Stirring method and stirring device

Abstract

The stirring method includes: a cell stirring step of stirring a cell suspension by circulating the cell suspension in a circulation path (130) including a bioreactor (24); an acquisition step for acquiring, over time, a predetermined physical quantity relating to the cell suspension at the site to be measured in the circulation path (130); and a determination step for determining, on the basis of the degree of variation of the physical quantity being less than a predetermined degree, that a uniform diffusion state, which is a state in which the cells are uniformly diffused in the cell suspension, has been reached.

Description

TECHNICAL FIELD

[0001] The present application relates to a stirring method and a stirring device for stirring a cell suspension. BACKGROUND

[0002] In order to efficiently culture cells, it is preferable to appropriately stir a cell suspension in cell culture. By stirring a cell suspension, it is thereby possible to supply nutrients to all cells in the cell suspension. A culture device of the type that stirs by aeration is disclosed in Japanese Patent Application Publication No. 2002-148258. SUMMARY

[0003] In a cell culture process, a series of processes including a cell stirring process and a cell culture process are repeatedly performed, for example, every 24 hours. In order to optimize the cell culture process, a technique that optimizes the cell stirring process is desired.

[0004] An object of the present application is to solve the above-described problems.

[0005] (1) The stirring method of the first application includes: a cell stirring process that stirs a cell suspension by circulating the cell suspension in a circulation path including a bioreactor; a process of acquiring a prescribed physical quantity related to the cell suspension at a measurement target site within the circulation path over time; and a determination process that determines that a state in which cells are uniformly dispersed in the cell suspension, that is, a uniform dispersion state, has been reached based on the fact that a degree of variation of the physical quantity is smaller than a degree determined in advance.

[0006] In the first application, in the determination process, the degree of variation is compared with the degree determined in advance. Thereby, in the determination process, it is possible to determine whether or not the uniform dispersion state has been reached. According to the first application, it is possible to execute the cell stirring process only for the necessary minimum time. According to the first application, it is possible to shorten the execution time of the cell stirring process, and it is possible to shorten the execution time of the cell culture process. Thus, according to the first application, it is possible to contribute to the optimization of the cell culture process.

[0007] (2) In the stirring method described in the above item (1), it can also be that the degree of variation of the physical quantity is grasped based on a standard deviation of a plurality of the physical quantities within a unit time determined in advance.

[0008] (3) In the stirring method described in the above item (1) or (2), it can also be that the physical quantity is acquired using an optical sensor.

[0009] (4) In the stirring method described in any one of the above items (1) to (3), it can also be that the physical quantity is at least one of an amount of light of transmitted light that transmits through the cell suspension, and an amount of light of scattered light that scatters in the cell suspension.

[0010] (5) In the agitation method according to any one of the above items (1) to (4), the agitation of the cell suspension can be stopped when it is determined in the determination step that the uniform diffusion state has been reached in the cell agitation step.

[0011] (6) In the agitation method according to any one of the above items (1) to (5), the cell suspension can be agitated by rotating the bioreactor using an actuator in the cell agitation step.

[0012] According to the structure of the above item (6), the cell suspension can be made into the uniform diffusion state more quickly.

[0013] (7) The agitation device of the second application includes a control section that agitates a cell suspension by circulating the cell suspension in a circulation path including a bioreactor; an acquisition section that acquires a predetermined physical quantity related to the cell suspension at a measurement target site in the circulation path over time; and a determination section that determines that a state in which cells are uniformly diffused in the cell suspension, that is, a uniform diffusion state, has been reached based on the fact that a variation degree of the physical quantity is smaller than a predetermined degree.

[0014] According to the second application, the same effects as the first application can be obtained.

[0015] (8) In the agitation device according to the above item (7), the variation degree of the physical quantity can be grasped based on a standard deviation of a plurality of the physical quantities in a predetermined unit of time.

[0016] (9) In the agitation device according to the above item (7) or (8), the physical quantity can be acquired using an optical sensor.

[0017] (10) In the agitation device according to any one of the above items (7) to (9), the physical quantity can be at least one of an amount of light of transmitted light that transmits through the cell suspension and an amount of light of scattered light that is scattered in the cell suspension.

