Culture system and culture medium delivery control method

Abstract

To provide a culture system each equipped with a system for controlling a pump to supply a set flow rate when the flow rate of the pump fluctuates due to errors in the size of the material constituting the pump, deterioration of the material, wear, and the like; and to provide a culture medium delivery control method.SOLUTION: A culture medium delivery control method is a culture medium delivery control method executed by a control unit in a culture system for culturing cell aggregates, and includes: a cell suspension detection step of detecting a cell suspension in a culture vessel; a drive state detection step of detecting a drive state of a delivery mechanism that delivers the culture medium by driving based on predetermined drive conditions; and a drive condition determination step of determining a drive condition of the delivery mechanism based on the cell suspension detected by the cell suspension detection step and the drive state detected by the drive state detection step.SELECTED DRAWING: Figure 7

Description

Technical Field

[0001] The present invention relates to a culture system and a culture medium feeding control method.

Background Art

[0002] As a method of cell culture, perfusion culture (continuous culture) can be mentioned. In perfusion culture, a bioreactor and a pump are used to supply and discharge the culture medium for culturing (see, for example, Patent Document 1). The supply and discharge of the culture medium may be performed separately in each process. At this time, it is desirable that the flow rate of the pump be set to an optimal value.

[0003] However, depending on the pump used, there are cases where the liquid is not correctly fed at the set flow rate. For this reason, there is also an automatic analyzer that uses a liquid level sensor to control the pump regarding the correction of the flow rate of the pump (see, for example, Patent Document 2). This automatic analyzer measures the supply time and, when the supply time exceeds a threshold value, controls the rotation speed of the pump so as to fall within the set supply time or issues an alarm.

[0004] In addition, there is known a liquid feeding device that measures the weight change of a container and a liquid and performs feedback control of a pump (see, for example, Patent Document 3).

[0005] Furthermore, there is known an infusion pump that performs feedback control of a pump based on the dropping interval or the number of drops of a drip sensor (see, for example, Patent Document 4).

[0006] Furthermore, there is known a body fluid balance control device that performs feedback control of a pump from a replenishing liquid sensor (see, for example, Patent Document 5).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

[0008] In the above-described culture system, many pumps called positive displacement pumps are used, including plunger pumps, gear pumps, tube pumps, and the like. Positive displacement pumps may not discharge the set flow rate due to the type, viscosity of the liquid to be pumped, size errors of pump members, deterioration and wear of members, etc.

[0009] Also, the above-described automatic analyzer (Patent Document 2) and liquid feeding device (Patent Document 3) converted the flow rate of the fluid from the measured time. When the fluid fluctuates such as pulsation due to the operation of the pump, a difference may occur in the converted flow rate depending on the timing of time measurement. For this reason, it has been difficult to use the flow rate converted from time as a criterion for control judgment.

[0010] Also, the infusion pump (Patent Document 4) and the body fluid balance control device (Patent Document 5) measured the number of droplets flowing discontinuously. Also in this case, those that measure the number of droplets do not always have a stable droplet volume per drop, and the liquid volume cannot always be accurately grasped. Also, depending on the timing of measurement, a difference may occur in the flow rate, and it has been difficult to use the flow rate as a criterion for control judgment.

[0011] The present invention has been made in view of the above points. Its object is to control a pump so that the pump delivers a set flow rate. When there are fluctuations in the flow rate of the pump due to errors in the material sizes of the components of the pump, material deterioration, wear, etc., a culture system and a culture medium delivery control method are provided that include a system for controlling the pump to deliver the set flow rate.

Means for Solving the Problems

[0012] The culture medium delivery control method according to the present invention is characterized in that it is a culture medium delivery control method executed by a control unit in a culture system for culturing cell aggregates, including a cell suspension detection step of detecting a cell suspension in a culture vessel, a drive state detection step of detecting the drive state of a liquid delivery mechanism that delivers a culture medium based on predetermined drive conditions, and a drive condition determination step of determining the drive conditions of the liquid delivery mechanism based on the cell suspension detected in the cell suspension detection step and the drive state detected in the drive state detection step of the liquid delivery mechanism.

Advantages of the Invention

[0013] Even if there is an error between the flow rate when a predetermined drive condition is set for the liquid delivery mechanism and the actual flow rate, by detecting the cell suspension in the culture vessel and the drive state of the liquid delivery mechanism and determining the drive conditions of the liquid delivery mechanism during the operation of the culture system, a system and method that can operate at an accurate flow rate are provided.

Brief Description of the Drawings

[0014]

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[0015] <<<<<<Outline of this embodiment>>>>> <<<<First embodiment>>>> DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment will be described with reference to the drawings. Fig. 1 is a diagram showing an example of a perfusion type culture system 10. Fig. 2 is a block diagram showing various sensors included in a monitoring device 300.

[0016] <<<Main components of the culture system 10>>> The culture system 10 mainly includes: The culture device 100, the path structure 200, the monitoring device 300, the adjustment device 400, and the control device 500. Note that the detailed configuration of the culture system 10 will be described later.

[0017] <<Path Structure 200>> The path structure 200 includes a supply system 220 and a circulation system 230. It has these.

[0018] <Supply System 220> By the supply system 220, the culture medium is introduced into the culture device 100.

[0019] <Circulation System 230> The circulation system 230 mainly includes a circulation pump 210, a circulation pipe 232, and a circulation pipe 238. The culture medium stored in the culture device 100 is led out to the circulation system 230. The culture medium led out to the circulation system 230 is returned (circulated) to the supply system 220.

[0020] <Circulation System 240> The circulation system 240 is constituted by the supply system 220 and the circulation system 230. Note that the oxygen supply module 420 exists between the supply system 220 and the circulation system 230.

[0021] <<Culture Device 100>> The culture device 100 has a culture vessel 110. The culture vessel 110 is a container for storing a cell suspension. The culture vessel 110 functions as a bioreactor for culturing cell aggregates (cells) (hereinafter simply referred to as cell aggregates). Note that the cell suspension is a system in which cell aggregates are dispersed in the culture medium. The cell suspension containing cell aggregates and the culture medium is stored in the culture vessel 110.

[0022] <<Monitoring Device 300>> The monitoring device 300 has detection devices such as various sensors. The detection signals emitted from the detection devices are transmitted to the control device 500.

[0023] As shown in FIG. 2, the monitoring device 300 has a liquid level sensor 318. The liquid level sensor 318 detects the cell suspension stored in the culture vessel 110. Details of the configuration and operation of the liquid level sensor 318 will be described later.

[0024] <<Adjustment Device 400>> The adjustment device 400 supplies the culture medium to the culture device 100.

[0025] <Circulation Pump 210> The circulation pump 210 draws out the culture medium contained in the cell suspension stored in the culture device 100 from the outlet 231 to the circulation pipe 232. Although the cell suspension is stored in the culture device 100 after the start of culturing the cell aggregates, the culture medium contained therein is drawn out to the circulation pipe 232 by the circulation pump 210. The circulation pump 210 can use a tube pump and will be described below as a tube pump.

[0026] A tube pump is a pump that uses a flexible tube and utilizes the restoring force of the tube. The tube used for the tube pump is configured separately from the tube pump detachably and is treated as a consumable. The tube used for the tube pump is a pump tube, which is separate from a normal pipe (such as the pipe constituting the path structure 200 described later) and is connected and communicated with the normal pipe. A part of the pump tube is detachably held in an arc shape by the tube pump.

[0027] The tube pump mainly has a rotating body and a plurality of rollers (not shown). The rotating body can rotate about the center of the arc. The rotating body has a plurality of long supports that extend radially along the radial direction. Each of the supports has a plurality of rollers at different positions of the end farthest from the rotation center (the position in contact with the pump tube arranged in an arc shape). Each of the plurality of rollers can rotate about the end of the support. As the rotating body rotates, each of the plurality of rollers rotates while pressing the pump tube held in an arc shape. The pump tube has a suction port and a discharge port. By the operation of releasing the pump tube from the pressing by the rotation operation of each of the plurality of rollers, a negative pressure is generated at the suction port of the pump tube, and due to the negative pressure, the medium is sucked into the inside of the pump tube from the suction port. The medium sucked into the inside of the pump tube is repeatedly guided to the discharge port and continuously fed by the operation of each of the plurality of rollers pressing the pump tube while rotating.

[0028] One rotation of the rotating body is one cycle of the tube pump. The rotation speed of the rotating body of the tube pump and the flow rate of the medium are in a proportional relationship. The flow rate of the medium can be determined by the rotation speed of the rotating body.

[0029] <<Control device 500>> Based on various detection signals issued from the monitoring device 300, the control device 500 executes various arithmetic processes, data processes, and judgment processes to control various devices such as the circulation pump.

[0030] The control device 500 mainly includes a processor (such as a CPU (Central Processing Unit)), a ROM (Read Only Memory), a RAM (Random Access Memory), an I / F (Interface Device), an auxiliary storage device (such as an HDD (Hard Disk Drive) or an SSD (Solid State Drive)), an input operation device (such as a keyboard, a mouse, a touch panel, etc.). For example, the control device 500 can be a PLC (Programmable Logic Controller), a personal computer, a tablet computer, a portable terminal device, or a device equivalent thereto.

[0031] The control device 500 transmits control signals to various devices such as the circulation pump 210 via an I / F (interface device).

[0032] <<Outlet 231 and classification nozzle 242>> As shown in FIG. 1, the circulation pipe 232 is connected to the classification nozzle 242. The classification nozzle 242 has a cylindrical and elongated shape. The classification nozzle 242 is installed toward the inside of the culture vessel 110. The classification nozzle 242 has an outlet 231 at its end.

[0033] The outlet 231 is located between the bottom surface and the upper end of the culture vessel 110 and is positioned so as to be able to contact the cell suspension stored in the culture vessel 110. The classification nozzle 242 is provided in communication with the circulation pipe 232. The medium contained in the cell suspension stored in the culture vessel 110 is led out to the circulation pipe 232 through the outlet 231 and the classification nozzle 242. The medium led out from the culture vessel 110 flows through the circulation pipe 232. The medium led out to the circulation pipe 232 is circulated or discarded.

[0034] <Function of classification nozzle 242> FIG. 4 is a schematic diagram showing the function of the classification nozzle 242. FIG. 5 is a diagram showing the correspondence between the gravitational sedimentation rate of cell aggregates and the size of cell aggregates. FIG. 6 is a schematic diagram showing the forces acting on the cell aggregate C in the classification nozzle 242. In FIG. 6, for simplicity, the cell aggregate C is shown as being spherical. The classification nozzle 242 extends in the vertical direction (±z direction). Although there is also a medium inside the classification nozzle 242, the medium is omitted in FIG. 6 for clarity. The cross-sectional area of the classification nozzle 242 described later is the cross-sectional area of the inner diameter portion when the classification nozzle extending in the vertical direction (±z direction) is cut horizontally.

[0035] As shown in Fig. 6, a gravitational force mg acts on the cell aggregate C in the -z direction (downward direction (direction of gravity)). Further, a buoyancy b from the culture medium and a resistance force f from the culture medium act on the cell aggregate C in the +z direction (upward direction (opposite direction of gravity)). The buoyancy b is generally determined by the density of the culture medium, the density of the cell aggregate C, etc. Also, the resistance force f is generally determined by the viscosity of the culture medium, the diameter R of the cell aggregate C, the gravitational sedimentation velocity v of the cell aggregate C, etc. From the condition that the cell aggregate C moves at a constant speed (the acceleration of the cell aggregate C is zero), the gravitational sedimentation velocity v of the cell aggregate C can be calculated. As shown in Fig. 5, the gravitational sedimentation velocity v of the cell aggregate C is proportional to the square of the diameter of the cell aggregate C (Stokes' law). If the size of the cell aggregate C increases, the gravitational sedimentation velocity v increases, and if the cell aggregate C becomes smaller, the gravitational sedimentation velocity v becomes smaller.

[0036] <Determination of the diameter of the cell aggregate> The gravitational sedimentation velocity of the cell aggregate can be determined from the density of the cell aggregate, the diameter of the cell aggregate, the density of the culture medium, and the viscosity of the culture medium. Among these, the diameter of the cell aggregate changes according to the growth of the cell aggregate. The cell aggregate can be imaged with an imaging device such as a camera, and the diameter of the cell aggregate can be determined from the imaging result. Also, in preliminary experiments or the like, the correspondence between the elapsed time from the start of culturing the cell aggregate and the diameter of the cell aggregate is determined in advance and stored in a storage device such as the ROM or RAM of the control device 500. By referring to the correspondence, the diameter of the cell aggregate corresponding to the time after the actual start of culturing can be determined. In the present embodiment, it is sufficient to be able to determine the diameter (size) of the cell aggregate at an arbitrary timing during the culturing process.

[0037] Note that the size of the cell aggregate may be anything that indicates the degree of growth of the cell aggregate, and is not limited to the diameter. In addition to the radius, etc., as long as it indicates a roughly range during the growth process, such as the density distribution or the occupied area of the cell aggregate. For example, according to various laws and equations to be referred to, the diameter, etc. can be used appropriately as needed. Hereinafter, when it is not particularly necessary to use the diameter or the like for explanation, for simplicity, simply the size is used.

[0038] <Determination of Flow Velocity in the Classification Nozzle 242> The gravitational sedimentation velocity of cell aggregates can be determined from the density of the cell aggregates, the size of the cell aggregates, the density of the culture medium, and the viscosity of the culture medium. The density of the cell aggregates can be determined in advance if the type of cell aggregates to be cultured is determined. Also, the density and viscosity of the culture medium can be determined in advance if the type of culture medium to be used is determined according to the type of cell aggregates to be cultured.

[0039] In this way, by determining the density of the cell aggregates, the size of the cell aggregates, the density of the culture medium, and the viscosity of the culture medium, the gravitational sedimentation velocity of the cell aggregates can be calculated. The culture medium stored in the culture vessel 110 is discharged into the circulation system 230. If the flow rate of the culture medium discharged from the culture vessel 110 is too high, there is a possibility of sucking cell aggregates into the classification nozzle 242. Therefore, it is preferable to discharge the culture medium at the maximum flow rate at which the cell aggregates can remain in the classification nozzle 242. That is, by setting the flow velocity (liquid feeding velocity) of the culture medium in the classification nozzle 242 to balance with the gravitational sedimentation velocity of the cell aggregates and flowing the culture medium, it is possible to discharge only the culture medium from the culture vessel 110 while keeping the cell aggregates in the classification nozzle 242. For example, the flow velocity (liquid feeding velocity) of the culture medium in the classification nozzle 242 can be calculated from various equations related to fluids. Even when the flow velocity of the culture medium in the classification nozzle 242 is set to balance with the gravitational sedimentation velocity of the cell aggregates, not only the culture medium but also small cell aggregates may be discharged. However, compared with the case of not balancing, the amount of cell aggregates discharged can be sufficiently reduced.

[0040] Also, a camera or the like may be provided outside the classification nozzle 242 to image the inside of the classification nozzle 242 and determine the flow velocity of the culture medium while observing the displacement of the cell aggregates.

[0041] In this way, according to the size of the cell aggregates to be classified, the flow velocity of the culture medium in the classification nozzle 242 can be accurately determined as the maximum flow velocity at which the cell aggregates are not discharged from the culture vessel 110.

[0042] Although the configuration using the classification nozzle 242 has been described, other members or methods may be used as long as they can classify cell aggregates based on the size of the cell aggregates.

[0043] Figure 4 is a conceptual diagram showing the size of cell aggregates and the displacement of cell aggregates in the classification nozzle 242.

[0044] <When the flow rate of the culture medium is low> As shown in FIG. 4a, the flow rate of the culture medium in the classification nozzle 242 is set to a flow rate corresponding to the size of small cell aggregates. Only small cell aggregates are lifted in the classification nozzle 242, and only small cell aggregates can be discharged from the culture vessel 110.

