JPWO2019022067A1 - Cell culture equipment - Google Patents

Abstract

The cell culture device images a culture vessel for culturing cells in the culture solution, a supply device for supplying the culture solution so that the culture solution forms an upward flow in the culture vessel, and a lower part of the culture vessel An imaging device that acquires a captured image, an image processing device that measures a maximum mass diameter of a cell mass in a culture tank based on the captured image, and a control device that controls supply of a culture solution by the supply device The culture tank has a shape that increases as the horizontal cross-sectional area of the culture tank increases upward, and the control device is provided with the culture tank per unit time when the maximum lump diameter is larger than a predetermined threshold value. The supply device is controlled so that the supply amount of the supplied culture solution repeatedly increases and decreases.

Description

  The present disclosure relates to a cell culture device.

  There is known an apparatus for observing a cell culture state by processing an image obtained by imaging a cell in a culture vessel. For example, in Patent Document 1, the incubator is photographed at each of the photographing positions recorded in the image position list, and the obtained image is processed to obtain the size, area, circumference, and the like of the colony. A cell culture device is described.

JP 2005-295818 A

  In the cell culture device described in Patent Literature 1, a camera or a drive device that moves the incubator is provided to capture images at each imaging position of the incubator. In this cell culture device, it is necessary to secure a space for installing the driving device and a space for moving the camera or the incubator. For this reason, there exists a possibility that a cell culture apparatus may enlarge.

  The present disclosure describes a cell culture device capable of observing a cell culture state without increasing the size of the device.

  A cell culture device according to one aspect of the present disclosure includes a culture vessel for culturing cells in a culture solution, a supply device that supplies the culture solution so that the culture solution forms an upward flow in the culture vessel, An imaging device that acquires a captured image by imaging the lower part of the culture tank, an image processing device that measures the maximum mass diameter of the cell mass in the culture tank based on the captured image, and supply of the culture solution by the supply device A control device for controlling. A culture tank has a shape which increases as the horizontal cross-sectional area of a culture tank goes upwards. The control device controls the supply device so that the supply amount of the culture solution supplied to the culture tank per unit time repeatedly increases and decreases when the maximum lump diameter is larger than a predetermined threshold value.

  According to the present disclosure, it is possible to observe the culture state of cells without increasing the size of the apparatus.

FIG. 1 is a diagram illustrating a schematic configuration of a cell culture device according to an embodiment. FIG. 2 is a flowchart showing an example of a series of processes in the maximum block diameter measurement mode. FIG. 3 is a flowchart illustrating an example of a series of processes in the lump diameter distribution measurement mode.

[1] Outline of Embodiment A cell culture device according to one aspect of the present disclosure includes a culture vessel for culturing cells in a culture solution, and a culture solution so that the culture solution forms an upward flow in the culture vessel. A supply device, an imaging device that acquires a captured image by imaging the lower part of the culture tank, an image processing device that measures a maximum mass diameter of a cell mass in the culture tank based on the captured image, and a supply And a control device for controlling supply of the culture solution by the device. A culture tank has a shape which increases as the horizontal cross-sectional area of a culture tank goes upwards. The control device controls the supply device so that the supply amount of the culture solution supplied to the culture tank per unit time repeatedly increases and decreases when the maximum lump diameter is larger than a predetermined threshold value.

  In this cell culture apparatus, the culture solution is supplied so that the culture solution forms an upward flow in the culture tank. Since the horizontal cross-sectional area of the culture tank increases as it goes upward, the flow rate of the culture solution (upward flow rate) decreases as it goes upward of the culture tank. For this reason, each cell mass in the culture tank is cultured while floating at a height where the sedimentation rate of the cell mass and the flow rate of the culture solution are balanced. That is, the cell mass is distributed such that the size of the cell mass increases toward the lower side of the culture tank and the size of the cell mass decreases toward the upper side of the culture tank. Therefore, by analyzing the captured image obtained by imaging the lower part of the culture tank, the maximum size of the cell mass in the culture tank can be obtained. Moreover, the cell mass in a culture tank starts sedimentation by reducing or making zero the supply amount of the culture solution supplied to a culture tank per unit time. For this reason, since the cell mass in the culture tank moves toward the lower part of the culture tank, more cell masses are imaged by repeatedly imaging the lower part of the culture tank after the cell mass starts to settle. obtain. Therefore, the size of each cell mass can be obtained by analyzing the captured image obtained by imaging the lower part of the culture tank. Thus, by analyzing the captured image obtained by imaging the lower part of the culture tank, the size of the cell mass can be measured without imaging the entire culture tank. Therefore, since it is not necessary to move the imaging device or the culture tank, it is possible to observe the cell culture state without increasing the size of the device.

