WO2019022067A1

WO2019022067A1 - Cell culture device

Info

Publication number: WO2019022067A1
Application number: PCT/JP2018/027669
Authority: WO
Inventors: 謙一 ▲濱▼口; 玄佐藤; 慎一坂井
Original assignee: 株式会社Ihi
Priority date: 2017-07-27
Filing date: 2018-07-24
Publication date: 2019-01-31
Also published as: JPWO2019022067A1

Abstract

This cell culture device comprises: a culture tank for culturing cells in a culture solution; a supply device for supplying the culture solution such that the culture solution forms an upward current in the culture tank; an imaging device for imaging a lower part of the culture tank to acquire a captured image; an image processing device for measuring the maximum mass diameter of a cell mass in the culture tank on the basis of the captured image; and a control device for controlling the supply of the culture solution by the supply device. The culture tank has a shape where the horizontal cross-sectional area of the culture tank increases going upward. The control device, in a case where the maximum mass diameter is greater than a predetermined threshold, controls the supply device such that the supply amount of the culture solution supplied to the culture tank per unit time is repeatedly increased and decreased.

Description

Cell culture device

The present disclosure relates to cell culture devices.

An apparatus for observing the culture state of cells by processing an image obtained by imaging the cells in a culture vessel is known. For example, in Patent Document 1, the incubator is photographed at each of the photographing positions recorded in the image position list, and the acquired image is processed to acquire the size, area, circumference, and the like of the colony. Cell culture devices have been described.

JP 2005-295818 A

The cell culture device described in Patent Document 1 is provided with a drive device for moving a camera or incubator for photographing at each photographing position of the incubator. In this cell culture apparatus, it is necessary to secure a space for installing a driving device and a space for moving a camera or an incubator. For this reason, there is a possibility that a cell culture apparatus may be enlarged.

The present disclosure describes a cell culture device that can observe the culture state of cells without upsizing the device.

A cell culture apparatus according to one aspect of the present disclosure includes a culture vessel for culturing cells in a culture solution, and a supply apparatus for supplying a culture solution so that the culture solution forms an ascending flow in the culture vessel, An image processing apparatus for acquiring a captured image by imaging the lower part of the culture tank, an image processing apparatus for measuring the maximum mass diameter of cell masses in the culture tank based on the captured image, and supply of a culture solution by a supply apparatus And a control device to control. The culture vessel has a shape which increases as the horizontal cross-sectional area of the culture vessel goes upward. The control device controls the supply device such that the supply amount of the culture solution supplied to the culture tank repeats increase and decrease per unit time when the maximum mass diameter is larger than the predetermined threshold value.

According to the present disclosure, the culture state of cells can be observed without increasing the size of the device.

FIG. 1 is a view showing a schematic configuration of a cell culture apparatus according to an embodiment. FIG. 2 is a flow chart showing an example of a series of processing of the largest block diameter measurement mode. FIG. 3 is a flow chart showing an example of a series of processing in the block size distribution measurement mode.

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

In this cell culture apparatus, the culture solution is supplied such that the culture solution forms an ascending flow in the culture tank. Since the horizontal cross-sectional area of the culture vessel increases as it goes upward, the flow rate of the culture solution (the speed of upflow) decreases as it goes upward. For this reason, each cell mass in the culture tank is cultured while floating at a height at which the sedimentation rate of the cell mass and the flow rate of the culture solution are in balance. That is, the cell mass is distributed such that the size of the cell mass increases as it goes downward in the culture vessel, and the size of the cell mass decreases as it goes upward in the culture bath. Therefore, the largest size of the cell mass in the culture tank can be obtained by analyzing the captured image obtained by imaging the lower part of the culture tank. In addition, the cell mass in the culture tank starts to sediment by reducing or zeroing the amount of culture solution supplied to the 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 clusters are imaged by repeatedly imaging the lower part of the culture tank after the cell mass starts to precipitate. 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 vessel. As described above, by analyzing a 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 an imaging device or a culture tank, it becomes possible to observe the culture condition of a cell, without enlarging a 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 vessel and the size of the cell mass decreases toward the upper side of the culture vessel. Therefore, the largest mass diameter of the cell mass in the culture tank can be obtained by analyzing the captured image obtained by imaging the lower part of the culture tank.