[0045] <When the flow rate of the culture medium is medium> As shown in FIG. 4b, the flow rate of the culture medium in the classification nozzle 242 is set to a flow rate corresponding to the sizes of small cell aggregates and medium-sized cell aggregates. Small cell aggregates and medium-sized cell aggregates are lifted in the classification nozzle 242, and small cell aggregates and medium-sized cell aggregates can be discharged from the culture vessel 110.

[0046] <When the flow rate of the culture medium is high> As shown in FIG. 4c, the flow rate of the culture medium in the classification nozzle 242 is set to a flow rate corresponding to the sizes of small cell aggregates, medium-sized cell aggregates, and large cell aggregates. Small cell aggregates, medium-sized cell aggregates, and large cell aggregates are lifted in the classification nozzle 242, and small cell aggregates, medium-sized cell aggregates, and large cell aggregates can be discharged from the culture vessel 110.

[0047] By using the classification nozzle 242, cell aggregates can be classified by utilizing the difference in gravitational sedimentation rates due to differences in the size of cell aggregates.

[0048] <Liquid level sensor 318 (high-position liquid level sensor 318H and low-position liquid level sensor 318L)> The liquid level sensor 318 detects the presence or absence of the cell suspension stored in the culture vessel 110. The liquid level sensor 318 outputs a detection signal indicating the presence or absence of the cell suspension. When the cell suspension increases or decreases, by turning the detection signal of the liquid level sensor 318 ON / OFF, it is possible to detect that the liquid level has passed the position of the liquid level sensor 318.

[0049] A plurality of liquid level sensors are provided as the liquid level sensor 318 at each of different liquid level positions of the culture vessel 110. Among the plurality of liquid level sensors, only the liquid level sensor 318 at the liquid level where the cell suspension is located outputs a signal indicating the presence of the cell suspension. Among the plurality of liquid level sensors, the liquid level sensor 318 where the cell suspension is not located outputs a signal indicating the absence of the cell suspension.

[0050] For example, as shown in FIG. 3, the liquid level sensor 318 has a high-position liquid level sensor 318H and a low-position liquid level sensor 318L. In FIG. 1, for simplicity, the high-position liquid level sensor 318H and the low-position liquid level sensor 318L are omitted. The high-position liquid level sensor 318H detects whether the cell suspension is at a position higher than the high position H. The low-position liquid level sensor 318L detects whether the cell suspension is at a position lower than the low position L. The low position L is higher than the outlet 231 of the classification nozzle 242. The high position H is higher than the low position L.

[0051] The liquid level sensor 318 is a sensor that detects the presence or absence of a cell suspension. The liquid level sensor 318 is used to detect the stop of supply when supplying the culture medium and the stop of discharge when discharging. The supply of the culture medium is carried out when the high-position liquid level sensor 318H does not detect the presence of the cell suspension, and the supply of the culture medium is stopped when the high-position liquid level sensor 318H detects the cell suspension. The discharge of the culture medium is carried out when the low-position liquid level sensor 318L detects the presence of the cell suspension, and the discharge of the culture medium is stopped when the low-position liquid level sensor 318L detects the absence of the cell suspension. That is, the high-position liquid level sensor 318H detects that the cell suspension has switched from a state where it does not exist to a state where it exists when the liquid level of the cell suspension rises. The low-position liquid level sensor 318L detects that the cell suspension has switched from a state where it exists to a state where it does not exist when the liquid level of the cell suspension drops. Let the volume of the cell suspension when the liquid level of the cell suspension is at the high position H be volume H, and the volume of the cell suspension when the liquid level of the cell suspension is at the low position L be volume L.

[0052] <Change in volume of cell suspension> The specific volume L of the cell suspension when the liquid level of the cell suspension is at the low position L is known in advance, and the specific volume H of the cell suspension when the liquid level of the cell suspension is at the high position H is known in advance. For example, volume L is 350 milliliters and volume H is 500 milliliters. Therefore, when the liquid level of the cell suspension increases from the low position L to the high position H, or when the liquid level of the cell suspension decreases from the high position H to the low position L, the change in the volume of the cell suspension is 150 milliliters (=500 - 350 milliliters).

[0053] <Flow rate by circulation pump 210> As the cell aggregate grows in the culture vessel 110, the cell aggregate becomes heavier and the rate of sedimentation in the cell suspension increases. In order to prevent the grown cell aggregate from being discharged from the culture vessel 110, the flow rate by the circulation pump 210 may be determined. Further, even assuming the case where the grown cell aggregate enters the classification nozzle 242 of the culture vessel 110, the flow rate by the circulation pump 210 may be determined so that the cell aggregate is not discharged from the classification nozzle 242.

[0054] The flow rate of the culture medium according to the size of the cell aggregate can be determined by referring to the corresponding relationship obtained from preliminary experiments or the like. Further, the size of the cell aggregate according to the elapsed time from the start of the culture can also be determined by preliminary experiments or the like. From these relationships, the flow rate of the culture medium can be associated with the elapsed time from the start of the culture and stored in advance in a storage unit such as an auxiliary storage device.

[0055] Based on the flow rate of the culture medium in the classification nozzle 242, in particular, it is preferable to determine the maximum flow rate of the culture medium in the classification nozzle 242 as the flow rate to such an extent that the cell aggregate is not derived from the circulation pipe 232. The flow rate can be obtained from the cross-sectional area and the flow rate of the portion where the culture medium flows, and the flow rate of the circulation pump 210 is determined. That is, the gravitational sedimentation rate is determined based on the size of the cell aggregate, the flow rate of the culture medium in the classification nozzle 242 is determined so that the cell aggregate of that size remains in the classification nozzle 242, and the flow rate of the circulation pump 210 is determined based on the flow rate of the culture medium in the classification nozzle 242.

[0056] <<<Pump Control Process>>> Hereinafter, it is assumed that the culture system 10 is operating steadily, the control device 500 is started and the processes such as initialization are completed, and the following pump control process is being executed steadily.

[0057] <<Pump Control Process 1>> FIG. 7 is a flowchart showing the pump control process 1. The pump control process 1 is called by the processor of the control device 500 and continuously executed.

[0058] The pump control process 1 is a process of updating the discharge amount per rotation of the drive unit of the circulation pump 210. From the determined flow rate and the discharge amount per rotation of the drive unit of the circulation pump 210, the rotational speed of the drive unit of the circulation pump 210 can be determined. That is, if the discharge amount per rotation of the drive unit of the circulation pump 210 is accurate, the rotational speed of the drive unit that accurately discharges the determined flow rate can be determined. The number of rotations of the circulation pump 210 is the number of times the circulation pump 210 rotates (one revolution). Note that it does not have to be one rotation, as long as it is an amount that can clearly define the amount driven by the circulation pump 210.

[0059] <Process of discharging the culture medium from the culture vessel 110> At the beginning of executing this process, the liquid level of the cell suspension stored in the culture vessel 110 is at the high position H. That is, at the beginning of executing this process, the amount of the cell suspension stored in the culture vessel 110 is the volume H.

[0060] First, the processor of the control device 500 sends a drive start signal to the circulation pump 210 to start driving the circulation pump 210 (step S711). The discharge amount per rotation of the circulation pump 210 at this time is set to a predetermined initial value. By driving the circulation pump 210, the culture medium is discharged from the culture vessel 110. As the culture medium is discharged, the liquid level of the cell suspension in the culture vessel 110 gradually decreases.

[0061] Next, the processor of the control device 500 starts counting the number of rotations of the circulation pump 210 (step S713). The number of rotations can be counted using the control signal supplied to the circulation pump 210. For example, when the power for driving the circulation pump 210 is a stepping motor, the number of rotations of the drive unit of the circulation pump 210 can be counted by counting the number of pulses of the pulse signal supplied to the stepping motor.

[0062] In addition, when the drive unit of the circulation pump 210 has a rotary encoder, the number of rotations of the drive unit of the circulation pump 210 may be counted by using the signal output from the rotary encoder without using the control signal supplied to the circulation pump 210. Further, regardless of the rotary encoder, a signal capable of counting the number of rotations of the drive unit of the circulation pump 210 may be used.

[0063] Next, the processor of the control device 500 determines whether the low-level liquid surface sensor 318L has detected the presence or absence of the cell suspension (step S715). When the low-level liquid surface sensor 318L detects the cell suspension, it emits a signal indicating the presence of the cell suspension. The processor of the control device 500 can determine whether the low-level liquid surface sensor 318L has detected the presence or absence of the cell suspension by receiving the signal from the low-level liquid surface sensor 318L.

[0064] When the processor of the control device 500 determines that the low-level liquid surface sensor 318L has detected the cell suspension (YES), the process returns to step S715. As a result, the liquid level of the cell suspension in the culture vessel 110 further decreases.

[0065] When the processor of the control device 500 determines that the low-level liquid surface sensor 318L has detected the absence of the cell suspension (NO), a drive stop signal is sent to the circulation pump 210 to stop the drive of the circulation pump 210 (step S717). As a result, the discharge of the culture medium from the culture vessel 110 ends.

[0066] At this point, the liquid level of the cell suspension stored in the culture vessel 110 is at the low position L. That is, at this point, the amount of the cell suspension stored in the culture vessel 110 is the volume L. By the operation of the circulation pump 210, the amount of the medium of volume H - volume L has been discharged from the culture vessel 110. In other words, from the state of volume H in which the high-position liquid level sensor 318H detects the cell suspension (the processes of steps S731 and 733), the medium is discharged by the circulation pump 210, the low-position liquid level sensor 318L detects the cell suspension, and when the circulation pump 210 stops, it means that the amount of the medium of volume H - volume L has been discharged from the culture vessel 110. The high-position liquid level sensor 318H and the low-position liquid level sensor 318L are provided at fixed positions. As described above, the volumes H and L are known, and the change amount, volume H - volume L, is also known. Therefore, by detecting the cell suspension with the high-position liquid level sensor 318H and the low-position liquid level sensor 318L, the change amount (volume H - volume L) can be immediately utilized.

[0067] Next, the processor of the control device 500 ends the counting of the number of rotations of the drive unit of the circulation pump 210 (step S719). The number of rotations at the time when the counting ends is the number of rotations of the drive unit of the circulation pump 210 while discharging the medium.

[0068] Next, the processor of the control device 500 calculates the discharge amount per rotation of the drive unit of the circulation pump 210 (step S723). The discharge amount per rotation of the drive unit of the circulation pump 210 can be calculated by dividing the amount of the medium discharged from the culture vessel 110 by the number of rotations of the drive unit of the circulation pump 210. The amount of the medium discharged from the culture vessel 110 is the amount of the medium discharged in the processes of steps S711 and S717. Specifically, the amount of the medium discharged from the culture vessel 110 is volume H - volume L. The number of rotations of the drive unit of the circulation pump 210 is the number of rotations obtained in the process of step S719.

[0069] Thus, since the change amount of the cell suspension (volume H - volume L) is the amount of the medium discharged from the culture vessel 110, the discharge amount per rotation of the drive unit of the circulation pump 210 is calculated from the change amount of the cell suspension (volume H - volume L) and the number of rotations of the drive unit of the circulation pump 210 corresponding to the change amount of the cell suspension. The number of rotations of the drive unit of the circulation pump 210 with respect to the change amount of the cell suspension (volume H - volume L) (= the amount of the medium discharged from the culture vessel 110) is preferably plural times.

[0070] By calculating and updating the discharge amount per rotation of the drive unit of the circulation pump 210, the drive unit of the circulation pump 210 can be driven at an accurate rotational speed in the subsequent medium discharge process.

[0071] The processes of steps S711 to S723 described above are executed when discharging the medium in the medium exchange circulation culture process. Specifically, the processes of steps S711 to S723 are executed in the medium discharge steps such as EX1 - 1, EX2 - 1, and EX3 - 1 of the medium exchange circulation culture process shown in FIG. 13 described later.

[0072] <Process of supplying the medium to the culture vessel 110> Next, the processor of the control device 500 sends a drive start signal to the medium supply pump 454 to start driving the medium supply pump 454 (step S729). By driving the medium supply pump 454, the medium is supplied to the culture vessel 110. As the medium is supplied, the liquid level of the cell suspension in the culture vessel 110 gradually rises.

[0073] Next, the processor of the control device 500 determines whether the high-level liquid surface sensor 318H has detected the cell suspension (step S731). Similar to the low-level liquid surface sensor 318L, when the high-level liquid surface sensor 318H detects the cell suspension, it emits a signal indicating the presence of the cell suspension. In the culture vessel 110, the high-level liquid surface sensor 318H is provided at a position higher than the low-level liquid surface sensor 318L. By receiving the signal from the high-level liquid surface sensor 318H, the processor of the control device 500 can determine whether the high-level liquid surface sensor 318H has detected the presence or absence of the cell suspension.

[0074] When the processor of the control device 500 determines that the high-level liquid surface sensor 318H has not detected the cell suspension (NO), the process returns to step S731. As a result, the liquid level of the cell suspension in the culture vessel 110 becomes even higher.

[0075] When the processor of the control device 500 determines that the high-level liquid surface sensor 318H has detected the cell suspension (YES), it sends a drive stop signal to the medium supply pump 454 to stop the drive of the medium supply pump 454 (step S733), and this subroutine ends. As a result, the supply of the medium to the culture vessel 110 ends.

[0076] At this time, the liquid level of the cell suspension stored in the culture vessel 110 is at the high level H. That is, at this time, the amount of the cell suspension stored in the culture vessel 110 is the volume H. By the operation of the medium supply pump 454, an amount of medium of volume H - volume L has been supplied to the culture vessel 110.

[0077] The processes of steps S729 to S733 described above are executed during the supply of the medium in the medium exchange and circulation culture process. Specifically, the processes of steps S729 to S733 are executed in the medium supply steps such as EX1-2, EX2-2, and EX3-2 of the medium exchange and circulation culture process shown in FIG. 13 described later.

[0078] <<Pump Control Process 2>> FIG. 8 is a flowchart showing the pump control process 2. The pump control process 2 is called and executed by the processor of the control device 500.

[0079] The pump control process 2 is a process of updating the discharge amount per rotation of the drive unit of the medium supply pump 454. From the determined flow rate and the discharge amount per rotation of the drive unit of the medium supply pump 454, the rotation speed of the drive unit of the medium supply pump 454 can be determined. That is, if the discharge amount per rotation of the drive unit of the medium supply pump 454 is accurate, the rotation speed of the drive unit that can accurately discharge the determined flow rate can be determined. Similar to the circulation pump 210 in the pump control process 1, the number of rotations of the medium supply pump 454 is the number of times the medium supply pump 454 rotates (one cycle). It does not have to be one rotation, as long as it is an amount that can clearly define the amount driven by the medium supply pump 454.

[0080] The pump control process 1 is a process executed when discharging the medium from the culture vessel 110. In contrast, the pump control process 2 is a process executed when supplying the medium to the culture vessel 110. When the rotation speed of the medium supply pump 454 is high and the flow rate of the medium is fast, the medium cannot be sufficiently heated by the medium heater 456. On the other hand, when the rotation speed of the medium supply pump 454 is low and the flow rate of the medium is slow, the medium will be overheated by the medium heater 456. Therefore, even when supplying the medium from the medium tank 452, it is preferable to appropriately adjust the rotation speed of the medium supply pump 454 to control the flow rate of the medium in the pump tube.

[0081] <Process of discharging the medium from the culture vessel 110> At the beginning of executing this process, the liquid level of the cell suspension stored in the culture vessel 110 is at the high position H. That is, at the beginning of executing this process, the amount of the cell suspension stored in the culture vessel 110 is the volume H.