  As described above, the cell mass is distributed such that the size of the cell mass increases toward the lower side of the culture tank, and the size of the cell mass decreases toward the upper side of the culture tank. Therefore, by analyzing the captured image obtained by imaging the lower part of the culture tank, the maximum mass diameter of the cell mass in the culture tank can be obtained.

  When the supply amount of the culture solution per unit time decreases, the flow rate of the upward flow in the culture tank decreases and the cell mass settles. Thereafter, when the supply amount of the culture solution per unit time starts to increase, the flow rate of the upward flow in the culture tank increases. At this time, the upward flow velocity increases toward the bottom of the culture tank, so that the cell mass is subjected to a greater shear stress. As a result, the cell mass is crushed and the entire cell mass diameter can be maintained below the threshold value.

  The control device may control the supply device such that the supply amount of the culture solution supplied to the culture tank per unit time decreases. The image processing apparatus may measure the distribution of the size of the cell mass in the culture tank based on the captured image. In this case, when the supply amount of the culture solution supplied to the culture tank per unit time decreases, the cell mass in the culture tank starts to settle. For this reason, since the cell mass in the culture tank moves toward the lower part of the culture tank, more cell masses are imaged by repeatedly imaging the lower part of the culture tank after the cell mass starts to settle. obtain. Therefore, by analyzing the captured image obtained by imaging the lower part of the culture tank, the distribution of the size of the cell mass in the culture tank can be obtained.

[2] Examples of Embodiments Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  FIG. 1 is a diagram illustrating a schematic configuration of a cell culture device according to an embodiment. A cell culture device 1 shown in FIG. 1 is a device for culturing cells to be cultured using a liquid medium (culture solution). The cell culture device 1 includes a culture tank 2, a pump 3, a camera 4, an image processing device 5, and a control device 6.

  The culture tank 2 is a container for culturing cells in a culture solution. The culture tank 2 has a shape in which the horizontal sectional area (horizontal sectional area) increases as it goes upward. In this embodiment, the shape of the culture tank 2 is an inverted truncated cone shape, but is not limited to an inverted truncated cone shape. For example, the shape of the culture tank 2 may be an inverted cone shape, an inverted polygonal pyramid shape, or an inverted polygonal frustum shape. The culture solution is filled in the culture tank 2. Moreover, in the culture tank 2, the cell or the carrier to which the cell adheres floats in a predetermined height range.

  Although the capacity | capacitance of the culture tank 2 is not specifically limited, For example, about 10 mL-10,000 mL may be sufficient. The capacity of the culture tank 2 can be appropriately selected based on the amount of cells to be cultured in one culture tank 2 or the size of the cell mass. The capacity of the culture tank 2 may be selected according to the handleability of the culture tank 2 and the like. For example, when the capacity of the culture tank 2 is about 50 mL to 500 mL, the handleability of the culture tank 2 alone is improved.

  A circulation line L <b> 1 is connected to the lower end of the culture tank 2. A circulation line L <b> 2 is connected to the upper end of the culture tank 2. The circulation line L1 and the circulation line L2 connect between the culture tank 2 and the pump 3, respectively. Each of the circulation line L1 and the circulation line L2 is, for example, a pipe.