When the amount of culture solution supplied per unit time decreases, the flow rate of the upflow in the culture vessel 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 upflow in the culture vessel increases. At this time, the cell mass is subjected to greater shear stress because the upward flow velocity increases as it goes downward in the culture vessel. As a result, the cell mass is disrupted, and the whole cell mass diameter can be maintained at or below the threshold.

The control device may control the supply device such that the amount of culture solution supplied to the culture tank per unit time decreases. The image processing apparatus may measure the size distribution of the cell mass in the culture tank based on the captured image. In this case, the amount of culture solution supplied to the culture tank decreases per unit time, whereby the cell mass in the culture tank starts to precipitate. For this reason, since the cell mass in the culture tank moves toward the lower part of the culture tank, more cell clusters are imaged by repeatedly imaging the lower part of the culture tank after the cell mass starts to precipitate. 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] Example of Embodiment Hereinafter, an embodiment of the present disclosure will be described with reference to the drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

FIG. 1 is a view showing a schematic configuration of a cell culture apparatus according to an embodiment. The cell culture apparatus 1 shown in FIG. 1 is an apparatus 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 vessel 2 has a shape in which the cross-sectional area in the horizontal direction (horizontal cross-sectional area) increases upward. In the present embodiment, the shape of the culture vessel 2 is an inverted truncated cone shape, but is not limited to the inverted truncated cone shape. For example, the shape of the culture vessel 2 may be an inverted cone, an inverted polygonal pyramid, or an inverted polygonal pyramid. The culture solution is filled in the culture vessel 2. Moreover, in the culture tank 2, the cell or the carrier to which the cell is attached floats in a predetermined height range.

The volume of the culture vessel 2 is not particularly limited, but may be, for example, about 10 mL to 10,000 mL. The volume of the culture tank 2 can be appropriately selected based on the amount of cells desired to be cultured in one culture tank 2, the size of a cell mass, and the like. The capacity of the culture tank 2 may be selected according to the handling property of the culture tank 2 or 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 L1 is connected to the lower end of the culture tank 2. A circulation line L2 is connected to the upper end of the culture vessel 2. Circulation line L1 and circulation line L2 connect between culture vessel 2 and 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 for supplying the culture solution to the culture tank 2 so that the culture solution forms an ascending flow in the culture tank 2. In the present embodiment, the pump 3 circulates (perfuses) the culture fluid by supplying the culture fluid into the culture tank 2. By operating the pump 3, the culture solution is supplied to the culture vessel 2 via the circulation line L1, and the culture solution is discharged from the culture vessel 2 via the circulation line L2. That is, when the pump 3 operates, the culture fluid 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 2a of the culture tank 2 to 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 constant temperature chamber for appropriately maintaining the culture solution at a temperature according to the culture environment. A window 10 a is provided on the side wall of the incubator 10. The window portion 10 a is a portion where the inside of the incubator 10 can be observed from the outside of the incubator 10. For example, a transparent glass or acrylic plate is used as the window 10a.

The camera 4 is an imaging device which acquires a captured image by imaging the culture tank 2. The camera 4 images at least the lower portion 2 a of the culture tank 2. In the present embodiment, since the camera 4 is disposed outside the incubator 10, the culture vessel 2 is imaged through the window 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 single-color background paper may be placed behind the culture vessel 2 as viewed from the camera 4. Thereby, in the captured image, the visibility of the cell mass is improved. The color of the background paper may be any color different from the color of the cells in the culture tank 2, and is, 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. The measurement items include the size (cell mass diameter) of the cell mass in the culture tank 2 and the distribution (average and dispersion) of the size. The cell mass diameter is an index indicating 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. When 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 major 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 sizes of cell masses in the culture tank 2. Details of measurement of cell mass will be described later. The image processing device 5 transmits 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 controller 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 storage devices include hard disks and semiconductor memories. The controller 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) as a main storage device, a communication module for communicating with other devices, and an auxiliary storage such as a hard disk. It is configured as a computer including hardware such as an apparatus. The operation of these components exerts the function of the control device 6 described later. The image processing device 5 is also configured as a computer similar to the control device 6.

The cell culture apparatus 1 may be additionally provided with a drainage line for discharging the culture solution to the outside, an introduction line for supplying a new culture solution, and the like.

