selected (S67). Then, the cell direction profiling process is executed for the region where the apparent number of cells is the maximum (S68). This cell direction profiling process is a process for obtaining the profile of the direction of the cells thinned in step S64. Here, the direction of the cell is obtained by the direction of a straight line connecting the coordinates of the start point and end point of the cell stored in the main memory 23 during the cell counting process. Further, the direction of the straight line is represented by, for example, an angle formed by the reference line and the cell direction, with the horizontal or vertical direction of the image as the reference line. Then, the number of appearances of the thinned cells at each angle is calculated and stored in the main memory 23. That is, when cells proliferate, they proliferate in various directions, but when the distance between adjacent cells decreases, the cells proliferate in the same direction. Therefore, as the cell density increases, the cells are aligned. Thus, the cell density can be grasped by obtaining the degree of alignment in the cell direction. Further, if the number of appearances of thinned cells is calculated for each angle, it is possible to grasp the cell density with higher accuracy. Further, when the number of appearances for each set angle range exceeds the set value, it is possible to determine execution of the passaging process.

  Here, the relationship between the direction of cells and the density will be described with reference to FIGS. FIG. 11 is an image showing a state of a cell when the cell density is small. FIG. 12 is an image showing the state of the cell when the cell density is high. FIG. 13 is a graph showing the direction of cells and the number of appearances of cells.

  As shown in FIG. 11, when the cell density is small, the cells proliferate in an irregular direction, whereas as shown in FIG. 12, when the cell density increases, the cells proliferate in the same direction. To do. For this reason, as shown in FIG. 13, when the density of the cells is small, the cells appear in a wide range of −90 ° to 90 ° as shown by a thin line, and the number of appearance of the cells in the direction of each cell is also shown. Few. On the other hand, when the density of the cells is large, as shown by a thick line, the cells appear only in the range of 0 ° to 90 °, and the number of appearance of the cells in the direction of each cell increases. That is, when the cell density is large, the cell direction angle converges to a certain range, so that the cell direction range becomes narrow. In addition, since the number of appearances of cells in the direction of each cell increases, the average value of the number of appearances increases. In addition, the angle of FIG. 13 is an angle of the direction of the cell with respect to a horizontal axis.

  When the cell image processing in FIG. 6 is completed, the processing returns to the processing in FIG. 5 and whether or not the apparent cell number obtained in step S66 in FIG. 6 has reached the target cell number set in the target cell number setting dialog 41. It is determined (S50). In this determination, when the apparent number of cells has reached the target number of cells, the culture is terminated. On the other hand, when the apparent target cell number has not been reached, the process proceeds to step S51, and it is determined whether or not the apparent cell number has reached a passage threshold (set value) to be passaged. In this determination, if the apparent number of cells is equal to or greater than the passage threshold value, the process proceeds to step S52 where a passage instruction is issued, and for example, passage processing is automatically performed based on this. Next, the passage threshold is updated to a high value (S55), the cell direction profile threshold is updated to a high value, and the process proceeds to step S54. Here, the reason for updating the passage threshold and the cell direction profile threshold to high values is to reduce the frequency of passage processing by the processing of FIG. 5 that is periodically executed.

  On the other hand, if the apparent number of cells is less than the passage threshold value, the process proceeds to step S53, and whether the profile in the cell direction has reached the profile threshold value (setting value) for determining whether or not to perform passage processing. Determine. In this determination, when the profile in the cell direction has reached the passage threshold value, the process proceeds to step S52, and a passage instruction is issued.

  On the other hand, if the profile in the cell direction has not reached the passage threshold value, the process proceeds to step S54 to determine whether or not the medium replacement interval has reached the replacement interval threshold value (set value) at which the medium replacement process should be performed. . If it is determined in this determination that the time for replacing the medium is reached, a medium replacement command is issued (S57), and a command to wait until the next image acquisition is issued (S58). On the other hand, when the time for exchanging the medium has not been reached, the pH image processing for processing the image of the medium in the incubator in which the pH indicator is dropped as shown in FIG. 10 is executed.

  When the pH image processing shown in FIG. 10 is started, the image data stored in the external storage device 24 is called (S71), and the image data is developed in the main memory 23. In this state, since the image is represented by an RGB color model, it is converted into a luminance and hue color model (S72). A color difference calculation process for obtaining a color difference value from the converted luminance and hue is executed (S73), the amount of change in pH is obtained, and the process returns to the process of FIG.

  In step S59 of FIG. 5, it is determined whether or not the pH change amount has reached the change amount threshold value. In this determination, if the pH change amount has reached the change amount threshold value, a medium replacement command is issued, and, for example, the medium replacement is automatically performed (S57). Then, a command to wait until the next image acquisition is issued (S58). On the other hand, if the pH change amount has not reached the change amount threshold value, a command to wait until the next image acquisition is issued (S58).