  The pump 3 functions as a supply device that supplies the culture solution to the culture vessel 2 so that the culture solution forms an upward flow in the culture vessel 2. In this embodiment, the pump 3 circulates (perfuses) the culture solution by supplying the culture solution into the culture tank 2. When the pump 3 operates, the culture solution is supplied to the culture tank 2 through the circulation line L1, and the culture solution is discharged from the culture tank 2 through the circulation line L2. That is, when the pump 3 operates, the culture solution circulates in the order of the circulation line L1, the culture tank 2, the circulation line L2, and the pump 3. In the culture tank 2, the culture fluid flows from the lower part 2 a of the culture tank 2 toward the upper part.

  The culture tank 2, the pump 3, the circulation line L1, and the circulation line L2 are accommodated in the incubator 10. The incubator 10 is a temperature-controlled room for appropriately maintaining the culture solution at a temperature corresponding to the culture environment. A window 10 a is provided on the side wall of the incubator 10. The window portion 10a is a portion where the inside of the incubator 10 can be observed from the outside of the incubator 10. As the window portion 10a, for example, transparent glass or an acrylic plate is used.

  The camera 4 is an imaging device that acquires a captured image by imaging the culture tank 2. The camera 4 images at least the lower part 2 a of the culture tank 2. In this embodiment, since the camera 4 is arrange | positioned outside the incubator 10, it images the culture tank 2 through the window part 10a. The captured image may be a still image or a moving image. The camera 4 transmits the captured image to the image processing device 5. In order to unify the background of the captured image, a monochrome background paper may be installed on the back side of the culture tank 2 when viewed from the camera 4. Thereby, the visibility of a cell cluster improves in a captured image. The color of the background paper may be a color different from the color of the cells in the culture tank 2, for example, green. The color of the background paper may be other than green.

  The image processing device 5 is a device that measures a cell mass in the culture tank 2 based on a captured image acquired by the camera 4. Examples of the measurement items include the size of the cell mass (cell mass diameter) in the culture tank 2 and the size distribution (average and variance). The cell mass diameter is an index representing the size of the cell mass, and is, for example, the maximum value of the distance between any two points in the shape of the cell mass. In the case where the cell mass is circular, the cell mass diameter indicates the diameter of the cell mass. When the cell mass is elliptical, the cell mass diameter indicates the length of the long axis of the cell mass. The image processing device 5 may estimate the total number of cells in the culture tank 2 from the distribution of the size of the cell mass in the culture tank 2. Details of the measurement of the cell mass will be described later. The image processing device 5 transmits the measurement data to the control device 6.

  The control device 6 is a controller that performs various controls related to the cell culture device 1. For example, the control device 6 controls the supply of the culture solution by the pump 3. The control device 6 transmits an imaging command to the camera 4 and causes the camera 4 to capture an image. The control device 6 stores the measurement data received from the image processing device 5 in a storage device (not shown). Examples of the storage device include a hard disk and a semiconductor memory. The control device 6 may perform temperature control of the incubator 10 and control of dissolved oxygen.

  The control device 6 includes a central processing unit (CPU), a random access memory (RAM) and a read only memory (ROM) that are main storage devices, a communication module that performs communication with other devices, and an auxiliary storage such as a hard disk. It is comprised as a computer provided with hardware, such as an apparatus. The function of the control device 6 described later is exhibited by the operation of these components. The image processing device 5 is also configured as a computer similar to the control device 6.

  Note that the cell culture apparatus 1 may be separately provided with a drain line for discharging the culture medium to the outside, an introduction line for supplying a new culture medium, and the like.

  In the cell culture device 1, the horizontal cross-sectional area of the culture tank 2 increases toward the upper side of the culture tank 2, so the flow rate of the culture solution (upward flow rate) decreases as it goes upward of the culture tank 2. Each cell is cultured while floating at a height where the sedimentation rate of the cell and the flow rate of the culture solution are balanced. Many cells are supported on a carrier, fixed, or attached by cell culture to form an aggregate (cell mass), and the three-dimensional culture of cells proceeds to grow the cell mass. As the cell mass grows and the cell mass diameter increases, the sedimentation rate of the cell mass increases. The cell mass further settles and floats at a height where the sedimentation rate of the cell mass and the flow rate of the culture solution are balanced. For this reason, the cell mass is distributed so that the cell mass diameter of the cell mass increases toward the lower side of the culture tank 2, and the cell mass diameter of the cell mass decreases toward the upper side of the culture tank 2. .