In the cell culture apparatus 1, the horizontal cross-sectional area of the culture tank 2 becomes larger as it goes upward of the culture tank 2, so the flow rate of the culture solution (the speed of upflow) decreases as it goes upward of the culture tank 2. Each cell is cultured while floating at a height at which the sedimentation rate of the cells and the flow rate of the culture medium are in balance. By culturing the cells, a large number of cells are supported, fixed or attached to a carrier to form an aggregate (cell mass), and the three-dimensional culture of the cells proceeds to grow the cell mass. As the cell mass diameter increases as the cell mass grows, the sedimentation speed of the cell mass increases. Then, the cell mass is further sedimented, and floats at a height at which the sedimentation speed of the cell mass and the flow rate of the culture solution are in balance. For this reason, the cell mass is distributed such that the cell mass diameter of the cell mass increases as going downward to the culture tank 2 and the cell mass diameter of the cell mass decreases as going upward to 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, a case where the cell culture device 1 performs the maximum mass diameter measurement mode and the mass diameter distribution measurement mode will be described. FIG. 2 is a flow chart showing an example of a series of processing of the largest block diameter measurement mode. The largest block size measurement mode is a mode for measuring the largest cell block size among the cell block sizes of the cell blocks present in the culture tank 2. The series of processes of the largest block diameter measurement mode shown in FIG. 2 is repeatedly performed, for example, once every 30 minutes.

First, the control device 6 transmits an imaging command to the camera 4 and transmits a maximum block size 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 of the cell mass becomes larger as it goes downward of the culture tank 2, The cell mass is distributed such 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 captures an image of the lower portion 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 portion 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, for example, by imaging the culture tank 2 before charging 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 differential image subjected to the filtering process into a binarized image (step S14). Specifically, the image processing device 5 compares the pixel value of each pixel of the differential image subjected to the filtering process with a predetermined binarization threshold, and the pixel value is larger than the binarization threshold. The difference image is converted into a binarized image by setting it to 0 (black) and setting it to 1 (white) if the pixel value is equal to or less than the binarization threshold.

Subsequently, the image processing apparatus 5 labels the binarized image (step S15). By this labeling, the same label number is assigned to the continuous part in the binarized image, and the background area and the area of each cell mass are extracted. Then, the image processing device 5 calculates the cell mass diameter of each cell mass (step S16). The image processing device 5 calculates, for example, the length of the major axis when the cell mass is regarded as elliptical, as the cell mass diameter, using the elliptical fitting. In elliptical fitting, the major axis may be long. Therefore, the image processing device 5 may calculate the cell mass diameter using circle fitting or rectangular fitting.

Subsequently, the image processing device 5 calculates the maximum mass diameter (step S17). Specifically, the image processing device 5 extracts the largest cell mass diameter among the cell mass diameters of all cell masses included in the captured image (binarized image), and the extracted cell mass diameter The largest block size of the cell mass in 2. Then, the image processing device 5 outputs measurement data including the largest block diameter (step S18). For example, the image processing device 5 transmits measurement data including the maximum mass diameter to the control device 6. Then, the control device 6 stores the measurement data in the storage device. In this way, the series of processes in the largest block diameter measurement mode is completed.

Thus, in the maximum block diameter measurement mode, since the pump 3 operates normally, the cell block diameter of the cell block increases toward the lower side of the culture tank 2 and as the cell block diameter increases toward the upper side of the culture tank 2 The cell mass is distributed such that the cell mass size of the cell mass is smaller. That is, the cell mass having the largest mass diameter is suspended in the lower portion 2 a of the culture tank 2. For this reason, the largest lump diameter is obtained by analyzing the captured image obtained by imaging the lower part 2a of the culture tank 2. As shown in FIG.

In addition, in the process shown by FIG. 2, when cell mass overlaps in a captured image, a cell mass larger than an actual cell mass may be detected. For this reason, the image processing apparatus 5 may repeat the processing of step S11 to step S17, and set the median value among the plurality of maximum block sizes obtained by the calculation of the fixed number of times as the maximum block size. In addition, the image processing apparatus 5 may calculate the maximum mass diameter using an image processing method other than the above.