  As described above, according to the present embodiment, the thinning process is performed on the cells, the number of cells and the cell profile (angle and number of appearances) are calculated, and the change of the culture schedule is supported. If this feature is utilized, the configuration of the culture apparatus and the setting of the culture schedule can be freely changed according to the user's implementation. For example, it can be considered that the color camera is fixed and the incubator is freely moved vertically and horizontally.

It is a figure which shows the structure of the cell culture apparatus which is one Embodiment of this invention. It is a block diagram of the operation terminal 8 of the cell culture apparatus which is one Embodiment of this invention. It is a figure which shows the inside of the thermostat of the cell culture apparatus which is one Embodiment of this invention. It is a figure of a target cell number setting screen. It is a flowchart at the time of performing culture | cultivation experiment with the cell culture apparatus which is one Embodiment of this invention. It is a flowchart which shows the image processing of a cell. It is a figure which shows an imaging | photography location. It is a figure which shows a thinning process. It is a figure which shows an area | region division process. It is a flowchart which shows pH image processing. It is the image which showed the mode of the cell when the density of a cell is small. It is the image which showed the mode of the cell when the density of a cell is large. It is the graph which showed the direction of a cell and the frequency | count of appearance of a cell.

Explanation of symbols

1: Cell culture device 2: Incubator 3: Constant temperature bath 4: Color camera 5: Camera drive device 6: Incubator drive device 7: Motor controller 8: Operation terminal 9: Converter 22: CPU
23: Main memory 24: External storage device 28: Monitor

Claims (10)

Cells in an image obtained by photographing the inside incubator for culturing cells treated thinning into a single line in the longitudinal direction of the cells, to calculate the number of cells apparently thin line ized cells as a single cell Dividing the image area into a plurality of areas, selecting an area having the maximum apparent cell number, and comparing the apparent cell number and the set cell number in the area to manage a culture schedule. And automatic culture method. Furthermore, the direction of the thinned cell in the region where the number of cells is maximum is obtained, the cell density of the maximum region is obtained based on the relationship between the degree of alignment of the direction of the cells and the cell density, and the cell density The automatic culturing method according to claim 1, wherein execution of the passaging treatment is determined when the set cell density is exceeded. The set number of cells is automatic culture method according to claim 1 or 2, characterized in that it comprises a set number of cells to determine the completion of the culture and setting the number of cells to perform passaging process. A thinning process is performed in which cells in an image taken in an incubator for culturing cells are converted into a single line in the longitudinal direction of the cells, the direction of the thinned cells is obtained, and the degree of alignment of the direction of the cells is determined. calculated fine 胞密 degree of the image based on the relation of the cell density by dividing the area of the image into a plurality, the cell density to select the area which is the maximum, set the cell density and fine 胞密 degree in the region An automatic culture method characterized in that the culture schedule is managed in comparison with The region where the cell density is maximum is also a region where the apparent cell number is calculated by dividing the area of the image into a plurality of areas by calculating the number of cells with a thinned cell as one cell. The automatic culture method according to claim 4. The angle of the thinned cell direction and the number of appearances of the thinned cell at each angle are calculated, the cell density is determined based on the number of appearances in a set angle range, and the cell density is the setting 6. The automatic culture method according to claim 2, 4, or 5, wherein the execution of the passaging treatment is determined when the cell density is exceeded.   A pH indicator is added to an incubator for culturing cells, a change in the pH indicator is detected based on an image obtained by photographing the inside of the incubator, and a medium replacement process is determined based on the change in the pH indicator. The automatic culture method according to any one of claims 1 to 6. In a cell culture apparatus provided with an incubator for culturing cells, a photographing means for photographing the inside of the incubator, and a control means,
Said control means, said cells in captured images by photographing means processes thinning into a single line in the longitudinal direction of the cell, calculate the number of cells apparently thin line ized cells as a single cell Dividing the region of the image into a plurality of regions, selecting the region having the maximum apparent cell number, and comparing the apparent cell number and the set cell number in the region to manage the culture schedule. A cell culture device. Further, the control means obtains the direction of the thinned cell in the maximum area, obtains the cell density in the maximum area based on the relationship between the degree of alignment of the cell direction and the cell density, and The cell culturing apparatus according to claim 8, wherein execution of the passage process is determined when the density exceeds a set cell density. In a cell culture apparatus provided with an incubator for culturing cells, a photographing means for photographing the inside of the incubator, and a control means,
  The control means performs a thinning process for converting cells in the image photographed by the photographing means into a single line in the longitudinal direction of the cells, obtains the direction of the thinned cells, and the degree of alignment of the cell directions The cell density of the image based on the relationship between the cell density and the cell density
  A cell culture device, wherein the image area is divided into a plurality of areas, the area having the maximum cell density is selected, and the culture schedule is managed by comparing the cell density and the set cell density in the area.

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