  Next, with reference to FIG.2 and FIG.3, the measuring method of the cell mass in the culture tank 2 is demonstrated. Here, the case where the cell culture apparatus 1 implements the maximum lump diameter measurement mode and the lump diameter distribution measurement mode will be described. FIG. 2 is a flowchart showing an example of a series of processes in the maximum block diameter measurement mode. The maximum clot diameter measurement mode is a mode for measuring the maximum cell clot diameter among the cell clot diameters of the cell clots existing in the culture tank 2. A series of processing in the maximum block diameter measurement mode shown in FIG. 2 is repeatedly performed at a frequency of about once every 30 minutes, for example.

  First, the control device 6 transmits an imaging command to the camera 4 and transmits a maximum block diameter measurement command to the image processing device 5. At this time, since the pump 3 supplies the culture solution to the culture tank 2 at a constant supply amount per unit time as usual, the cell mass diameter of the cell mass increases toward the lower side of the culture tank 2 and the culture is performed. The cell mass is distributed so that the cell mass diameter of the cell mass decreases toward the upper side of the tank 2. When the camera 4 receives an imaging command from the control device 6, the camera 4 images the lower part 2 a of the culture tank 2 and transmits the captured image to the image processing device 5. Then, the image processing device 5 acquires a captured image from the camera 4 (step S11).

  Subsequently, the image processing device 5 generates a difference image using the captured image and the background image stored in advance (step S12). Specifically, the image processing device 5 generates a difference image by calculating the difference between the pixel value of each pixel included in the captured image and the pixel value of the pixel of the background image corresponding to the pixel. The background image is an image obtained by imaging the lower part 2a of the culture tank 2 in which the culture solution is circulated in a state where the cells are not cultured. The background image is acquired by, for example, imaging the culture tank 2 before introducing the cells at the time of initial culture.

  Subsequently, the image processing device 5 performs filter processing on the difference image (step S13). The image processing device 5 performs, for example, noise processing and edge processing as filter processing. Then, the image processing device 5 converts the difference image that has been subjected to the filter processing into a binarized image (step S14). Specifically, the image processing device 5 compares the pixel value of each pixel of the difference image subjected to the filter process with a predetermined binarization threshold value, and the pixel value is larger than the binarization threshold value. Is set to 0 (black), and is set to 1 (white) when the pixel value is equal to or less than the binarization threshold value, thereby converting the difference image into a binarized image.

  Subsequently, the image processing apparatus 5 performs labeling on the binarized image (step S15). By this labeling, the same label number is assigned to a continuous portion in the binarized image, and the background region and each cell cluster region are extracted. Then, the image processing device 5 calculates the cell mass diameter of each cell mass (step S16). The image processing apparatus 5 calculates, for example, the length of the long axis when the cell mass is regarded as an ellipse as the cell mass diameter by using ellipse fitting. In the ellipse fitting, there is a possibility that the long axis becomes long. For this reason, the image processing apparatus 5 may calculate the cell mass diameter using circular fitting or rectangular fitting.

  Subsequently, the image processing apparatus 5 calculates the maximum lump diameter (step S17). Specifically, the image processing device 5 extracts the maximum cell mass diameter among the cell mass diameters of all the cell masses included in the captured image (binarized image), and uses the extracted cell mass diameter as a culture tank. 2 is the maximum mass of the cell mass within 2. Then, the image processing device 5 outputs measurement data including the maximum lump diameter (step S18). For example, the image processing device 5 transmits measurement data including the maximum lump diameter to the control device 6. And the control apparatus 6 memorize | stores measurement data in a memory | storage device. In this way, a series of processes in the maximum lump diameter measurement mode is completed.