[0082] First, the processor of the control device 500 sends a drive start signal to the circulation pump 210 to start driving the circulation pump 210 (step S811). At this time, the discharge volume per rotation of the circulation pump 210 is set to a preset initial value. Due to the driving of the circulation pump 210, the culture medium is discharged from the culture vessel 110. As the culture medium is discharged, the liquid level of the cell suspension in the culture vessel 110 gradually decreases.

[0083] Next, the processor of the control device 500 determines whether the low-position liquid level sensor 318L has detected the cell suspension (step S813).

[0084] When the processor of the control device 500 determines that the low-position liquid level sensor 318L has not detected the cell suspension (NO), the process returns to step S813. As a result, the liquid level of the cell suspension in the culture vessel 110 further decreases.

[0085] When the processor of the control device 500 determines that the low-position liquid level sensor 318L has detected the cell suspension (YES), it sends a drive stop signal to the circulation pump 210 to stop driving the circulation pump 210 (step S815). As a result, the discharge of the culture medium from the culture vessel 110 ends.

[0086] At this point, the liquid level of the cell suspension stored in the culture vessel 110 is at the low position L. That is, at this point, the amount of the cell suspension stored in the culture vessel 110 is the volume L. By the operation of the circulation pump 210, an amount of the culture medium equal to the volume H - volume L has been discharged from the culture vessel 110.

[0087] The processes of steps S811 to S815 described above are executed when discharging the culture medium during the culture medium exchange and circulation culture process. Specifically, the processes of steps S811 to S815 are executed in the culture medium discharge steps such as EX1-1, EX2-1, and EX3-1 of the culture medium exchange and circulation culture process shown in FIG. 13 described later.

[0088] <Process of supplying the culture medium to the culture vessel 110> The processor of the control device 500 sends a drive start signal to the medium supply pump 454 to start driving the medium supply pump 454 (step S817). At this time, the discharge amount per rotation of the medium supply pump 454 is set to a preset initial value. By driving the medium supply pump 454, the medium is supplied to the culture vessel 110. As the medium is supplied, the liquid level of the cell suspension in the culture vessel 110 gradually rises.

[0089] Next, the processor of the control device 500 starts counting the number of rotations of the medium supply pump 454 (step S819). The number of rotations can be counted using the control signal supplied to the medium supply pump 454. For example, when the power for driving the medium supply pump 454 is a stepping motor, the number of rotations of the drive unit of the medium supply pump 454 can be counted by counting the number of pulses of the pulse signal supplied to the stepping motor.

[0090] When the drive unit of the medium supply pump 454 has a rotary encoder, the number of rotations of the drive unit of the medium supply pump 454 may be counted using the signal output from the rotary encoder without using the control signal supplied to the medium supply pump 454. Also, regardless of the rotary encoder, a signal capable of counting the number of rotations of the drive unit of the medium supply pump 454 may be used.

[0091] Next, the processor of the control device 500 determines whether the high-position liquid level sensor 318H has detected the presence or absence of the cell suspension (step S821). When the high-position liquid level sensor 318H detects the cell suspension, it emits a signal indicating the presence of the cell suspension. The processor of the control device 500 can determine whether the high-position liquid level sensor 318H has detected the presence or absence of the cell suspension by receiving the signal from the high-position liquid level sensor 318H.

[0092] When the processor of the control device 500 determines that the high-level liquid surface sensor 318H has not detected the cell suspension (NO), the process returns to step S821. As a result, the liquid level of the cell suspension in the culture vessel 110 becomes even higher.

[0093] When the processor of the control device 500 determines that the high-level liquid surface sensor 318H has detected the absence of the cell suspension (YES), it sends a drive stop signal to the medium supply pump 454 to stop the drive of the medium supply pump 454 (step S823). As a result, the supply of the medium to the culture vessel 110 ends.

[0094] Next, the processor of the control device 500 ends the counting of the number of rotations of the drive unit of the medium supply pump 454 (step S825). The number of rotations at the time when the counting ends is the number of rotations of the drive unit of the medium supply pump 454 while discharging the medium.

[0095] At this time, the liquid level of the cell suspension stored in the culture vessel 110 is at the high level H. That is, at this time, the amount of the cell suspension stored in the culture vessel 110 is the volume H. By the operation of the medium supply pump 454, an amount of medium of volume H - volume L is supplied to the culture vessel 110. In other words, in the processes of steps S813 and 815, after the low-level liquid surface sensor 318L detected the cell suspension and the circulation pump 210 stopped, the medium was supplied by the medium supply pump 454, the high-level liquid surface sensor 318H detected the cell suspension, and when the medium supply pump 454 stopped, it means that an amount of medium of volume H - volume L was supplied to the culture vessel 110. As described above, the high-level liquid surface sensor 318H and the low-level liquid surface sensor 318L are provided at fixed positions, the volumes H and L are known, and the amount of the change in volume, volume H - volume L, is also known. Therefore, by detecting the cell suspension with the high-level liquid surface sensor 318H and the low-level liquid surface sensor 318L, the change amount (volume H - volume L) can be immediately utilized.

[0096] Next, the processor of the control device 500 calculates the discharge amount per rotation of the drive unit of the medium supply pump 454 (step S829). The discharge amount per rotation of the drive unit of the medium supply pump 454 can be calculated by dividing the amount of the medium supplied to the culture vessel 110 by the number of rotations of the drive unit of the medium supply pump 454. The amount of the medium supplied to the culture vessel 110 is the amount of the medium discharged in the processes of steps S817 and S823. The number of rotations of the drive unit of the medium supply pump 454 is the number obtained in the process of step S825.

[0097] In this way, since the change amount of the cell suspension (volume H - volume L) is the amount of the medium supplied to the culture vessel 110, the discharge amount per rotation of the drive unit of the medium supply pump 454 is calculated from the change amount of the cell suspension (volume H - volume L) and the number of rotations of the drive unit of the medium supply pump 454 corresponding to the change amount of the cell suspension. The number of rotations of the drive unit of the medium supply pump 454 with respect to the change amount of the cell suspension (volume H - volume L) (= the amount of the medium supplied to the culture vessel 110) is preferably plural.

[0098] By calculating and updating the discharge amount per rotation of the drive unit of the medium supply pump 454, the drive unit of the medium supply pump 454 can be driven at an accurate rotation speed in the medium supply process after the next time.

[0099] The processes of steps S817 to S829 described above are executed when supplying the medium in the medium exchange and circulation culture process. Specifically, the processes of steps S817 to S829 are executed in the medium supply steps such as EX1 - 2, EX2 - 2, and EX3 - 2 of the medium exchange and circulation culture process shown in FIG. 13 described later.

[0100] <<Pump Control Process 3>> Figures 9A and 9B are flowcharts showing the pump control process 3. The pump control process 3 is a process for determining and updating the discharge amount per rotation of the drive unit of the circulation pump 210 and the discharge amount per rotation of the drive unit of the medium supply pump 454. The pump control process 3 is called and executed by the processor of the control device 500. The pump control process 3 is executed during the discharge and supply of the medium in the medium exchange circulation culture process.

[0101] The pump control process 3 executes processes similar to the processes of steps S711 to S723 in FIG. 7 and processes similar to the processes of steps S817 to S829 in FIG. 8.

[0102] The processes of steps S711 to S723 in FIG. 7 are processes for discharging the medium. Specifically, they are executed in the medium discharge steps such as EX1-1, EX2-1, and EX3-1 of the medium exchange circulation culture process shown in FIG. 13 described later. Also, the processes of steps S817 to S829 in FIG. 8 are processes for supplying the medium. Specifically, they are executed in the medium supply steps such as EX1-2, EX2-2, and EX3-2 of the medium exchange circulation culture process shown in FIG. 13 described later.

[0103] Also in the pump control process 3, when the high-position liquid level sensor 318H detects the cell suspension in the state of volume H (processes of steps S731 and 733), the medium is discharged by the circulation pump 210, the low-position liquid level sensor 318L detects the cell suspension, and when the circulation pump 210 stops, it means that the amount of medium of volume H - volume L has been discharged from the culture vessel 110. Also, after the low-position liquid level sensor 318L detects the cell suspension and the circulation pump 210 stops, when the medium is supplied by the medium supply pump 454, the high-position liquid level sensor 318H detects the cell suspension, and when the medium supply pump 454 stops, it means that the amount of medium of volume H - volume L has been supplied to the culture vessel 110. Volume H and volume L are known, and the change amount volume H - volume L is also known. Therefore, by detecting the cell suspension with the high-position liquid level sensor 318H and the low-position liquid level sensor 318L, the change amount (volume H - volume L) can be immediately utilized.

[0104] <<<Results of pump control process>>> FIG. 10 is a first example showing the result of the flow rate when the discharge amount per rotation of the drive unit of the circulation pump 210 is updated by the pump control process 1 of FIG. 7. FIG. 10 is an example of updating the pump drive conditions with the flow rate at the time when the culture elapsed time is T0 as the target flow rate. FIG. 10 shows an example in which the pump control process 1 is carried out between the culture elapsed times T0 and T1, the discharge amount per rotation of the drive unit of the circulation pump 210 is updated at the time point of T1, and after the culture elapsed time is T1, the pump is driven in a state where the discharge amount per rotation of the drive unit of the circulation pump 210 is updated, and the flow rate is measured.

[0105] Between the culture elapsed times T0 and T1, the pump 210 is driven with the discharge amount per rotation of the drive unit of the circulation pump 210 being an initially determined initial value. As the pump tube of the circulation pump 210 constituted by the tube pump deteriorates over time, the flow rate gradually decreases as the culture elapsed time increases. The pump control process 1 is carried out between the culture elapsed times T0 and T1, and the discharge amount per rotation of the drive unit of the circulation pump 210 is determined and updated.

[0106] At the time when the culture elapsed time is T1, the discharge amount per rotation of the drive unit of the circulation pump 210 is updated (drive condition update) by the process of step S723 described above. By this drive condition update, after T1, the flow rate can be made closer to the target flow rate (the flow rate when the culture elapsed time is T0).

[0107] FIG. 11 shows a second example of the result of the flow rate when the drive condition of the circulation pump 210 is updated by the pump control process 1 of FIG. 7. FIG. 11 is an example of setting first to third target flow rates that are different from each other as the culture elapsed time elapses, and updating the discharge amount per rotation of the drive unit of the circulation pump 210 so as to achieve the set target flow rates. As described above, the size of the cell aggregate increases as the culture progresses. The first to third target flow rates are determined according to the size of the cell aggregate.

[0108] <When the culture elapsed time is T0 to T1> When the elapsed culture time is between T0 and T1, the circulation pump 210 is driven with the discharge amount per rotation of the drive unit of the circulation pump 210 being the preset initial value, and at the same time, pump control process 1 is performed. When the elapsed culture time is between T0 and T1, as the elapsed culture time passes, the flow rate gradually decreases. As the pump tube of the circulation pump 210 deteriorates over time, the flow rate gradually decreases as the elapsed culture time approaches T1.

[0109] When the elapsed culture time reaches T1, the discharge amount per rotation of the drive unit of the circulation pump 210 is updated by the process of step S723 described above (first drive condition update). Between the elapsed culture times T1 and T2, the circulation pump 210 is stopped.

[0110] <When the elapsed culture time is between T2 and T3> When the elapsed culture time is between T2 and T3, the discharge amount per rotation of the drive unit of the circulation pump 210 starts to be driven using the value updated at T1. The flow rate at T2 is set as the first target flow rate, which is the same as the flow rate when the elapsed culture time is T0. Since the rotation speed of the drive unit for the first target flow rate is determined based on the updated discharge amount per rotation of the drive unit of the circulation pump 210, the discharge starts at an accurate flow rate. As described above, as the pump tube of the circulation pump 210 deteriorates over time, the flow rate gradually decreases as the elapsed culture time approaches T3. During the period from T2 to T3, pump control process 1 is performed.

[0111] When the elapsed culture time reaches T3, the discharge amount per rotation of the drive unit of the circulation pump 210 is updated by the process of step S723 described above (second drive condition update). Between the elapsed culture times T3 and T4, the circulation pump 210 is stopped.

[0112] <When the elapsed culture time is between T4 and T5> Similarly, when the culture elapsed time is between T4 and T5, the discharge volume per rotation of the drive unit of the circulation pump 210 starts driving using the value updated at T3. The flow rate at T4 is set as the second target flow rate. Since the rotation speed of the drive unit that becomes the second target flow rate is determined based on the updated discharge volume per rotation of the drive unit of the circulation pump 210, the discharge starts at an accurate flow rate. As the pump tube of the circulation pump 210 deteriorates over time, the flow velocity gradually decreases as the culture elapsed time approaches T5. During the period of T4 to T5, the pump control process 1 is performed.

[0113] When the culture elapsed time is T5, the discharge volume per rotation of the drive unit of the circulation pump 210 is updated by the process of step S723 described above (third drive condition update). The circulation pump 210 is stopped during the culture elapsed time between T5 and T6.

[0114] <When the culture elapsed time is T6~> Similarly, when the culture elapsed time is between T6 and, the discharge volume per rotation of the drive unit of the circulation pump 210 starts driving using the value updated at T5. The flow rate at T6 is set as the third target flow rate. Since the rotation speed of the drive unit that becomes the third target flow rate is determined based on the updated discharge volume per rotation of the drive unit of the circulation pump 210, the discharge starts at an accurate flow rate.

[0115] In this way, as the culture elapsed time passes, the rotation speed of the circulation pump 210 is adjusted based on the size of the cell aggregate. By doing so, an environment that is not derived from the circulation pipe 232 can be maintained, and the cell aggregate can be grown.

[0116] <<<Other configurations of the culture system 10>>> FIG. 1 is a diagram showing an example of a perfusion type culture system 10. Hereinafter, mainly, among the configurations of the culture system 10, the configurations that have not been described yet will be described.

[0117] The culture system 10 mainly includes a culture device 100, a path structure 200, The monitoring device 300, the adjustment device 400, the control device 500, and the stirring device 600 are provided.

[0118] <<Path structure 200>> The path structure 200 has a supply system 220 and a circulation system 230. are provided.

[0119] <Supply system 220> The supply system 220 has a supply pipe 222, a supply pipe 224, and a supply pipe 226. The medium is introduced into the culture device 100 by the supply system 220.

[0120] <Circulation system 230> The circulation system 230 has a circulation pump 210, a sampling port, a circulation pipe 232, and a circulation pipe 238. Of the cell suspension stored in the culture device 100, the medium is led out to the circulation system 230. The medium led out to the circulation system 230 is returned (circulated) to the supply system 220.

[0121] <Circulation system 240> The circulation system 240 is constituted by the supply system 220 and the circulation system 230.

[0122] <<Overview of culturing cell aggregates in culture system 10>> In a perfusion-type cell culture system (culture system 10), a culture medium and cell aggregates (cell suspension) are stored in a culture vessel (bioreactor) 110, and the cell aggregates are cultured while the culture medium and cell aggregates are stirred by a stirring device 600. The cell aggregates consume nutrients, oxygen, etc. in the culture medium in the culture vessel 110, metabolize waste products, grow larger and increase in number, and also aggregate to form aggregates. Therefore, when the nutrients and oxygen in the culture medium decrease, the culture medium is added (medium overlay) or part or all of the culture medium is replaced with a new culture medium (medium exchange).

[0123] The supply of a new culture medium to the culture vessel 110 uses a culture medium supply pump 454, a three-way valve 250, and liquid transfer pipes 224, 226, which will be described later. The discharge of the old culture medium from the culture vessel uses a circulation pump 210, a three-way valve 250, liquid transfer pipes 232, 238, etc., which will be described later. Also, the pH and dissolved oxygen concentration of the culture medium contained in the cell suspension in the culture vessel are measured to adjust the pH and control the concentration of dissolved oxygen. Furthermore, glucose is added as necessary.