[0018] (11) In the agitation device according to any one of the above items (7) to (10), the control section can stop the agitation of the cell suspension when it is determined by the determination section that the uniform diffusion state has been reached.

[0019] (12) In the agitation device according to any one of the above items (7) to (11), the control section can agitate the cell suspension by rotating the bioreactor using an actuator.

[0020] According to the structure of the above item (12), the same effect as that of the above item (6) can be obtained.

[0021] According to the present application, it is possible to contribute to optimization of the cell stirring process. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a schematic diagram showing a cell culture device.

[0023] Figure 2 is a block diagram of a culture control device (stirring device) provided in the cell culture device.

[0024] Figure 3 is a flowchart of a cell culture process.

[0025] Figure 4 is a schematic diagram showing a cell input process.

[0026] Figure 5 is a schematic diagram showing a cell stirring process.

[0027] Figure 6 is a schematic diagram showing a cell filling process.

[0028] Figure 7 is a schematic diagram showing a cell culture process.

[0029] Figure 8 is a flowchart of a cell stirring process.

[0030] Figure 9A 、 Figure 9B is a timing chart showing a time change in the light receiving amount of transmitted light detected by the sensor section. DETAILED DESCRIPTION

[0031] [1 Configuration of cell culture device 10]

[0032] Figure 1 is a schematic diagram showing a cell culture device 10. The cell culture device 10 is provided with a cell culture circuit 12 and a support device 14. The cell culture device 10 cultures cells separated from a biological tissue in a culture medium. The cells cultured in the cell culture device 10 are, for example, adherent cells or suspension cells. As the cells cultured in the cell culture device 10, for example, ES cells, iPS cells, mesenchymal stem cells, and the like can be given. Note that the cells cultured in the cell culture device 10 are not limited to the above-mentioned cells.

[0033] [1-1 Cell culture circuit 12]

[0034] A liquid flows in the cell culture loop 12. As the liquid, a cell suspension, a culture medium, a cleaning solution, a peeling solution, and the like can be given. The cell suspension is a solution containing cells. The culture medium is a culture solution for proliferating cells. The culture medium can be selected depending on the cells to be cultured. As the culture medium, for example, MEM (Minimum Essential Medium) is used. The cleaning solution is a solution for cleaning the inside of the cell culture loop 12. As the cleaning solution, for example, water, a buffer solution, physiological saline, and the like can be given. As the buffer solution, PBS (Phosphate Buffered Salts), TBS (Tris-Buffered Saline), and the like can be given. The peeling solution is a solution for peeling cells from the bioreactor 24 of the cell culture loop 12. As the peeling solution, for example, a trypsin solution, an EDTA solution (Ethylene Diamine Tetraacetic Acid solution), and the like can be given. Note that the culture medium, the cleaning solution, and the peeling solution are not limited to the above-mentioned liquids.

[0035] The cell culture loop 12 is a disposable article that is replaced each time a cell culture process is performed. The cell culture loop 12 includes a liquid supply section 16, a cell recovery section 18, a waste liquid storage section 20, and a culture main body 22.

[0036] The liquid supply section 16 includes a plurality of medical bags (not shown). The medical bags are filled with liquids to be supplied to the culture main body 22. For example, the first medical bag is filled with a cell suspension. The second medical bag is filled with a culture medium. The third medical bag is filled with a cleaning solution. The fourth medical bag is filled with a peeling solution. Note that a plurality of kinds of culture media can be separately filled in the medical bags.

[0037] The cell recovery section 18 and the waste liquid storage section 20 each include a medical bag (not shown). The cell recovery section 18 recovers cells after being cultured by the culture main body 22. The waste liquid storage section 20 stores waste liquid generated by the culture main body 22.

[0038] The culture main body 22 includes a bioreactor 24, a flow path 26, a sensor section 28, and a gas exchange section 30.