  Thus, in the maximum clot diameter measurement mode, since the pump 3 is operating normally, the cell clot diameter of the cell clot increases as it goes downward of the culture tank 2, and as it goes upward of the culture tank 2, The cell mass is distributed so that the cell mass diameter of the cell mass becomes small. That is, the cell mass having the maximum mass diameter is suspended in the lower part 2 a of the culture tank 2. For this reason, the maximum lump diameter is obtained by analyzing the captured image obtained by imaging the lower part 2a of the culture tank 2.

  In the process shown in FIG. 2, when the cell clusters overlap in the captured image, a cell cluster larger than the actual cell cluster may be detected. For this reason, the image processing apparatus 5 may repeatedly perform the processing of Steps S11 to S17, and set the median value among the plurality of maximum block diameters obtained by a fixed number of calculations as the maximum block diameter. Further, the image processing apparatus 5 may calculate the maximum lump diameter using an image processing method other than the above.

  Next, the lump diameter distribution measurement mode will be described. FIG. 3 is a flowchart illustrating an example of a series of processes in the lump diameter distribution measurement mode. The lump diameter distribution measurement mode is a mode for measuring the distribution of the cell lump diameter of the cell lump existing in the culture tank 2. A series of processing in the lump size distribution measurement mode shown in FIG. 3 is repeatedly performed at a frequency of about once a day, for example.

  First, the control device 6 stops the operation of the pump 3 (step S21). In addition, the control apparatus 6 may control the pump 3 so that the supply amount of the culture solution supplied to the culture tank 2 per unit time decreases. Thereby, since the flow rate of the culture solution (upflow) in the culture tank 2 starts to decrease, the cell mass in the culture tank 2 cannot float in the culture solution and starts to settle. Then, the control device 6 transmits an imaging command to the camera 4 and transmits a lump diameter distribution measurement command to the image processing device 5. Since the process of step S22-step S27 is the same as the process of step S11-step S16, description is abbreviate | omitted here.

  Subsequently, the image processing device 5 determines whether or not all cell clusters have settled (step S28). For example, the image processing device 5 may determine whether or not all cell clusters have settled depending on whether or not a circular object is present in the captured image. Specifically, the image processing device 5 determines that not all cell clusters have settled when a circular object exists in the captured image, and all cells when the circular object does not exist in the captured image. It is determined that the lump has settled. Further, the image processing device 5 may determine whether or not all cell clusters have settled based on the elapsed time from the start of sedimentation of the cell clusters. Specifically, the image processing device 5 determines that not all cell masses have settled when a predetermined time has not elapsed since receiving the mass size distribution measurement command, and the mass size distribution measurement command When a predetermined time has elapsed after receiving the signal, it is determined that all cell clusters have settled. The predetermined time is the time required for all the cell clusters to settle, and is measured and set in advance.

  In step S28, when the image processing apparatus 5 determines that all the cell clusters have not settled (step S28; NO), the processing of step S22 to step S28 is performed again. Note that the image processing device 5 calculates the cell mass diameter of each cell mass included in the captured image for each of the plurality of captured images. At this time, the image processing apparatus 5 calculates one cell mass diameter with respect to the same cell mass which exists over two or more captured images by tracking each cell mass sequentially. Tracking is performed by, for example, the Mean-shift method.

  On the other hand, in step S28, when it is determined that all the cell clusters have settled (step S28; YES), the image processing apparatus 5 outputs measurement data including the cell cluster diameter (cluster diameter distribution) of each cell cluster (step S28; YES). Step S29). For example, the image processing device 5 transmits measurement data including the cell mass diameter (cluster diameter distribution) of each cell mass to the control device 6. And the control apparatus 6 memorize | stores measurement data in a memory | storage device. And the control apparatus 6 restarts the driving | operation of a pump (step S30). Specifically, the control device 6 controls the pump 3 so as to return the flow rate of the culture solution (upflow) in the culture tank 2 to the state before the execution of the lump size distribution measurement mode. Then, a series of processing in the lump diameter distribution measurement mode ends.