[0124] <<New Culture Medium and Circulated Culture Medium>> The culture medium includes a new culture medium or a circulated culture medium.

[0125] <New Culture Medium> The new culture medium is a new culture medium stored in a culture medium tank 452, which will be described later. The new culture medium is supplied from the culture medium tank 452 to the culture device 100 via the culture medium supply pump 454 and the three-way valve 250.

[0126] <Circulated Culture Medium> The circulated culture medium is the culture medium that has already been stored in the culture device 100 and has been used for culturing cell aggregates. The circulated culture medium is led out from the culture medium tank 452 to the circulation system 230 and circulated to the supply system 220. The circulated culture medium is returned to the culture medium tank 452 again (circulated) or discarded to a waste liquid tank 260, which will be described later.

[0127] <<Culture Device 100>> The culture device 100 has a culture vessel 110. The culture vessel 110 is a container for storing a cell suspension. The culture vessel 110 functions as a bioreactor for culturing cell aggregates. Inside the culture vessel 110, the cell aggregates divide and grow.

[0128] <Supply pipe 226 and circulation pipe 232> As shown in FIG. 1, a supply pipe 226 and a circulation pipe 232 are arranged in the culture vessel 110.

[0129] <Inlet 227> The supply pipe 226 has an inlet 227. The inlet 227 opens toward the culture vessel 110. The culture medium supplied to the culture vessel 110 flows through the supply pipe 226. Both the fresh culture medium and the circulated culture medium flow through the supply pipe 226. The culture medium that has flowed through the supply pipe 226 is introduced into the culture vessel 110 from the inlet 227. The introduced culture medium is stored in the culture vessel 110.

[0130] When the liquid level of the cell suspension is controlled to be lower than the outlet 231, the culture medium (circulated culture medium) and the cell aggregates are not derived into the circulation pipe 232. On the other hand, when the liquid level of the cell suspension is made higher than the outlet 231, the circulation pump 210 is operated so that cell aggregates having a desired size are not derived into the circulation pipe 232. By controlling in this way, it is possible to make it difficult for cell aggregates to be derived into the circulation pipe 232.

[0131] In addition, when there are cell aggregates near the outlet 231 of the circulation pipe 232, there is a possibility of being derived from the outlet 231. For this reason, a cell aggregate - culture medium separation filter (not shown) described later may be provided. Even when cell aggregates are derived into the circulation pipe 232, it is possible to prevent the cell aggregates from flowing out after the circulation pipe 232.

[0132] <Positions of the inlet 227 and the outlet 231> As shown in FIG. 1, the outlet 231 is arranged at a position lower than the inlet 227.

[0133] <Height of the liquid surface of the cell suspension> When the liquid surface of the cell suspension is higher than the outlet 231, the medium contained in the cell suspension can be discharged from the outlet 231 by driving the circulation pump 210 described later. During the medium replacement circulation culture process described later, the liquid surface of the cell suspension can be made higher than the outlet 231 to circulate the medium.

[0134] On the other hand, when the liquid surface of the cell suspension is lower than the outlet 231, the medium cannot be discharged from the outlet 231. During the intermittent stirring culture process and the medium addition culture process described later, the liquid surface of the cell suspension is made lower than the outlet 231 so that the medium and cell aggregates are not discharged from the outlet 231.

[0135] In this way, the discharge of the medium from the culture vessel 110 can be controlled by driving the circulation pump 210 and the height of the liquid surface of the cell suspension.

[0136] The inlet 227 is located at the upper part of the culture vessel 110. Even when the liquid surface of the cell suspension is at its highest, the inlet 227 does not come into contact with the cell suspension. Thereby, the inlet 227 can be kept clean.

[0137] <<Stirring device 600>> <Configuration of the stirring device 600> The stirring device 600 stirs the cell suspension stored in the culture vessel 110. The stirring device 600 has a magnetic stirrer 610. The magnetic stirrer 610 has a stirring bar 612 and a drive motor 614. The stirring bar 612 rotates when the rotational motion of the drive motor 614 is transmitted.

[0138] <Stirring by the stirring device 600> Due to the rotation of the stirrer 612, the cell suspension stored in the culture vessel 110 is stirred, and accordingly, the medium contained in the cell suspension is stirred. By stirring the medium, the cell aggregates contained in the cell suspension are displaced. When the cell aggregates are displaced, the cell aggregates are more likely to come into contact with each other, and the formation of cell aggregates is promoted by the contact between the cell aggregates. When the cell aggregates are displaced, they are in a state where they are likely to come into contact with the cell aggregates, and the cell aggregates become larger due to the contact with the cell aggregates. Note that the cell aggregates themselves also proliferate by cell division to form cell aggregates.

[0139] <Rotation speed of the drive motor 614> The rotation speed of the drive motor 614 is controlled by the control device 500. By rotating the drive motor 614, the medium can be made to flow.

[0140] The minimum rotation speed of the drive motor 614 (corresponding to the minimum flow rate of the medium) is preferably set such that the cell aggregates do not contact the wall surface of the culture vessel 110. The wall surface of the culture vessel 110 is all the surfaces where the cell aggregates may come into contact. For example, when the culture vessel 110 has a cylindrical or tubular shape, the wall surface is the inner surface of the bottom surface and the side surface. On the other hand, the maximum rotation speed of the drive motor 614 (corresponding to the maximum flow rate of the medium) is preferably set such that the cell aggregates displaced within the culture vessel 110 do not float up and are not discharged from the outlet 231 due to the flow caused by the operation of the circulation pump 210. As the cell aggregates grow, they become heavier and tend to sink, and are more likely to come into contact with the bottom surface of the culture vessel 110. Therefore, it is preferable that the control device 500 controls the rotation speed of the drive motor 614 to increase according to the growth of the cell aggregates (such as the size of the cell aggregates).

[0141] <Rotation direction of the drive motor 614> The drive motor 614 rotates in a certain direction. For example, the drive motor 614 rotates clockwise or counterclockwise in a plan view. Note that the drive motor 614 may be reversed as appropriate. By reversing the rotation direction, the possibility of contact between the cell aggregates can be increased.

[0142] <<Adjustment device 400>> The adjustment device 400 supplies a fresh medium and a recycled medium to the culture device 100. The adjustment device 400 has a recycling system 230 and a supply system 220.

[0143] <Circulation pump 210> The circulation pump 210 draws the medium contained in the cell suspension stored in the culture device 100 from the outlet 231 into the circulation pipe 232. The recycled medium drawn into the circulation pipe 232 is guided by the circulation pump 210 through the circulation pipes 232 - 238 to the oxygen supply module 420. After passing through the oxygen supply module 420, the recycled medium is guided to the supply pump drive unit 410A. By operating the circulation pump 210, the recycled medium is guided as follows: outlet 231 → circulation pipe 232 → circulation pump 210 → circulation pipe 238 → oxygen supply module 420 → ···

[0144] The circulation pump 210 is operated to such an extent that the cell aggregates stored in the culture device 100 are not drawn into the circulation pipe 232. For example, by appropriately reducing the rotation speed of the circulation pump 210, the flow rate of the recycled medium flowing through the circulation pipes 232 - 238 is decreased. By decreasing the flow rate in the circulation pipes 232 - 238, the gravitational sedimentation rate of the cell aggregates is exceeded, and the cell aggregates stored in the culture device 100 can be prevented from being drawn into the circulation pipe 232.

[0145] In this embodiment, the circulation pump 210 is composed of a tube pump. Therefore, even for tube pumps of the same specification, the liquid delivery volume per rotation of the circulation pump 210 may differ from one tube pump to another. Also, when the tube pump is used over a long period, the pump tube deteriorates and the elastic force weakens, and the liquid delivery volume per rotation of the circulation pump 210 may decrease.

[0146] <Sampling port> The circulation system 230 has a sampling port (not shown). At the sampling port, the flowing circulation medium is sampled. The sampled medium is analyzed by various analyzers and the like, and the state of the medium can be obtained.

[0147] <Supply pump drive unit 410A> The supply pump drive unit 410A has an oxygen supply module 420, a pH adjuster supply unit 430, a glucose concentrate supply unit 440, a medium supply unit 450, a circulation heater 460, and supply pipes 222 to 226 (supply system 220).

[0148] <Oxygen supply module 420> The oxygen supply module 420 adds oxygen to the circulating circulation medium. The oxygen supply module 420 is connected to an oxygen cylinder 424 via a pressure regulator (not shown) and a valve 422. The valve 422 opens and closes in response to a control signal from the control device 500. When the valve 422 is in the open state, oxygen is supplied from the oxygen cylinder 424 to the circulation medium through the oxygen supply module 420. When the valve 422 is in the closed state, the supply of oxygen to the circulation medium is blocked.

[0149] <pH adjuster supply unit 430> The pH adjuster supply unit 430 has a pH adjuster tank 432 and a supply pump 434. The pH adjuster is stored in the pH adjuster tank 432. The supply pump 434 operates in response to a control signal from the control device 500. When the supply pump 434 operates, the pH adjuster stored in the pH adjuster tank 432 is led out to the supply pipe 222. The led-out pH adjuster is added to the circulation medium flowing through the supply pipe 222.

[0150] In the present embodiment, the supply pump 434 is composed of a tube pump.

[0151] <Glucose concentrate supply unit 440> The glucose concentrate supply unit 440 includes a glucose concentrate tank 442 and a supply pump 444. The glucose concentrate tank 442 stores the glucose concentrate. The supply pump 444 operates according to a control signal from the control device 500. When the supply pump 444 operates, the glucose concentrate stored in the glucose concentrate tank 442 is led out to the supply pipe 222. The led-out glucose concentrate is added to the circulating medium flowing in the supply pipe 222.

[0152] In the present embodiment, the supply pump 444 is composed of a tube pump.

[0153] <Medium supply unit 450> The medium supply unit 450 includes a medium tank 452, a medium supply pump 454, and a medium heater 456. The medium tank 452 stores a fresh medium. The medium supply pump 454 operates according to a control signal from the control device 500. When the medium supply pump 454 operates, the fresh medium stored in the medium tank 452 is led out to the supply pipe 222 via the medium heater 456.

[0154] In the present embodiment, the medium supply pump 454 is composed of a tube pump. Therefore, even for tube pumps with the same specifications, the liquid feeding amount per rotation of the medium supply pump 454 may be different for each tube pump. Also, when the tube pump is used over a long period, the pump tube may deteriorate and the elastic force may weaken, and the liquid feeding amount per rotation of the medium supply pump 454 may decrease.

[0155] The medium heater 456 warms the fresh medium to a desired temperature. Note that the fresh medium is also warmed by the circulation heater 460 described later. The medium tank 452 stores the fresh medium at a low temperature. Therefore, with only the circulation heater 460, it is likely that the warming will be insufficient for supplying the culture vessel 110. The medium heater 456 preliminarily warms the fresh medium to a certain temperature. The warmed fresh medium is added to the circulating medium flowing in the supply pipe 222.

[0156] <Circulation Heater 460> The circulation heater 460 heats the circulated medium, the fresh medium, the pH adjuster, and the glucose concentrate (hereinafter, for simplicity, without distinguishing between the circulated medium and the fresh medium, simply referred to as the medium. Also, including the pH adjuster and the glucose concentrate, simply referred to as the medium). The circulation heater 460 heats the passing medium to a temperature suitable for supply to the culture vessel 110. Specifically, the circulation heater 460 heats the passing medium to a temperature suitable for culturing the cell aggregates in the culture vessel 110.

[0157] The circulation heater 460 operates in response to the control signal of the control device 500. The circulation heater 460 heats the passing medium according to the control signal.

[0158] <<Three-way Valve 250 and Waste Liquid Tank 260>> The three-way valve 250 controls the flow of the medium that has passed through the circulation heater 460. The three-way valve 250 can be switched to either a medium supply state or a medium disposal state.

[0159] <Medium Supply State> The medium supply state is a state in which the medium is supplied to the culture vessel 110. The medium supply state is a state in which the supply pipe 224 and the supply pipe 226 are in communication. When the three-way valve 250 is in the medium supply state, the medium is supplied to the culture vessel 110 via the supply pipe 224 and the supply pipe 226.

[0160] <Medium Disposal State> The medium disposal state is a state in which the medium is discharged to the waste liquid tank 260. The medium disposal state is a state in which the supply pipe 224 and the waste pipe 262 are in communication. When the three-way valve 250 is in the medium disposal state, the medium is discarded into the waste liquid tank 260 via the supply pipe 224 and the waste pipe 262. The discarded medium is led to the waste liquid tank 260.

[0161] The three-way valve 250 operates according to the control signal of the control device 500. The three-way valve 250 assumes a medium supply state or a medium discard state according to the control signal.

[0162] <<Monitoring device 300>> The monitoring device 300 has detection devices such as various sensors. The detection signal emitted from the detection device is transmitted to the control device 500.

[0163] Figure 2 is a block diagram showing various sensors included in the monitoring device 300. As shown in Figure 2, the monitoring device 300 has a temperature sensor 312, a dissolved oxygen sensor 314, a pH sensor 316, and a liquid level sensor 318.

[0164] <Temperature sensor 312> The temperature sensor 312 detects the temperature of the cell suspension stored in the culture vessel 110. The temperature sensor 312 outputs a detection signal indicating the detected temperature.

[0165] <Dissolved oxygen sensor 314> The dissolved oxygen sensor 314 detects the amount of dissolved oxygen in the cell suspension stored in the culture vessel 110. The dissolved oxygen sensor 314 outputs a detection signal indicating the detected amount of dissolved oxygen.

[0166] <pH sensor 316> The pH sensor 316 detects the pH value of the cell suspension stored in the culture vessel 110. The pH sensor 316 outputs a detection signal indicating the detected pH value.

[0167] <Camera> The monitoring device 300 may have a camera. The camera images the cell aggregates being cultured in the culture vessel 110. The camera outputs a detection signal indicating the captured image. The image to be captured may be a still image or a moving image. Any image that can acquire the state (such as size) of the cell aggregates is acceptable.

[0168] ​ <<Control Device 500>> Based on various detection signals emitted from the monitoring device 300, the control device 500 executes various arithmetic processes, data processes, and judgment processes to control pumps and the like.

[0169] The control device 500 transmits control signals to each of the following via an I / F (interface device): a stirring device 600, a valve 422, a three-way valve 250, a circulation pump 210, a supply pump 434, a supply pump 444, a medium supply pump 454, a medium heater 456, a circulation heater 460, and transmits a control signal to each of them.

[0170] <Control of Magnetic Stirrer 610> The control device 500 transmits a control signal indicating the rotational speed of the drive motor 614 to the stirring device 600.

[0171] The rotational speed is such that within the culture vessel 110, cell aggregates are likely to come into contact with each other, the cell aggregates do not contact the wall portion, and they do not float up to the outlet 231. The wall surface of the culture vessel 110 is all the surfaces where cell aggregates may come into contact. For example, when the culture vessel 110 has a cylindrical shape, the wall surface is the inner surfaces of the bottom surface and the side surfaces. This rotational speed is determined by preliminary experiments and the like and is stored in advance in an auxiliary storage device or the like.

[0172] Also, as the cell aggregates grow, they become heavier and tend to sink, and are likely to contact the bottom surface of the culture vessel 110. This rotational speed is also determined by preliminary experiments and the like, is associated with the elapsed time since the start of the culture, and is stored in advance in an auxiliary storage device or the like. The control device 500 reads out the rotational speed corresponding to the elapsed time since the start of the culture from an auxiliary storage device or the like and transmits it to the magnetic stirrer 610.