Next, the block size distribution measurement mode will be described. FIG. 3 is a flow chart showing an example of a series of processing in the block size distribution measurement mode. The block size distribution measurement mode is a mode for measuring the distribution of cell block sizes of cell blocks present in the culture tank 2. The series of processes in the block size distribution measurement mode shown in FIG. 3 is repeatedly performed, for example, at a frequency of about once a day.

First, the control device 6 stops the operation of the pump 3 (step S21). The controller 6 may control the pump 3 so that the amount of culture solution supplied to the culture tank 2 decreases per unit time. As a result, the flow rate of the culture solution (upflow) in the culture tank 2 starts to decrease, so the cell mass in the culture tank 2 can not float in the culture solution, and sedimentation starts. Then, the control device 6 transmits an imaging command to the camera 4 and also transmits a chunk diameter distribution measurement command to the image processing device 5. The processes of step S22 to step S27 are the same as the processes of step S11 to step S16, and thus the description thereof is omitted here.

Subsequently, the image processing device 5 determines whether or not all cell aggregates have been sedimented (step S28). For example, the image processing device 5 may determine whether or not all cell clusters have been sedimented based 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 been sedimented when a circular object is present in the captured image, and all cells are present when no circular object is present in the captured image. Determine that the mass has settled. In addition, the image processing device 5 may determine whether all the cell clusters have been sedimented or not based on the elapsed time from the start of the cell mass sedimentation. Specifically, the image processing device 5 determines that not all cell aggregates have been sedimented when a predetermined time has not elapsed since receiving the mass diameter distribution measurement command, and the mass diameter distribution measurement command It is determined that all cell clusters have sedimented when a predetermined time has elapsed since the reception of. The predetermined time is a time required for settling of all cell masses, and is previously measured and set.

When it is determined in step S28 that the image processing apparatus 5 does not mean that all cell clusters have been sedimented (step S28; NO), the process of step S22 to step S28 is performed again. The image processing device 5 calculates, for each of the plurality of captured images, the diameter of the cell mass of each cell mass included in the captured image. At this time, the image processing device 5 calculates one cell block diameter for the same cell block existing over two or more captured images by sequentially tracking each cell block. Tracking is performed, for example, by the Mean-shift method.

On the other hand, when it is determined in step S28 that the image processing apparatus 5 determines that all the cell masses have been sedimented (step S28; YES), the image processing apparatus 5 outputs measurement data including cell mass size (mass size distribution) of each cell mass ( Step S29). For example, the image processing device 5 transmits measurement data including the cell mass diameter (mass diameter distribution) of each cell mass to the control device 6. Then, the control device 6 stores the measurement data in the storage device. Then, the control device 6 resumes the operation of the pump (step S30). Specifically, the control device 6 controls the pump 3 to return the flow rate of the culture solution (upflow) in the culture tank 2 to the state before the execution of the mass distribution measurement mode. Then, the series of processing in the mass distribution measurement mode ends.

As described above, in the mass distribution measurement mode, the flow rate of the pump 3 is decreased or made zero at the start of measurement, and thus the cell mass in the culture tank 2 starts to precipitate. Therefore, since all cell clusters 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 from the start of sedimentation to the end of sedimentation, All cell masses can be imaged. For this reason, by analyzing a plurality of captured images obtained by imaging the lower portion 2a of the culture tank 2, cell mass diameters (mass diameter distribution) of all cell masses can be obtained.

In addition, even if imaging the lower part 2a of the culture tank 2 after a cell mass finishes settling, a cell mass may have flowed in into the circulation line L1, and it can not image all cell masses. In addition, even if all cell clusters are 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 contour of each cell cluster. Therefore, by imaging the lower portion 2a of the culture tank 2 from the start of sedimentation to the end of sedimentation, all cell masses can be observed.

As described above, in the cell culture apparatus 1, the culture solution is supplied such that the culture solution forms an ascending flow in the culture tank 2. As the horizontal cross-sectional area of the culture vessel 2 increases as it goes upward, the flow rate of the culture solution (the speed of the upflow) decreases as it goes upward. For this reason, each cell in the culture tank 2 is cultured while floating at a height at which the settling speed of the cells and the flow rate of the culture solution are balanced. That is, the cell mass (cell mass diameter) becomes larger as it goes downward in the culture tank 2, and the cell mass size (cell mass diameter) becomes smaller as it goes upward in the culture tank The mass is distributed. Therefore, by analyzing the captured image obtained by imaging the lower portion 2a of the culture tank 2, the maximum size (maximum mass diameter) of the cell mass in the culture tank 2 can be obtained.