  Thus, in the lump diameter distribution measurement mode, the flow rate of the pump 3 is reduced or zeroed at the start of measurement, so that the cell lump in the culture tank 2 starts to settle. Therefore, since all the cell masses in the culture tank 2 reach the lower part 2a of the culture tank 2, by imaging the lower part 2a of the culture tank 2 until all the cell masses start to settle and settle down, All cell masses can be imaged. For this reason, by analyzing a plurality of captured images obtained by imaging the lower part 2a of the culture tank 2, the cell mass diameter (the mass diameter distribution) of all the cell masses can be obtained.

  Even if the lower part 2a of the culture tank 2 is imaged after the cell mass has settled, there is a possibility that the cell mass has flowed into the circulation line L1, and the entire cell mass cannot be imaged. Moreover, even if all the cell clusters have accumulated at the bottom of the culture tank 2, the distance between the cell clusters is short in the captured image, and it is difficult to specify the outline of each cell cluster. For this reason, all the cell masses can be observed by imaging the lower part 2a of the culture tank 2 until all the cell masses start sedimentation and finish sedimentation.

  As described above, in the cell culture device 1, the culture solution is supplied so that the culture solution forms an upward flow in the culture tank 2. Since the horizontal cross-sectional area of the culture tank 2 increases as it goes upward, the flow rate of the culture solution (upward flow rate) decreases as it goes upward of the culture tank 2. For this reason, each cell in the culture tank 2 is cultured while floating at a height in which the sedimentation rate of the cell and the flow rate of the culture solution are balanced. That is, the size of the cell mass (cell mass diameter) increases toward the lower side of the culture tank 2, and the size of the cell mass (cell mass diameter) decreases as it goes above the culture tank. The lumps are distributed. Therefore, by analyzing the captured image obtained by imaging the lower part 2a of the culture tank 2, the maximum size (maximum mass diameter) of the cell mass in the culture tank 2 can be obtained.

  Moreover, the cell mass in the culture tank 2 starts sedimentation by reducing or making the supply amount of the culture solution supplied to the culture tank 2 per unit time zero. For this reason, since the cell mass in the culture tank 2 moves toward the lower part 2a of the culture tank 2, it is possible to capture more images by repeatedly imaging the lower part 2a of the culture tank 2 after the cell mass starts to settle. Cell mass can be imaged. Therefore, by analyzing the captured image obtained by imaging the lower part 2a of the culture tank 2, the size of each cell mass can be obtained, and the mass diameter distribution can be obtained.

  Thus, by analyzing the captured image obtained by imaging the lower part 2a of the culture tank 2, the size of the cell mass (in this embodiment, the maximum mass diameter is obtained without imaging the entire culture tank 2). And lump size distribution) can be measured. Therefore, since it is not necessary to move the camera 4 or the culture tank 2, it becomes possible to observe the culture state of a cell, without enlarging an apparatus. Moreover, since it is not necessary to move the camera 4 or the culture tank 2, the number of components can be reduced, and the manufacturing cost and the maintenance cost can be reduced.

  Conventionally, in order to measure the number of cells in culture in a culture tank, some of the cells in the culture tank are taken out and visually measured using a microscope and a blood cell counter. However, since the cells come into contact with the air in the process of removing the cells from the culture tank, there is a risk that unintended bacteria may enter the culture tank. In addition, since the cells used for measurement cannot be returned to the culture tank and cultured, the cell culture rate is reduced. On the other hand, since the cell culture device 1 measures the maximum cell diameter and distribution of the cell mass by analyzing the captured image, the cell culture state can be changed without removing the cell mass from the culture tank 2. It becomes possible to observe in real time.

  Moreover, in the cell culture apparatus 1, the camera 4 does not need to image the whole culture tank 2, and should just image the lower part 2a of the culture tank 2 at least. For this reason, a high-performance (high resolution and wide-angle) camera may not be used as the camera 4.

  As mentioned above, although embodiment of this indication was described, this invention is not limited to the said embodiment.

  For example, the kind of cell cultured by the cell culture apparatus 1 is not specifically limited. For example, stem cells and the like in animals are cultured. The culture medium used as a culture medium in cell culture is appropriately selected according to the type of cells to be cultured.