[0173] <Control of Valve 422> The control device 500 transmits a control signal indicating the open or closed state of the valve 422 to the valve 422. When the control device 500 transmits a control signal indicating the open state to the valve 422, the valve 422 becomes open. When the valve 422 is open, the oxygen cylinder 424 and the oxygen supply module 420 communicate with each other. Oxygen is supplied to the circulating medium through the oxygen supply module 420.

[0174] When the control device 500 transmits a control signal indicating the closed state to the valve 422, the valve 422 becomes closed. When the valve 422 is closed, the oxygen cylinder 424 and the oxygen supply module 420 no longer communicate with each other, and the supply of oxygen to the circulating medium stops.

[0175] The control device 500 obtains the amount of dissolved oxygen in the cell suspension in the culture vessel 110 from the detection signal output by the dissolved oxygen sensor 314. The control device 500 controls the valve 422 so as to achieve a predetermined target dissolved oxygen concentration. For example, the valve 422 is controlled by PID control (Proportional-Integral-Differential control) based on the obtained amount of dissolved oxygen so as to achieve the target dissolved oxygen concentration. Note that the supply of oxygen is performed in the medium exchange and circulation culture process described later.

[0176] <Control of Three-way Valve 250> The control device 500 transmits a control signal indicating the medium supply state or the medium discard state to the three-way valve 250. When the control device 500 transmits a control signal indicating the medium supply state to the three-way valve 250, the supply pipe 224 and the supply pipe 226 are communicated with each other. By the communication between the supply pipe 224 and the supply pipe 226, the medium is supplied to the culture vessel 110 through the supply pipe 224 and the supply pipe 226.

[0177] When the control device 500 transmits a control signal indicating a culture medium disposal state to the three-way valve 250, the supply pipe 224 and the disposal pipe 262 are communicated with each other. Due to the communication between the supply pipe 224 and the disposal pipe 262, the culture medium is discarded into the waste liquid tank 260 via the supply pipe 224 and the disposal pipe 262.

[0178] <Control of the circulation pump 210> The control device 500 transmits a control signal indicating operation or stop to the circulation pump 210. When the control device 500 transmits a control signal indicating operation to the circulation pump 210, the circulation pump 210 operates. Thereby, the culture medium can be circulated. When the control device 500 transmits a control signal indicating stop to the circulation pump 210, the circulation pump 210 stops. Thereby, the circulation of the culture medium can be stopped.

[0179] The control device 500 can also transmit the information indicating the rotation speed of the circulation pump 210 to the circulation pump 210 included in the control signal. The circulation pump 210 operates at the rotation speed indicated by the control signal. By changing the rotation speed of the circulation pump 210, the flow rate (flow velocity) of the culture medium per unit time can be adjusted. The rotation speed of the circulation pump 210 is determined by preliminary experiments or the like, is associated with the flow rate per unit time, and is stored in advance in an auxiliary storage device or the like.

[0180] When the control device 500 transmits a control signal indicating a culture medium supply state to the three-way valve 250 and a control signal indicating operation to the circulation pump 210, the three-way valve 250 is in a culture medium supply state and the circulation pump 210 operates. By controlling in this way, the culture medium can be circulated in the order of the culture vessel 110 → the outlet 231 → the circulation pipe 232 → the circulation pump 210 → the circulation pipe 238 → the oxygen supply module 420 → the supply pipe 222 → the circulation heater 460 → the supply pipe 224 → the supply pipe 226 → the inlet 227 → the culture vessel 110.

[0181] When the control device 500 sends a control signal indicating the medium disposal state to the three-way valve 250 and a control signal indicating operation to the circulation pump 210, the three-way valve 250 enters the medium disposal state and the circulation pump 210 operates. By controlling in this way, the medium can be discarded in the order of the culture vessel 110 → the outlet 231 → the circulation pipe 232 → the circulation pump 210 → the circulation pipe 238 → the oxygen supply module 420 → the supply pipe 222 → the circulation heater 460 → the supply pipe 224 → the waste pipe 262 → the waste liquid tank 260.

[0182] <Control of the supply pump 434> The control device 500 sends a control signal indicating operation or stop to the supply pump 434. When the control device 500 sends a control signal indicating operation to the supply pump 434, the supply pump 434 operates. By the operation of the supply pump 434, the pH adjuster stored in the pH adjuster tank 432 is led out to the supply pipe 222.

[0183] When the control device 500 sends a control signal indicating stop to the supply pump 434, the supply pump 434 stops. By the stop of the supply pump 434, the leading-out of the pH adjuster to the supply pipe 222 stops.

[0184] The control device 500 can also send the information indicating the rotation speed of the supply pump 434 to the supply pump 434 included in the control signal. The supply pump 434 operates at the rotation speed indicated by the control signal. By changing the rotation speed of the supply pump 434, the flow rate (flow velocity) of the pH adjuster per unit time can be adjusted. The rotation speed of the supply pump 434 is determined by preliminary experiments etc., is associated with the flow rate per unit time, and is stored in advance in an auxiliary storage device etc.

[0185] <Control of the supply pump 444> The control device 500 transmits a control signal indicating operation or stop to the supply pump 444. When the control device 500 transmits a control signal indicating operation to the supply pump 444, the supply pump 444 operates. By the operation of the supply pump 444, the glucose concentrate stored in the glucose concentrate tank 442 is led out to the supply pipe 222.

[0186] When the control device 500 transmits a control signal indicating stop to the supply pump 444, the supply pump 444 stops. By the stop of the supply pump 444, the leading-out of the glucose concentrate to the supply pipe 222 stops.

[0187] The control device 500 can also transmit to the supply pump 444 a control signal including information indicating the rotation speed of the supply pump 444. The supply pump 444 operates at the rotation speed indicated by the control signal. By changing the rotation speed of the supply pump 444, the flow rate (flow velocity) of the glucose concentrate per unit time can be adjusted. The rotation speed of the supply pump 444 is determined by preliminary experiments or the like, is associated with the flow rate per unit time, and is stored in advance in an auxiliary storage device or the like.

[0188] <Control of the medium supply pump 454> The control device 500 transmits a control signal indicating operation or stop to the medium supply pump 454. When the control device 500 transmits a control signal indicating operation to the medium supply pump 454, the medium supply pump 454 operates. By the operation of the medium supply pump 454, the fresh medium stored in the medium tank 452 is led out to the supply pipe 222.

[0189] When the control device 500 transmits a control signal indicating stop to the medium supply pump 454, the medium supply pump 454 stops. By the stop of the medium supply pump 454, the leading-out of the fresh medium to the supply pipe 222 stops.

[0190] The control device 500 can also transmit information indicating the rotation speed of the medium supply pump 454 to the medium supply pump 454 included in the control signal. The medium supply pump 454 operates at the rotation speed indicated by the control signal. By changing the rotation speed of the medium supply pump 454, the flow rate (flow velocity) of the fresh medium per unit time can be adjusted. The rotation speed of the medium supply pump 454 is determined by a preliminary experiment or the like, is associated with the flow rate per unit time, and is stored in advance in an auxiliary storage device or the like.

[0191] <Control of the medium heater 456> The control device 500 transmits a control signal indicating operation or stop to the medium heater 456. When the control unit transmits a control signal indicating operation to the medium heater 456, the medium heater 456 operates. When the medium heater 456 operates, the fresh medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0192] When the control device 500 transmits a control signal indicating stop to the medium heater 456, the medium heater 456 stops.

[0193] The control device 500 can also transmit information indicating the current value of the medium heater 456 to the medium heater 456 included in the control signal. The medium heater 456 operates at the current value indicated by the control signal. By changing the current value of the medium heater 456, the temperature for heating the fresh medium can be adjusted. The current value of the medium heater 456 is determined by a preliminary experiment or the like, is associated with the temperature, and is stored in advance in an auxiliary storage device or the like.

[0194] <Control of the circulation heater 460> The control device 500 transmits a control signal indicating operation or stop to the circulation heater 460. When the control unit transmits a control signal indicating operation to the circulation heater 460, the circulation heater 460 operates. When the circulation heater 460 operates, the medium passing through the circulation heater 460 is heated by the circulation heater 460.

[0195] When the control device 500 sends a control signal indicating stop to the circulation heater 460, the circulation heater 460 stops.

[0196] The control device 500 can also send information indicating the current value of the circulation heater 460 to the circulation heater 460 included in the control signal. The circulation heater 460 operates at the current value indicated by the control signal. By changing the current value of the circulation heater 460, the temperature of the culture medium passing through the circulation heater 460 can be adjusted. The current value of the circulation heater 460 is determined by preliminary experiments or the like, is associated with the temperature, and is stored in advance in an auxiliary storage device or the like.

[0197] <<<Processing of the culture system 10>>> FIG. 13 is a time chart showing an overview of the processing of the culture system 10.

[0198] The processing of the culture system 10 seeding process and intermittent stirring culture process and culture medium addition culture process and culture medium exchange circulation culture process and has.

[0199] <<Seeding process>> The seeding process is a process of preparing a cell suspension in the culture vessel 110. By the seeding process, a cell suspension in which cell aggregates are dispersed in the culture medium can be generated. The seeding process may be a process by the control device 500 or manual work by an operator. In order to make it easier for cell aggregates to come into contact with each other, the liquid level of the cell suspension in the culture vessel 110 may be prepared to be lower than the outlet 231.

[0200] The intermittent stirring culture process, the culture medium addition culture process, and the culture medium exchange circulation culture process will be described according to the time chart shown in FIG. 13.

[0201] <<Intermittent stirring culture process>> The intermittent stirring culture process is a cell aggregation process in the initial stage. Cell aggregates are generated by the intermittent stirring culture process. The intermittent stirring culture process performs stirring and stopping at least once. The intermittent stirring culture process preferably repeats stirring and stopping multiple times. By stirring the cell suspension, the cell aggregates are brought into a state where they easily come into contact with each other, promoting aggregation. By stopping the stirring, the state where the cell aggregates are in contact with each other is maintained, and stable cell aggregates are generated.

[0202] In the example shown in FIG. 13, the intermittent stirring culture process has process numbers IN1 to IN4, ···. In the example shown in FIG. 13, stirring and stopping are repeated multiple times. In the intermittent stirring culture process, the amount of the cell suspension in the culture vessel 110 is constant.

[0203] <Control of Supply Pump 434, Supply Pump 444, Valve 422, Medium Heater 456, and Circulation Heater 460> In the intermittent stirring culture process, the control device 500 transmits a control signal indicating stop to the supply pump 434 and the supply pump 444. Thereby, in the intermittent stirring culture process, the pH adjuster and the glucose concentrate are not supplied to the medium.

[0204] Also, in the intermittent stirring culture process, the control device 500 transmits a control signal indicating a closed state to the valve 422. Thereby, in the intermittent stirring culture process, oxygen is not supplied to the medium.

[0205] In the intermittent stirring culture process, the control device 500 transmits a control signal indicating the medium supply state to the three-way valve 250. Thereby, in the intermittent stirring culture process, the three-way valve 250 is in the medium supply state.

[0206] In the intermittent stirring culture process, the control device 500 transmits a control signal indicating stop to the medium heater 456 and the circulation heater 460. Thereby, in the intermittent stirring culture process, the medium heater 456 and the circulation heater 460 are in a non-operating and stopped state.

[0207] <Stirring Control (Process Numbers IN1, IN3, ···)> The control device 500 operates the drive motor 614 of the magnetic stirrer 610 to rotate the stir bar 612. As a result, the stir bar 612 rotates and the stirring of the cell suspension is started.

[0208] <Stop control (processing numbers IN2, IN4, ···)> After the stirring time has elapsed since the start of stirring, the control device 500 sends a stop control signal to the magnetic stirrer 610. As a result, the drive motor 614 stops and the stir bar 612 comes to rest. Due to the stoppage of the stir bar 612, the cell suspension gradually decelerates and stops.

[0209] <Repeated stirring and stopping after the second time> The stirring after the second time (processing numbers IN3 etc. in FIG. 13) starts after the stop time has elapsed since the stirring was stopped. Stirring is started by executing the stirring control.

[0210] <Main features of the intermittent stirring culture process> In the intermittent stirring culture process, stirring and stopping are performed at least once. It is preferable that stirring and stopping are repeatedly performed a plurality of times. By repeating stirring and stopping, stable cell aggregates can be gradually grown.

[0211] <Stirring time and stop time> The stirring time during which the drive motor 614 is rotating and the stop time during which the drive motor 614 is stopped are also based on the type of cell aggregates and the desired size of the cell aggregates. The stirring time and the stop time are determined by preliminary experiments etc. and are stored in advance in an auxiliary storage device etc. The control device 500 reads out the stirring time and the stop time and determines whether the stirring time or the stop time has been reached.

[0212] Based on the image captured by the camera, the size and number of the cell aggregates may be obtained, and the stirring time, the stop time, and whether to repeat may be determined. By performing various image processes on the captured image, it is preferable to convert it into an image in which the size and number of the cell aggregates are easily obtained.

[0213] <<Medium addition culture process and medium addition preparation process>> The medium addition culture process is a cell aggregation process in the intermediate stage. By the medium addition culture process, cell aggregates are grown by sequentially adding the medium.

[0214] By sequentially adding the medium, the cell aggregates are split to increase the number of cell aggregates and enlarge the cell aggregates. When cell aggregates not contained in the cell aggregates split, they come into contact with the new cell aggregates to enlarge the cell aggregates. When the cell aggregates contained in the cell aggregates split, the contained cell aggregates are enlarged.

[0215] At the start of the medium addition culture process, the cell aggregates are still small. For this reason, when the medium is circulated, the cell aggregates may enter the circulation pipe 232 or the like together with the medium, and the medium is not circulated in the medium addition culture process. By the medium addition culture process, the cell aggregates are grown to a size such that the cell aggregates do not enter the circulation pipe 232.

[0216] In the example shown in FIG. 13, the medium addition culture process has process numbers AD1 to AD2,.... As shown in FIG. 13, before the medium addition culture process, a medium addition preparation process (process number PA1) is executed. The medium addition preparation process is a medium priming process.

[0217] Note that in the medium addition preparation process and the medium addition culture process, following the intermittent stirring culture process, the pH adjuster, the glucose concentrate, and oxygen are not supplied to the medium.

[0218] <Medium addition preparation process (process number PA1)> The control device 500 transmits a control signal indicating operation to the medium heater 456 and the circulation heater 460. Thereby, before adding the medium, the medium heater 456 and the circulation heater 460 are operated. When adding the medium, the medium can be accurately heated.

[0219] The control device 500 operates the drive motor 614 of the magnetic stirrer 610 to rotate the stir bar 612. As a result, the stir bar 612 rotates at a predetermined rotational speed, and stirring is started again.

[0220] The control device 500 transmits a control signal indicating the medium discard state to the three-way valve 250. As a result, the three-way valve 250 connects the supply pipe 224 and the discard pipe 262.

[0221] The control device 500 transmits a control signal indicating operation to the medium supply pump 454. As a result, the fresh medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0222] The fresh medium led out to the supply pipe 222 passes through the circulation heater 460, flows through the supply pipe 224, and reaches the three-way valve 250. The fresh medium that reaches the three-way valve 250 is guided toward the discard pipe 262 and discarded into the waste liquid tank 260. As a result, the supply pipe to which the circulation heater 460 is attached, the supply pipe 224, and the three-way valve 250 can be washed (medium priming) with the fresh medium.

[0223] The medium priming is performed as follows. The fresh medium stored in the medium tank 452 is made to flow into the supply pipe 222 using the medium supply pump 454. The flowing medium reaches the waste liquid tank 260 through the discard pipe 262 via the supply paths of the medium heater 456 and the circulation heater 460 and the three-way valve 250. Washing these pipes and the like with the flowing medium is referred to as medium priming.