In addition, the cell mass in the culture tank 2 starts to be sedimented by reducing or zeroing the supply amount of the culture solution supplied to the culture tank 2 per unit time. For this reason, since the cell mass in the culture tank 2 moves toward the lower part 2a of the culture tank 2, more cells can be collected by repeatedly imaging the lower part 2a of the culture tank 2 after sedimentation starts. Cell clusters 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.

As described above, by analyzing a captured image obtained by imaging the lower portion 2a of the culture tank 2, without imaging the entire culture tank 2, the size of the cell mass (in the present embodiment, the maximum mass diameter) And mass 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 cells without upsizing the apparatus. Moreover, since it is not necessary to move the camera 4 or the culture tank 2, it is possible to reduce the number of components and to reduce the manufacturing cost and the maintenance cost.

Conventionally, in order to measure the number of cells in culture in a culture tank, some cells in the culture tank are taken out and visual measurement using a microscope and a hemocytometer is performed. However, in the process of removing the cells from the culture tank, unintended contaminants may contaminate the culture tank because the cells come in contact with the atmosphere. In addition, since the cells used for measurement can not be cultured again by returning them to the culture tank, the cell culture rate is reduced. On the other hand, in the cell culture apparatus 1, since the largest block diameter and distribution of the cell mass are measured by analyzing the captured image, the culture state of the cells can be obtained without taking out 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 at least the lower part 2a of the culture tank 2. Therefore, 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 type of cells cultured by the cell culture apparatus 1 is not particularly limited. For example, stem cells and the like in animals are cultured. A culture solution to be 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 disposed outside the incubator 10. However, some or all of the camera 4, the image processing device 5, and the control device 6 are incubators. It may be arranged inside of ten. The pump 3, the circulation line L 1, and part or all of the circulation line L 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. Cell mass shape, roundness, aspect ratio, circularity, and area may be measured.

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

The controller 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 whole culture may be adversely affected. Therefore, the control device 6 may control the pump 3 by comparing the maximum mass diameter measured by the image processing device 5 with a predetermined threshold value. Specifically, the control device 6 controls the pump 3 to supply the culture solution to the culture tank 2 at a constant supply amount per unit time when the maximum mass diameter is equal to or less than the threshold value. On the other hand, when the maximum mass diameter is larger than the threshold, 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 repeats increase and decrease with time. . The threshold is, for example, 500 μm.

For example, the control device 6 controls the pump 3 so that the driving time for turning on the pump 3 and the stopping time for turning off the pump 3 are alternately repeated. Then, the control device 6 controls the pump 3 to supply the culture solution to the culture tank 2 at a constant supply amount per unit time when the maximum mass diameter becomes equal to or less than the threshold. Thereby, when the pump 3 is stopped, the amount of supply of the culture solution per unit time decreases, so that the flow rate of the culture solution in the culture tank 2 is reduced, 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 is suspended during normal operation. Then, as the flow rate of the culture solution increases toward the lower side of the culture tank 2, each cell mass is subjected to a shear stress larger than that during normal operation. As a result, the cell mass is disrupted, and the whole cell mass diameter can be maintained at or below the threshold.

The controller 6 may control the pump 3 so that the supply amount of the culture solution supplied to the culture tank 2 is increased per unit time in order to apply a larger shear stress to the cell mass than during normal operation. The pump 3 may be controlled so that the amount of culture solution supplied to the culture tank 2 per unit time changes sinusoidally.

1 cell culture apparatus 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 part L1 Circulation line L2 Circulation line

Claims

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 upflow in the culture vessel;
An imaging device for acquiring a captured image by imaging a lower portion of the culture vessel;
An image processing apparatus for measuring the maximum mass diameter of the cell mass in the culture tank based on the captured image;
A control device that controls the supply of the culture solution by the supply device;
Equipped with
The culture vessel has a shape that increases as the horizontal cross-sectional area of the culture vessel goes upward,
The control device controls the supply device such that the supply amount of the culture fluid supplied to the culture tank repeats increase and decrease per unit time 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 distribution of sizes of cell clusters in the culture tank based on the captured image.