  In the above embodiment, the camera 4, the image processing device 5, and the control device 6 are arranged outside the incubator 10. However, the camera 4, the image processing device 5, and the control device 6 are partially or entirely in the incubator. 10 may be disposed inside. Some or all of the pump 3, the circulation line L <b> 1, and the circulation line L <b> 2 may be disposed outside the incubator 10.

  In the above embodiment, the image processing device 5 and the control device 6 are configured by different computers, but the image processing device 5 and the control device 6 may be configured by one computer.

  In the above embodiment, the cell mass diameter and the distribution of the cell mass diameter are measured, but the measurement items are not limited to these. The shape, roundness, aspect ratio, circularity, and area of the cell mass may be measured.

  Further, the image processing device 5 may extract a cell mass region from a captured image by causing a model such as a neural network to learn a cell mass image to be measured.

  The control device 6 may control the pump 3 based on the measurement data. For example, if a large cell mass is generated in the lower part 2a of the culture tank 2, the entire culture may be adversely affected. Therefore, the control device 6 may control the pump 3 by comparing the maximum lump diameter measured by the image processing device 5 with a predetermined threshold value. Specifically, when the maximum lump diameter is equal to or smaller than the threshold value, the control device 6 controls the pump 3 so as to supply the culture solution to the culture tank 2 at a constant supply amount per unit time. On the other hand, when the maximum lump diameter is larger than the threshold value, the control device 6 controls the pump 3 so that the supply amount of the culture solution supplied to the culture tank 2 per unit time repeatedly increases and decreases over time. . The threshold value is, for example, 500 μm.

  For example, the control device 6 controls the pump 3 so as to alternately repeat a drive time for turning on the pump 3 and a stop time for turning off the pump 3. And the control apparatus 6 will control the pump 3 so that a culture solution may be supplied to the culture tank 2 by the fixed supply amount per unit time, when the largest lump diameter becomes below a threshold value. Thereby, when the pump 3 is stopped, the supply amount of the culture solution per unit time is decreased, so that the flow rate of the culture solution in the culture tank 2 is lowered and the cell mass is sedimented. Thereafter, when the pump 3 is driven, the supply amount of the culture solution per unit time starts to increase, and the flow rate of the culture solution in the culture tank 2 increases. At this time, each cell mass is sedimented at a position lower than the height at which the cell mass floats during normal operation. And since it goes to the downward direction of the culture tank 2, the flow rate of a culture solution becomes large, Therefore Each cell mass receives a bigger shear stress than the time of normal operation. As a result, the cell mass is crushed and the entire cell mass diameter can be maintained below the threshold value.

  The control device 6 may control the pump 3 so that the supply amount of the culture solution supplied to the culture tank 2 per unit time increases in order to give the cell mass a greater shear stress than during normal operation. The pump 3 may be controlled so that the supply amount of the culture solution supplied to the culture tank 2 per unit time changes in a sine wave shape.

1 Cell culture device 2 Culture tank 2a Lower part 3 Pump (supply device)
4 Camera (imaging device)
5 Image Processing Device 6 Control Device 10 Incubator 10a Window L1 Circulation Line L2 Circulation Line

Claims (2)

A culture vessel for culturing cells in a culture solution;
A supply device for supplying the culture solution so that the culture solution forms an upward flow in the culture tank;
An imaging device for acquiring a captured image by imaging the lower part of the culture tank;
Based on the captured image, an image processing device that measures the maximum mass diameter of the cell mass in the culture tank,
A control device for controlling the supply of the culture solution by the supply device;
With
The culture tank has a shape that increases as the horizontal cross-sectional area of the culture tank goes upward,
The control device controls the supply device so that the supply amount of the culture solution supplied to the culture tank per unit time repeatedly increases and decreases when the maximum mass diameter is larger than a predetermined threshold value. Cell culture equipment. The control device controls the supply device such that the supply amount of the culture solution supplied to the culture tank per unit time decreases,
The cell culture device according to claim 1, wherein the image processing device measures a size distribution of a cell mass in the culture tank based on the captured image.

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