[0224] After a predetermined time has elapsed, the control device 500 transmits a control signal indicating stop to the medium supply pump 454. As a result, the lead-out of the fresh medium to the supply pipe 222 stops, and the medium priming ends.

[0225] <Medium additional culture process> After the medium addition preparation process (PA1), the medium addition culture process is executed. The medium addition culture process is the process numbers AD1 to AD2, ··· shown in FIG. 13.

[0226] <Medium addition process (process number AD1)> The control device 500 transmits a control signal indicating the medium supply state to the three-way valve 250. As a result, the three-way valve 250 enters the medium supply state. Note that the medium heater 456 and the circulation heater 460 are already operating.

[0227] The control device 500 transmits a control signal indicating operation to the medium supply pump 454. As a result, the fresh medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0228] The fresh medium led out to the supply pipe 222 is heated by the circulation heater 460, flows through the supply pipe 224, and reaches the three-way valve 250. The fresh medium that has reached the three-way valve 250 is guided toward the supply pipe 226 and introduced into the culture vessel 110 from the introduction port 227. In this way, fresh medium is added to the culture vessel 110.

[0229] After a predetermined time has elapsed, the control device 500 transmits a control signal indicating stop to the medium supply pump 454. As a result, the first addition of fresh medium to the culture vessel 110 is completed.

[0230] The control device 500 also transmits a control signal indicating stop to the medium heater 456 and the circulation heater 460. As a result, the medium supply pump 454, the medium heater 456, and the circulation heater 460 stop.

[0231] <Medium addition process (process number AD2)> The medium addition process (process number AD2) is a process of adding medium again after a predetermined time has elapsed after the medium has been added by the above-described medium addition process (process number AD1).

[0232] After a predetermined time has elapsed since the addition of the new medium to the culture vessel 110 is completed, the control device 500 transmits a control signal indicating activation to the medium heater 456 and the circulation heater 460. Thereby, the medium heater 456 and the circulation heater 460 are activated before adding the next medium. When adding the medium, the medium can be accurately heated.

[0233] Note that the three-way valve 250 is already in a state of discarding the medium, and the supply pipe 224 and the waste pipe 262 are in communication.

[0234] The control device 500 transmits a control signal indicating activation to the medium supply pump 454. Thereby, the new medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0235] The new medium led out to the supply pipe 222 is heated by the circulation heater 460, flows through the supply pipe 224, passes through the three-way valve 250, and is introduced into the culture vessel 110 from the inlet 227 of the supply pipe 226. In this way, the new medium is added to the culture vessel 110 again.

[0236] After a predetermined time has elapsed, the control device 500 transmits a control signal indicating stop to the medium supply pump 454. Thereby, the second addition of the new medium to the culture vessel 110 is completed.

[0237] <Medium addition process (after process number AD2)> In the same manner as process numbers AD1 and AD2, the addition of the medium can be repeated a plurality of times.

[0238] <Position of the liquid level and end of the medium addition and culture process> Each time the new medium is added to the culture vessel 110, the liquid level of the cell suspension stored in the culture vessel 110 gradually rises. When the liquid level of the cell suspension becomes higher than the outlet 231 due to the addition of the medium, the medium addition and culture process is terminated.

[0239] By setting the liquid level of the cell suspension higher than the outlet 231, the medium can be led out from the outlet 231 to the circulation pipe 232.

[0240] <Features of the medium addition culture process> In the medium addition culture process, by adding the medium, the cell aggregates are further grown and divided to enlarge the cell aggregates. In the medium addition culture process, the medium is added at least once. Preferably, the medium is added multiple times in the medium addition culture process.

[0241] Based on the image captured by the camera, the size and number of the cell aggregates can be obtained, and the amount of the medium to be added and the number of additions can be determined. By performing various image processes on the captured image, it is preferable to convert it into an image in which the size and number of the cell aggregates can be easily obtained.

[0242] <<Medium exchange circulation culture process and medium circulation preparation process>> The medium exchange circulation culture process is the cell aggregation process in the final stage. By the medium exchange circulation culture process, the cell aggregates are grown (enlarged) to the target final stage. By sequentially exchanging the medium, a new medium is supplied, and the cell aggregates are further divided to increase the cell aggregates and enlarge the cell aggregates. Furthermore, while circulating the medium and adding a pH adjuster, etc., not only the medium but also the nutrients necessary for the growth of the cell aggregates are supplied, and the cell aggregates are further divided to increase the cell aggregates and enlarge the cell aggregates.

[0243] At the final stage of the medium addition culture process, the cell aggregates have grown large enough not to enter the circulation pipe 232, etc. Therefore, in the medium exchange circulation culture process, nutrients insufficient for the supply of the medium are added by circulating the medium, such as a pH adjuster, a glucose concentrate, and the amount of dissolved oxygen. Finally, cell aggregates of a desired size can be obtained.

[0244] In the example shown in FIG. 13, the medium exchange circulation culture process has process numbers EX1-1 to EX1-3, EX2-1 to EX2-3, EX3-1 to EX3-3, and so on. In the medium exchange circulation culture process, the discharge, supply, and circulation of the medium are repeated multiple times.

[0245] As shown in FIG. 13, before the medium exchange circulation culture process, a medium circulation preparation process (process numbers PE1 to PE3) is executed. The medium circulation preparation process is a circulation priming process.

[0246] <Medium Circulation Preparation Process (Process Numbers PE1 to PE3)> The medium heater 456 and the circulation heater 460 are already operating. Also, in the medium circulation preparation process, following the medium additional culture process, the pH adjuster, glucose concentrate, and oxygen are not supplied to the medium.

[0247] <Circulation Priming (Process Number PE1)> The control device 500 transmits a control signal indicating the medium waste state to the three-way valve 250. As a result, the three-way valve 250 enters the medium waste state.

[0248] The control device 500 transmits a control signal indicating operation to the circulation pump 210. As a result, the circulation pump 210 operates. Due to the operation of the circulation pump 210, the medium stored in the culture vessel 110 is led out to the circulation pipe 232 through the outlet 231. The medium led out to the circulation pipe 232 is discarded into the waste liquid tank 260 in the order of culture vessel 110 → outlet 231 → circulation pipe 232 → circulation pump 210 → circulation pipe 238 → oxygen supply module 420 → supply pipe 222 → circulation heater 460 → supply pipe 224 → waste pipe 262.

[0249] When the control device 500 determines from the detection signal output from the low-level liquid surface sensor 318L that the liquid surface of the cell suspension has become lower than the outlet 231, it transmits a control signal indicating stop to the circulation pump 210. As a result, the circulation pump 210 stops.

[0250] This completes the circulation priming. Through the circulation priming, the circulation pipes 232-238, the circulation pump 210, the oxygen supply module 420, the supply pipes 222-224, the circulation heater 460, and the three-way valve 250 can be washed with the medium (circulation medium) contained in the cell suspension accommodated in the culture vessel 110.

[0251] <Addition (Process number PE2)> The control device 500 transmits a control signal indicating the medium supply state to the three-way valve 250. As a result, the three-way valve 250 enters the medium supply state.

[0252] The control device 500 transmits a control signal indicating stop to the circulation heater 460. As a result, the circulation heater 460 stops. Note that the medium heater 456 is already operating.

[0253] The control device 500 transmits a control signal indicating operation to the medium supply pump 454. As a result, the fresh medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0254] The fresh medium led out to the supply pipe 222 is heated by the circulation heater 460, flows through the supply pipe 224, and reaches the three-way valve 250. The fresh medium that reaches the three-way valve 250 is guided toward the supply pipe 226 and introduced into the culture vessel 110 from the introduction port 227. In this way, fresh medium is added to the culture vessel 110.

[0255] When the liquid level of the cell suspension becomes higher than the outlet 231 based on the detection signal output from the high-position liquid level sensor 318H, the control device 500 transmits a control signal indicating stop to the medium supply pump 454. As a result, the medium supply pump 454 stops.

[0256] The control device 500 transmits a control signal indicating stop to the medium heater 456. As a result, the medium heater 456 stops.

[0257] In this way, a new medium can be added to the culture vessel 110.

[0258] <Circulation 1 (Processing number PE3)> The control device 500 transmits a control signal indicating operation to the circulation heater 460. As a result, the circulation heater 460 operates. Thus, the circulation heater 460 operates before circulating the medium. When circulating the medium, the medium can be accurately heated. Note that the medium heater 456 is stopped.

[0259] The control device 500 transmits a control signal indicating the open state of the valve 422 to the valve 422. By opening the valve 422, oxygen is supplied from the oxygen supply module 420 to the circulating medium.

[0260] The control device 500 transmits a control signal indicating operation to the supply pump 434. By operating the supply pump 434, the pH adjuster stored in the pH adjuster tank 432 is supplied to the circulating medium.

[0261] The control device 500 transmits a control signal indicating operation to the supply pump 444. By operating the supply pump 444, the glucose concentrate stored in the glucose concentrate tank 442 is supplied to the circulating medium.

[0262] The control device 500 transmits a control signal indicating operation to the circulation pump 210. As a result, the circulation pump 210 operates. Note that in the above-mentioned addition (processing number PE2), the three-way valve 250 is in the medium supply state.

[0263] Due to the operation of the circulation pump 210, the medium contained in the cell suspension stored in the culture vessel 110 is led out to the circulation pipe 232 through the outlet 231. The medium (circulating medium) led out to the circulation pipe 232 circulates as follows: culture vessel 110 → outlet 231 → circulation pipe 232 → circulation pump 210 → circulation pipe 238 → oxygen supply module 420 → supply pipe 222 → circulation heater 460 → supply pipe 224 → supply pipe 226 → inlet 227 → culture vessel 110.

[0264] In subsequent processing, the supply of oxygen to the circulating medium, the supply of the pH adjuster, and the supply of the glucose concentrate are continued.

[0265] Based on the detection signal output from the low-level liquid surface sensor 318L, when the liquid surface of the cell suspension becomes lower than the outlet 231, the control device 500 transmits a control signal indicating stop to the circulation pump 210. As a result, the circulation pump 210 stops.

[0266] <Medium Exchange Circulation Culture Process> After the above-described medium circulation preparation process (processing numbers PE1 to PE3), the medium exchange circulation culture process is executed.

[0267] <Discharge (Processing Number EX1-1)> Before discharging the medium, the control device 500 transmits a control signal indicating operation to the medium heater 456. The medium heater 456 operates. As a result, before circulating the medium, the medium heater 456 operates. When circulating the medium, the medium can be accurately heated. Note that the circulation heater 460 is already operating. Note that the medium heater 456 is stopped.

[0268] Next, the control device 500 transmits a control signal indicating the medium waste state to the three-way valve 250. As a result, the three-way valve 250 is in the medium waste state.

[0269] Next, the control device 500 transmits a control signal indicating operation to the circulation pump 210. As a result, the circulation pump 210 operates. Due to the operation of the circulation pump 210, the medium contained in the cell suspension stored in the culture vessel 110 is led out to the circulation pipe 232 through the outlet 231. The medium led out to the circulation pipe 232 is discarded into the waste liquid tank 260 via the culture vessel 110 → outlet 231 → circulation pipe 232 → circulation pump 210 → circulation pipe 238 → oxygen supply module 420 → supply pipe 222 → circulation heater 460 → supply pipe 224 → waste pipe 262. That is, the medium used for the growth of cell aggregates is discarded.

[0270] Next, when the liquid level of the cell suspension becomes lower than the outlet 231 from the detection signal output from the low-level liquid level sensor 318L, the control device 500 transmits a control signal indicating stop to the circulation pump 210. As a result, the circulation pump 210 stops.

[0271] <Supply (processing number EX1)> The control device 500 transmits a control signal indicating the medium supply state to the three-way valve 250. As a result, the three-way valve 250 enters the medium supply state.

[0272] The control device 500 transmits a control signal indicating operation to the medium heater 456. As a result, the medium heater 456 operates. Note that the circulation heater 460 is already operating.

[0273] Next, the control device 500 transmits a control signal indicating operation to the medium supply pump 454. As a result, the fresh medium stored in the medium tank 452 is heated by the medium heater 456 and led out to the supply pipe 222.

[0274] The fresh medium led out to the supply pipe 222 passes through the circulation heater 460, flows through the supply pipe 224, and reaches the three-way valve 250. The fresh medium that reaches the three-way valve 250 is guided toward the supply pipe 226 and introduced into the culture vessel 110.

[0275] When the liquid level of the cell suspension becomes higher than the outlet 231 from the detection signal output from the high-level liquid level sensor 318H, the control device 500 stops the medium supply pump 454 and the medium heater 456. In this way, the fresh medium can be supplied to the culture vessel 110.

[0276] <Circulation 2 (processing number EX1-3)> The control device 500 transmits a control signal indicating operation to the circulation pump 210. As a result, the circulation pump 210 operates. Note that in the above-described addition (processing number PE2), the three-way valve 250 is in the medium supply state. Due to the operation of the circulation pump 210, the medium contained in the cell suspension stored in the culture vessel 110 is led out to the circulation pipe 232 through the outlet 231. The medium led out to the circulation pipe 232 circulates as follows: culture vessel 110 → outlet 231 → circulation pipe 232 → circulation pump 210 → circulation pipe 238 → oxygen supply module 420 → supply pipe 222 → circulation heater 460 → supply pipe 224 → supply pipe 226 → inlet 227 → culture vessel 110.

[0277] <Discharge (processing number EX1-1), supply (processing number EX1), processes after circulation 2 (processing number EX1-3)> In the same manner as discharge (processing number EX1-1), supply (processing number EX1), and circulation 2 (processing number EX1-3), the processes of discharge, supply, and circulation are repeated multiple times (EX2-1 to EX2-3, EX3-1 to EX3-3, ···).

[0278] By repeating the processes of discharge, supply, and circulation multiple times, cell aggregates can be grown (enlarged) to the final stage for the desired purpose.

[0279] <Repeated discharge of the medium, supply of the medium, and circulation of the medium> Repeat the discharge of the medium, supply of the medium, and circulation of the medium. The number of repetitions is based on the size and number of cell aggregates finally desired, etc. The number of repetitions can be determined through preliminary experiments, etc.

[0280] Note that the cell aggregates may be imaged with a camera, and the size of the cell aggregates may be determined from the imaging results.

[0281] The control device 500 reads the rotation speed of the circulation pump 210 according to the elapsed time since the start of cultivation and controls the circulation pump 210. According to the growth of cell aggregates (such as the size of cell aggregates), a drive motor (not shown) is rotated. The state in which the cell aggregates are suspended can be maintained and they can be grown accurately.

[0282] The pH adjuster, glucose concentrate, and dissolved oxygen amount can be determined according to the type of cell aggregates and the desired size of the aggregates, etc. The pH adjuster, glucose concentrate, and dissolved oxygen amount can also be determined by preliminary experiments, etc.

[0283] Data captured by the camera may be used for image analysis, etc. to obtain the size and number of cell aggregates, and to determine whether to end. By performing various image processes on the captured image, it is preferable to convert it into an image in which the size and number of cell aggregates are easily obtained.

[0284] <<<<Second Embodiment>>>> In the above-described first embodiment, an example was shown in which the rotation speeds of the circulation pump 210 and the medium supply pump 454 were updated to adjust the flow rate in the discharge and supply of the medium in the medium exchange circulation culture process shown in FIG. 13. It is not limited to this, and the rotation speeds of the circulation pump 210 and the medium supply pump 454 may be updated in other processes of culturing cell aggregates.

[0285] When layering (adding) the medium at the initial stage of culturing cell aggregates, for example, also at the start of the medium addition culture process shown in FIG. 13, the rotation speed of the medium supply pump 454 may be updated to adjust the flow rate of the medium. At the time of this initial layering of the culture, the rotation speed of the medium supply pump 454 can be determined from the specifications of the medium supply pump 454 and the amount to be layered. For this reason, the rotation speed of the motor of the medium supply pump 454 is not optimized. Therefore, there is a possibility that the desired amount of the medium cannot be layered when layering. When starting the layering, optimize the rotation speed of the medium supply pump 454 to enable layering of the desired amount.

[0286] As shown in FIG. 12, a minimum position liquid level sensor 318LL that detects an amount less than that of the low position liquid level sensor 318L is added to the monitoring device 300. Similar to the low position liquid level sensor 318L and the high position liquid level sensor 318H, when the liquid level of the cell suspension is at the minimum position LL, the minimum position liquid level sensor 318LL outputs a signal indicating the presence of the cell suspension. Similarly, when the liquid level of the cell suspension is not at the minimum position LL, the minimum position liquid level sensor 318LL outputs a signal indicating the absence of the cell suspension. The specific volume LL of the cell suspension when the liquid level of the cell suspension is at the minimum position LL is known. For example, the volume LL is 200 milliliters.

[0287] The culture of cell aggregates starts from a state where cell aggregates are placed in a small amount of medium. The position of the liquid level of the cell suspension in this state is the initial position L0, and the specific volume L0 of the cell suspension is also known. For example, the volume L0 is 150 milliliters. After that, layering is started and layering is performed until the liquid level of the cell suspension reaches the minimum position LL. As a result, the medium is added by an amount of volume LL - volume L0. By counting the number of rotations of the medium supply pump 454 during this layering, the discharge amount per rotation of the medium supply pump 454 can be calculated. By adding the medium, the rotation speed of the medium supply pump 454 when increasing the cell suspension from the minimum position LL to the low position L can be optimized.

[0288] In this way, even in the layering of the medium at the initial stage of culture, the rotation speed of the medium supply pump 454 can be optimized to culture cell aggregates.

[0289] <<<<Modification Example 1>>>> The culture system 10 shown in FIG. 1 includes a pH adjuster supply unit 430 and a glucose concentrate supply unit 440. The pH adjuster supply unit 430 adds a pH adjuster to the circulated medium. The glucose concentrate supply unit 440 adds a glucose concentrate to the circulated medium.

[0290] On the other hand, a dialysis module and a dialysis fluid tank (not shown) may be provided in the middle of the circulation pipe 238. The dialysis fluid stored in the dialysis fluid tank can be supplied to the dialysis module to supply the components necessary for the growth of cell aggregates to the culture medium.

[0291] <<<<Modification 2>>>> In the first and second embodiments, a configuration using a tube pump for the circulation pump 210 and the culture medium supply pump 454 is shown. However, when using a tube pump, it is not limited. As long as the culture medium can be fed by the operation of the circulation pump 210 and the culture medium supply pump 454 and the discharge amount per rotation of the drive unit can be detected. When the discharge amount per rotation of the drive unit of the pump changes due to deterioration of the pump over time, etc., the rotation speed of the pump can be adjusted.

[0292] <<<<Modification 3>>>> In the first and second embodiments, a configuration is shown in which the flow rate (feeding rate) of the culture medium is determined from the discharge amount per rotation of the culture medium supply pump 454 using the change amount of the cell suspension that changes during the culture medium exchange of cell culture. However, it is not limited to the time of culture medium exchange of cell culture. When the amount of the cell suspension in the culture vessel changes, the flow rate (feeding rate) of the culture medium may be determined.

[0293] <<<<Modification 4>>>> In the first and second embodiments, a configuration is shown in which the cell suspension in the culture vessel is acquired using a liquid level sensor. However, it is not limited to this. Any non-contact method such as a weighing scale or a capacitance type sensor that can acquire the cell suspension in the culture vessel may be used.

[0294] <<<<Scope of Embodiment>>>> As described above, the first and second embodiments have been described. However, the descriptions and drawings forming part of this disclosure should not be understood as limiting. Various embodiments not described here are included.

[0295] <<<<Embodiment of the Invention>>>> <<<Pump and Medium Exchange Used in Culture System 10>>> When culturing cell aggregates using a perfusion-type cell culture system such as culture system 10 (see Fig. 1), a pump is used for transferring the medium and the like. For example, in culture system 10, pumps such as the circulation pump 210 and the medium supply pump 454 shown in Fig. 1 described later are used. The flow rate of the pump is one of the conditions for perfusion culture and is required to be accurate.

[0296] For medium exchange, a determined amount of the medium contained in the cell suspension in the culture vessel is discharged, and when the amount of the medium in the cell suspension decreases by discharging the medium in the culture vessel, the density of the cell aggregates increases, and there is a possibility that the cell aggregates approach each other and undesirably bind. Therefore, it is preferable that the time when the amount of the medium in the culture vessel is small is as short as possible, and the medium exchange needs to be completed in a short time. However, if the flow rate of the medium discharged from the culture vessel is too high, there is also a possibility of sucking the cell aggregates. Due to this suction, the cell aggregates are discarded together with the old medium, so the medium must be discharged from the culture vessel at an optimal flow rate.

[0297] Furthermore, when classifying by utilizing the difference in the gravitational sedimentation rate due to the difference in the aggregate size, the size of the cell aggregates that can be classified is determined by the flow velocity in the classification nozzle. Therefore, the flow velocity of the medium in the classification nozzle must be accurate. Here, the flow velocity is obtained by dividing the flow rate by the cross-sectional area of the portion through which the flow passes.

[0298] In addition, in a perfusion-type cell culture system, a tube pump (peristaltic pump) is often used for the pump. The liquid delivery volume of the tube pump depends on physical properties such as the inner diameter of the pump tube used, the hardness of the pump tube, and the elastic force. The materials of the pump tube are PVC, silicon, Pharmed, etc. Even for pump tubes of the same specification, the inner diameter and physical properties may vary depending on the lot. Therefore, for tube pumps, even when using pump tubes of the same specification and even when the rotational speed of the motor used in the tube pump is the same, the liquid delivery volume per rotation of the tube pump may vary from pump to pump.

[0299] Furthermore, when a tube pump is used over a long period of time, the pump tube may deteriorate and the elastic force may weaken, resulting in a decrease in the liquid delivery volume. That is, even if a specified amount of liquid can be delivered at the start of using the tube pump, when used over a long time, even if the rotational speed of the motor used in the tube pump does not change, the flow rate of the tube pump may decrease compared to the specified amount at the start of use.

[0300] Note that not only tube pumps, but also positive displacement pumps such as plunger pumps, gear pumps, and tube pumps can be used in a perfusion-type cell culture system.

[0301] <<Control of Pump Flow Rate>> As a method of controlling the pump flow rate, there is also a method of measuring the flow rate using a flow meter and performing feedback control of the flow rate. However, generally, since tube pumps generate pulsations in the flow, the method of measuring the flow rate is limited. Furthermore, under the conditions of culturing cell aggregates, sterilization such as autoclaving of the flow meter section in contact with the liquid is required. Additionally, since it is also necessary to minimize the portions where the medium etc. stagnates as much as possible, the use of a flow meter becomes difficult.

[0302] In addition, a method of measuring the flow rate of the culture medium from the outer surface of the pump tube using ultrasonic waves or the like is easy to sterilize and suitable. However, it requires a straight tube portion at the portion where the flow rate is measured, and the outer diameter of the pump tube that can be applied becomes large, or pulsatile flow affects the measurement.

[0303] <<Method for Controlling the Liquid Feeding of the Culture Medium in the Culture System 10>> In such a situation of culturing cell aggregates, in the present embodiment, a method for controlling the liquid feeding of the culture medium in the culture system 10 using a pump such as a tube pump is provided.

[0304] In the culture system 10, the culture medium contained in the cell suspension is periodically exchanged in the culture vessel 110. For discharging the culture medium, a circulation pump 210 and a three-way valve 250 are used to feed the culture medium to the waste liquid tank 260. The flow rate of the circulation pump 210 is adjusted in the discharging process of the culture medium during the exchange of the culture medium, or the flow rate of the culture medium supply pump 454 is adjusted in the supply process of the culture medium. The discharging process and the supply process of the culture medium can be utilized.

[0305] According to the method for controlling the liquid feeding of the culture medium according to the present embodiment, since the maximum flow rate at which cell aggregates are not discharged from the culture vessel can be set according to the size of the cell aggregates, the culture medium can be exchanged in the shortest time. Further, in the classification of separating cell aggregates according to the size of the cell aggregates, classification can be performed at a flow rate suitable for the size of the cell aggregates to be classified.

[0306] <<Tube Pump>> In a perfusion-type cell culture system, a tube pump is often used for transferring and exchanging the culture medium and the like. A tube pump is a pump that uses a flexible pump tube and utilizes the restoring force of the pump tube. The pump tube used for the tube pump is configured separately from the tube pump detachably and is treated as a consumable. The wetted part is only the inner surface of the tube, and the outer surface of the pump tube is not wetted. That is, it is only necessary that the inner surface of the pump tube is sterilized. Further, since the pump tube is disposable, cleanliness can be maintained.

[0307] <Configuration and Operation of Tube Pump> The tube used in the tube pump is a pump tube, which is separate from a normal pipe (such as the pipe forming the path structure 200 described later) and is connected to and communicates with the normal pipe. A part of the pump tube is detachably held in an arc shape by the tube pump.

[0308] The tube pump mainly has a rotating body and a plurality of rollers (not shown). The rotating body can rotate around the center of the arc. The rotating body has a plurality of long supports extending radially along the radial direction. Each of the supports has a plurality of rollers at different positions of the end farthest from the rotation center (the position in contact with the pump tube arranged in an arc shape). Each of the plurality of rollers can rotate around the end of the support. As the rotating body rotates, each of the plurality of rollers rotates while pressing the pump tube held in an arc shape. The pump tube has a suction port and a discharge port. By the operation of releasing the pump tube from the pressing by the rotation operation of each of the plurality of rollers, a negative pressure is generated at the suction port of the pump tube, and due to the negative pressure, the medium is sucked into the inside of the pump tube from the suction port. The medium sucked into the inside of the pump tube is repeatedly guided to the discharge port and continuously fed by the operation of each of the plurality of rollers pressing the pump tube while rotating.

[0309] One rotation of the rotating body is one cycle of the tube pump. Generally, the rotation speed of the rotating body of the tube pump and the flow rate of the medium are in a proportional relationship. The flow rate of the medium can be determined by the rotation speed of the rotating body.

[0310] The flow rate of the medium needs to be accurate, and it is necessary to improve the accuracy of the flow rate (the liquid feeding amount per unit time) of the tube pump.

[0311] <<Accuracy of Flow Rate by Tube Pump>> A tube pump is required to have an accurate flow rate (liquid delivery volume). Specifically, an accurate flow rate (liquid delivery volume) of the medium is required at the time of each of the following medium exchanges and after the completion of the culture.

[0312] <During medium exchange in the culture system 10> In the culture system 10, the medium is exchanged periodically. The medium exchange involves discharging the old medium in which nutrient components and the like have been consumed by culturing and metabolic components such as waste products have increased from the culture vessel, and supplying the new medium to the culture vessel, so that part or all of the medium is exchanged. When the amount of the medium contained in the cell suspension in the culture vessel decreases during the medium exchange, the density of the cell aggregates with respect to the medium increases. A state where the density of the cell aggregates is high may result in insufficient amounts of oxygen and nutrients such as glucose required for culturing the cell aggregates. Furthermore, in a state where the density of the cell aggregates is high, the chance of contact between the cell aggregates increases. As a result, binding of the cell aggregates is likely to occur, and it is necessary to finish the medium exchange in as short a time as possible. However, it is desirable to maintain the state where the cell aggregates are left in the culture vessel when discharging the medium from the culture vessel. For this reason, it is necessary to discharge the medium at a rate that does not suck up the cell aggregates. That is, in order to set the rate of discharging the medium from the culture vessel to an optimal rate, it is necessary to make the flow rate of the pump accurate.

[0313] <After the completion of the culture in the culture system 10> After the completion of the culture in the culture system 10, a classification process may be performed to select only the cell aggregates of the required size. The classification process utilizes the correspondence between the size of the cell aggregates and the gravitational sedimentation rate of the cell aggregates. That is, it utilizes the phenomenon that small cell aggregates have a slow gravitational sedimentation rate and large cell aggregates have a fast gravitational sedimentation rate. Since the classification process in the culture system 10 utilizes the gravitational sedimentation rate and the flow rate inside the member (classification nozzle) when discharging the medium and the like, accurately setting the flow rate of the medium and the like affects the separation accuracy of the cell aggregates of the desired size. The flow rate of the medium can be determined from the area of the classification nozzle through which the medium flows and the flow rate of the medium. The accuracy of the flow rate of the medium affects the classification accuracy.

[0314] <<<Overview of the culture system 10>>> The culture system 10 shown in FIG. 1 is a culture system for culturing cell aggregates. The culture system 10 is a perfusion system. Specifically, the culture system 10 forms cell aggregates by causing cell aggregates to divide and grow while the cell aggregates come into contact with each other.

[0315] <<Cell aggregates>> Cell aggregates are the objects to be cultured by the culture system 10. By culturing cell aggregates, the cell aggregates are divided to increase the number of cell aggregates. Cell aggregates are formed when cell aggregates come into contact with each other. The culture system 10 is a system that grows cells to form cell aggregates.

[0316] The cells may be any cells that can form cell aggregates in a culture medium and are not particularly limited. Examples of cells include cells used in research related to regenerative medicine and cells used as cell preparations. Specifically, pluripotent stem cells such as ES cells and iPS cells, various progenitor cells such as nephron progenitor cells, ureteric bud cells, and stromal progenitor cells, various stem cells such as mesenchymal stem cells, neural stem cells, and adipose stem cells can be mentioned.

[0317] <<Culture medium>> The culture medium can be selected and used appropriately according to the cell aggregates and is not particularly limited. In addition, conventionally known materials and additives useful for cell culture can be used as appropriate. Examples of basal media include DMEM, DMEMHG, and EMEM. As additives, it is preferable to add amino acids, vitamins, proteins (growth factors), glucose, antibiotics, etc.

[0318] <<Cell suspension>> A cell suspension is a system in which cell aggregates are dispersed in a culture medium. In the culture vessel 110, cell aggregates and a culture medium are stored as a cell suspension.

[0319] <<Stirring>> Stirring means displacing cell aggregates and the culture medium contained in the cell suspension. By stirring, the cell aggregates are displaced along with the displacement of the culture medium. Mainly, the cell aggregates are displaced along with the displacement of the culture medium. Note that the operation on the cell suspension only needs to be able to displace the cell aggregates, and it may be not only stirring but also operations such as shaking.

[0320] <<Floating>> Floating mainly means a state in which cell aggregates are not in contact with the wall surface of the culture vessel (for example, the culture vessel 110 described later, etc.). The wall surface of the culture vessel is all the surfaces where cell aggregates may come into contact. For example, when the culture vessel has a cylindrical or rectangular tube shape, the wall surface is the inner surface of the bottom and side surfaces. When the culture vessel has a spherical shape, it is the inner surface of the spherical surface. Note that even in a state where a part of the cell aggregates is in contact with the wall surface of the culture vessel, a state where it can be easily separated from the wall surface due to the displacement of the culture medium is also included in floating.

[0321] <<Aggregation>> Aggregation means that cell aggregates gather together to form a large mass. Note that even if they have aggregated once, the cell aggregates may separate during the culture process. Even if the case of separation is included, it only needs to be possible to culture the cell aggregates to become larger finally. Note that the size of the cell aggregates does not matter. A state where at least two cell aggregates are aggregated is included in the cell aggregates. The state where cell aggregates are aggregated may also be referred to as a cell aggregate.

[0322] <<First Aspect>> According to the first aspect, a culture medium feeding control method executed by a control unit (for example, the control device 500 described above, etc.) in a culture system 10 for culturing cell aggregates, a cell suspension detection step of detecting a cell suspension in a culture vessel (for example, the culture vessel 110 described above, etc.) (for example, the processing of steps S715, S731, S813, S821 by the high-position liquid level sensor 318H, low-position liquid level sensor 318L, and lowest-position liquid level sensor 318LL described above), and A driving state detection step (such as the processes in steps S713, S719, S819, and S825 described above) for detecting the driving state (such as the transfer amount of the culture medium by the circulation pump 210 and the medium supply pump 454, or the rotation speed (rotational speed) of the circulation pump 210 and the medium supply pump 454) of a liquid feeding mechanism (such as the circulation pump 210 and the medium supply pump 454 mentioned above) that drives based on predetermined driving conditions to feed the culture medium, and A driving condition determination step (such as the processes in steps S723 and S829 described above) for determining the driving conditions (such as the discharge amount of the culture medium by the circulation pump 210 and the medium supply pump 454 mentioned above) of the liquid feeding mechanism based on the cell suspension detected in the cell suspension detection step and the driving state detected in the driving state detection step are provided.

[0323] The culture medium liquid feeding control method is executed by a control unit. The culture medium liquid feeding control method includes a cell suspension detection step, a driving state detection step, and a driving condition determination step.

[0324] The cell suspension detection step detects the cell suspension in the culture vessel. The detection of the cell suspension may be optical, may detect the weight of the cell suspension, or may detect it by capacitance. The detection of the cell suspension may be any method that can detect the cell suspension from the outside of the culture vessel and various tubes. The cell suspension can be detected while maintaining a clean state. Since the cell suspension detection step detects the cell suspension, when the amount of the cell suspension in the culture vessel changes, the change amount of the cell suspension can be determined based on the detected cell suspension.

[0325] The driving state detection step detects the driving state of a liquid feeding mechanism that feeds the culture medium. The liquid feeding mechanism may be various pumps such as a tube pump, as long as it can feed the culture medium while maintaining a clean state. The liquid feeding mechanism preferably has an operation that repeats, such as a rotational operation, a reciprocating operation, or a vibrating operation. In particular, the liquid feeding mechanism more preferably has a periodic operation.

[0326] Note that "driving" mainly refers to transmitting power to and moving the devices and components that make up the culture system 10. The object to be driven may be a motor that constitutes a circulation pump 210, a medium supply pump 454, etc., or a rotor, etc., or it may be a device or component or a part thereof that repeatedly displaces.

[0327] The driving state of the liquid feeding mechanism may be information indicating the operating state and operating conditions of the liquid feeding mechanism. For example, the driving state can be information directly indicating the state of the liquid feeding mechanism such as the rotation speed (rotational speed), period, frequency, vibration frequency, etc., or the number of sliding times, angular velocity, etc. Also, it may be information indirectly indicating the state of the liquid feeding mechanism such as the amount of medium flowed by the liquid feeding mechanism.

[0328] The driving condition determination step determines the driving conditions of the liquid feeding mechanism based on the cell suspension and the driving state. The cell suspension is detected by the cell suspension detection step. The driving state is detected by the driving state detection step. The driving conditions of the liquid feeding mechanism are determined based on the cell suspension and the driving state. The driving conditions of the liquid feeding mechanism may be conditions required for driving the liquid feeding mechanism. The driving conditions of the liquid feeding mechanism may be direct conditions or indirect conditions for the liquid feeding mechanism. In particular, the driving conditions of the liquid feeding mechanism preferably include information regarding the amount of medium. For example, the driving conditions of the liquid feeding mechanism can be the discharge amount of the medium per unit (e.g., per unit operation, per unit time, etc.).

[0329] The cell suspension preferably has a change amount of the cell suspension. As a result of driving the liquid feeding mechanism, the amount of the cell suspension changes. From the change amount of the cell suspension and the driving state of the liquid feeding mechanism, the driving conditions of the liquid feeding mechanism can be determined. By detecting the cell suspension in the culture vessel, without depending on the liquid feeding state of the medium, the cross-sectional area of the part where the medium is fed, etc., the change in the liquid feeding speed of the medium throughout the culture system 10 can be obtained and judged. That is, by detecting the cell suspension in the culture vessel, it is possible to judge not the change in the local liquid feeding amount of the medium or the change in the liquid feeding amount of the medium in a short time, but the overall change in the liquid feeding amount of the medium in the culture system 10.

[0330] Instead of directly obtaining the flow rate of the flowing medium, the cell suspension in the culture vessel is detected, and the driving state of the liquid feeding mechanism is also detected to determine the driving conditions of the liquid feeding mechanism. Therefore, even when the flow of the medium fluctuates, the driving conditions of the liquid feeding mechanism can be stably determined.

[0331] <<Second Aspect>> The second aspect is, in the first aspect, the liquid feeding mechanism can repeat a unit operation (for example, one rotation of the circulation pump 210 or the medium supply pump 454 described above) that causes the medium to flow at a predetermined flow rate, and the driving state of the liquid feeding mechanism is the number of times (for example, the number of rotations of the circulation pump 210 or the medium supply pump 454 described above) that the liquid feeding mechanism performs the unit operation (for example, the operation of one rotation of the circulation pump 210 or the medium supply pump 454 described above).

[0332] The liquid feeding mechanism repeats the unit operation. The unit operation is an operation that continuously flows the medium at a predetermined flow rate. By the unit operation of the liquid feeding mechanism, the number of times the unit operation is performed can be accurately defined, and the driving conditions of the liquid feeding mechanism can be accurately determined.

[0333] <<Third Aspect>> The third aspect is, in the first aspect or the second aspect, the driving condition determination step is Based on the flow rate of the derivation (for example, the discharge process of the medium described above) or introduction (for example, the supply process of the medium described above) of the medium to the culture vessel based on the change amount of the cell suspension in the culture vessel detected in the cell suspension detection step, and the number of times the liquid feeding mechanism performs the unit operation, the unit driving condition per unit operation of the liquid feeding mechanism is determined as the driving condition (for example, the discharge amount of the medium per rotation of the circulation pump 210 or the medium supply pump 454 described above), including a unit driving condition determination step.

[0334] Since the unit driving conditions of the liquid feeding mechanism per unit time are determined, even when the flow of the culture medium fluctuates, the driving conditions of the liquid feeding mechanism can be stably determined.

[0335] <<Fourth Aspect>> The fourth aspect is in the first to third aspects, the unit driving conditions are the amount of the culture medium fed by the unit operation of the liquid feeding mechanism (for example, the rotation angle (= rotation speed) per unit time of the circulation pump 210 or the culture medium supply pump 454), the amount of the culture medium fed by the unit operation of the liquid feeding mechanism is updated based on the flow amount of the culture medium derived or introduced into the culture vessel and the number of times the liquid feeding mechanism has performed the unit operation.

[0336] Since the amount of displacement per unit time of the driving unit is determined, even when the flow of the culture medium fluctuates, the driving conditions of the liquid feeding mechanism can be stably determined.

[0337] <<Fifth Aspect>> The fifth aspect is in the first to fourth aspects, a cell aggregate size acquisition step of acquiring the size of the cell aggregate (for example, the above-mentioned "Among these, the diameter of the cell aggregate changes according to the growth of the cell aggregate. The cell aggregate can be imaged with an imaging device such as a camera, and the diameter of the cell aggregate can be determined from the imaging result. Also, in a preliminary experiment or the like, the correspondence between the elapsed time from the start of culturing the cell aggregate and the diameter of the cell aggregate is determined in advance and stored in a storage device such as the ROM or RAM of the control device 500. By referring to the correspondence, the diameter of the cell aggregate corresponding to the time after the actual start of culturing can be determined." etc.) and, A sedimentation rate determination step of determining the sedimentation rate of cell aggregates in the cell suspension based on the size of the cell aggregates (for example, as described above, "The gravitational sedimentation rate of cell aggregates can be determined from the density of the cell aggregates, the size of the cell aggregates, the density of the medium, and the viscosity of the medium. The density of the cell aggregates can be determined in advance if the type of cell aggregates to be cultured is determined. Also, the density and viscosity of the medium can be determined in advance if the type of medium to be used is determined according to the type of cell aggregates to be cultured." etc.), and A flow rate determination step of determining the flow rate of the medium in which the cell aggregates are not derived from the classification nozzle from the cross-sectional area of the classification nozzle for deriving the cell suspension from the culture vessel and the sedimentation rate (for example, as described above, "Based on the flow rate of the medium in the classification nozzle 242, in particular, it is preferable to determine the maximum flow rate of the medium in the classification nozzle 242 as the flow rate to such an extent that cell aggregates are not derived from the circulation pipe 232. The flow rate can be obtained from the cross-sectional area of the portion where the medium flows and the flow rate, and the flow rate of the circulation pump 210 is determined." etc.), and A liquid feeding mechanism control step of controlling the liquid feeding mechanism based on the flow rate and the driving conditions (for example, as described above, "Based on the determined flow rate and the discharge amount per rotation of the driving part of the circulation pump 210, the rotation speed of the driving part of the circulation pump 210 can be determined." etc.), and further includes.

[0338] Since the cell aggregates are not derived from the classification nozzle and only the medium is derived, the cell aggregates can be stably cultured.

[0339] <<Sixth Aspect>> The sixth aspect is in the first aspect to the fifth aspect, The cell suspension detection step, the driving state detection step, and the driving condition determination step are executed in at least one of the medium discharge step and the medium supply step in the medium exchange in the culture process of the cell aggregates. The medium feeding control method according to claim 1.

[0340] By effectively utilizing the medium discharge step and the medium supply step required for culturing the cell aggregates, the liquid feeding rate can be determined.

[0341] <<Aspect 7>> According to the 7th aspect, a cell suspension detection unit (for example, the above-described high-position liquid level sensor 318H, low-position liquid level sensor 318L, lowest-position liquid level sensor 318LL, etc.) that detects a cell suspension in a culture vessel (for example, the above-described culture vessel 110, etc.) for culturing cell aggregates, and a drive state detection unit (for example, the above-described control device 500, etc.) that detects the drive state (for example, the transfer amount of the culture medium by the circulation pump 210 and the culture medium supply pump 454, the rotation speed (rotation speed) of the circulation pump 210 and the culture medium supply pump 454, etc.) of a liquid feeding mechanism (for example, the above-described circulation pump 210, culture medium supply pump 454, etc.) that feeds the culture medium based on predetermined drive conditions, and a drive condition determination unit (for example, the above-described control device 500, etc.) that determines the drive conditions (for example, the discharge amount of the culture medium by the circulation pump 210 and the culture medium supply pump 454, etc.) of the liquid feeding mechanism based on the cell suspension detected by the cell suspension detection unit and the drive state detected by the drive state detection unit are provided, and a culture system is provided.

[0342] Since the cell suspension in the container is detected and the drive state of the liquid feeding mechanism is used, even when the flow of the culture medium fluctuates, the flow rate of the culture medium fed by the liquid feeding mechanism can be accurately obtained, and if necessary, the drive conditions of the liquid feeding mechanism can be stably determined.

[0343] <<Aspect 8>> According to the 8th aspect, a cell suspension detection unit (for example, the above-described high-position liquid level sensor 318H, low-position liquid level sensor 318L, lowest-position liquid level sensor 318LL, etc.) that detects the amount of cell suspension stored in a culture vessel (for example, the above-described culture vessel 110, etc.), and an operation unit (for example, the motors of the above-described circulation pump 210 and culture medium supply pump 454, etc.) that repeats a unit operation, and a culture medium flow unit (for example, the above-described circulation pump 210, culture medium supply pump 454, etc.) that causes the culture medium to flow according to the unit operation, A unit operation state detection unit (such as the aforementioned control device 500) that detects information regarding the operation state of the operation unit (for example, the discharge amount and liquid feed amount per rotation described above), An operation control unit (such as the aforementioned control device 500) that determines the frequency of repetition of the unit operation of the operation unit based on the change amount of the cell suspension in the culture vessel detected by the cell suspension detection unit and the information regarding the operation state detected by the unit flow rate detection unit. A culture system including the above is provided.

[0344] Since the cell suspension in the container is detected and the driving state of the liquid feed mechanism is used, even when the flow of the culture medium fluctuates, the flow rate of the culture medium can be adjusted for the liquid feed mechanism by determining the operation of the liquid feed mechanism.

[0345] <<Aspect 9>> Aspect 9 is, in Aspect 8, The information regarding the operation state is the number of times the operation unit has performed the unit operation.

[0346] <<Aspect 10>> Aspect 10 is, in Aspect 8 or Aspect 9, The operation control unit Based on the change amount of the cell suspension in the culture vessel detected by the cell suspension detection unit and The number of times the operation unit has performed the unit operation, Determines the operation of the operation unit per unit time.

[0347] <<Aspect 11>> Aspect 11 is, in Aspect 8 to Aspect 10, The operation control unit Determines the cycle of repetition of the unit operation of the operation unit.

Explanation of Reference Numerals

[0348] 10 Culture system 110 Culture vessel 210 Circulation Pump 318H, 318L, 318LL Liquid Level Sensors 454 Medium Supply Pump 500 Control Device

Claims

1. A medium feeding control method executed by a control unit in a culture system for culturing cell aggregates, comprising: a cell suspension detection step of detecting a cell suspension in a culture vessel; a driving state detection step of detecting a driving state of a liquid feeding mechanism that drives and feeds a medium based on predetermined driving conditions; a driving condition determination step of determining driving conditions of the liquid feeding mechanism based on the cell suspension detected in the cell suspension detection step and the driving state detected in the driving state detection step.

2. The liquid feeding mechanism is capable of repeating a unit operation of flowing a medium at a predetermined flow rate, and the driving state of the liquid feeding mechanism is the number of times the liquid feeding mechanism has performed the unit operation. The medium feeding control method according to claim 1.

3. The driving condition determination step includes: a unit driving condition determination step of determining, as the driving condition, a unit driving condition per unit operation of the liquid feeding mechanism based on the flow rate of derivation or introduction of the medium to / from the culture vessel based on the change amount of the cell suspension in the culture vessel detected in the cell suspension detection step and the number of times the liquid feeding mechanism has performed the unit operation. The medium feeding control method according to claim 2.

4. The unit driving condition is the amount of the medium fed by the unit operation of the liquid feeding mechanism, and the amount of the medium fed by the unit operation of the liquid feeding mechanism is updated based on the flow rate of derivation or introduction of the medium to / from the culture vessel and the number of times the liquid feeding mechanism has performed the unit operation. The medium feeding control method according to claim 3.

5. a cell aggregate size determination step of determining the size of cell aggregates; a sedimentation rate determination step of determining the sedimentation rate of cell aggregates in the cell suspension based on the size of the cell aggregates; a flow rate determination step of determining the flow rate of the medium in which cell aggregates are not derived from the classification nozzle based on the cross-sectional area of the classification nozzle for deriving the cell suspension from the culture vessel and the sedimentation rate; and a liquid feeding mechanism control step of controlling the liquid feeding mechanism based on the flow rate and the driving conditions. The medium feeding control method according to claim 4.

6. The cell suspension detection step, the driving state detection step, and the driving condition determination step are executed in at least one of medium discharge and medium supply in medium exchange during the culture process of cell aggregates. The medium feeding control method according to claim 1.

7. A cell suspension detection unit that detects a cell suspension in a culture vessel for culturing cell aggregates, A drive state detection unit that detects the drive state of a liquid feeding mechanism that feeds a culture medium based on predetermined drive conditions, A culture system comprising: a drive condition determination unit that determines the drive conditions of the liquid feeding mechanism based on the cell suspension detected by the cell suspension detection unit and the drive state detected by the drive state detection unit.

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