[0039] The bioreactor 24 has a plurality of hollow fiber membranes 32 and a cylindrical housing 34. The plurality of hollow fiber membranes 32 are housed inside the housing 34. Each of the hollow fiber membranes 32 extends along the length direction of the bioreactor 24. The hollow fiber membranes 32 are composed of, for example, a high molecular material. The hollow fiber membranes 32 have a plurality of pores (not shown). A first end portion of each of the hollow fiber membranes 32 is fixedly joined to a first end portion 34a of the housing 34 in the length direction. A second end portion of each of the hollow fiber membranes 32 is fixedly joined to a second end portion 34b of the housing 34 in the length direction.

[0040] The bioreactor 24 has a first region 36 and a second region 38. The first region 36 is a space inside each of the hollow fiber membranes 32. The second region 38 is a space between the outer peripheral surface of each of the hollow fiber membranes 32 and the inner peripheral surface of the housing 34. The first region 36 and the second region 38 communicate with each other via the plurality of pores of each of the hollow fiber membranes 32.

[0041] The housing 34 has a first port 40, a second port 42, a third port 44, and a fourth port 46. The first port 40 is disposed at the first end portion 34a of the housing 34. The first port 40 is connected to the first end portion of each of the hollow fiber membranes 32. Thus, the first port 40 communicates with the first region 36. The second port 42 is disposed at the second end portion 34b of the housing 34. The second port 42 is connected to the second end portion of each of the hollow fiber membranes 32. Thus, the second port 42 communicates with the first region 36.

[0042] The third port 44 and the fourth port 46 are disposed at the outer peripheral surface of the housing 34. The third port 44 is disposed between the first port 40 and the central portion in the length direction of the housing 34. The fourth port 46 is disposed between the second port 42 and the central portion in the length direction of the housing 34. The third port 44 and the fourth port 46 respectively communicate with the second region 38.

[0043] The flow path 26 is composed of a plurality of tubes (piping) through which a liquid flows. Each of the tubes is composed of a soft resin material. The flow path 26 has a first supply flow path 48, a first circulation flow path 50, a second supply flow path 52, a second circulation flow path 54, a recovery flow path 56, and a waste liquid flow path 58.

[0044] The first supply flow path 48 introduces various liquids of the liquid supply portion 16 to the first circulation flow path 50. The first supply flow path 48 has a plurality of first upstream flow paths 48a and one first downstream flow path 48b. One first upstream flow path 48a is provided with respect to one medical bag of the liquid supply portion 16. The first upstream flow path 48a is connected to the medical bag of the liquid supply portion 16. In addition, each of the first upstream flow paths 48a is connected to the first downstream flow path 48b. The first downstream flow path 48b is connected to a first confluence portion 60 in the first circulation flow path 50.

[0045] The first circulation flow path 50 introduces the liquid introduced from the first supply flow path 48 to the bioreactor 24. In addition, the first circulation flow path 50 introduces the liquid discharged from the bioreactor 24 to the bioreactor 24 again. The first end portion 50a of the first circulation flow path 50 is connected to the first port 40 of the bioreactor 24. The second end portion 50b of the first circulation flow path 50 is connected to the second port 42 of the bioreactor 24. The first circulation flow path 50 communicates with the inner holes (the first region 36) of the respective hollow fiber membranes 32. The first confluence portion 60 is provided in the first circulation flow path 50. The portion of the first circulation flow path 50 divided by the first end portion 50a and the first confluence portion 60 is referred to as a first flow path 51a (a first pipe). The portion of the first circulation flow path 50 divided by the second end portion 50b and the first confluence portion 60 is referred to as a second flow path 51b (a second pipe). The recovery branch portion 64 is provided in the second flow path 51b. Further, the first branch portion 72 is provided in the portion of the second flow path 51b between the recovery branch portion 64 and the second end portion 50b.

[0046] The second supply flow path 52 introduces the various liquids of the liquid supply portion 16 to the second circulation flow path 54. The second supply flow path 52 has a plurality of second upstream flow paths 52a and one second downstream flow path 52b. One second upstream flow path 52a is provided with respect to one medical bag of the liquid supply portion 16. The second upstream flow path 52a is connected to the medical bag of the liquid supply portion 16. In addition, the respective second upstream flow paths 52a are connected to the second downstream flow path 52b. The second downstream flow path 52b is connected to the second confluence portion 62 in the second circulation flow path 54.

[0047] The second circulation flow path 54 introduces the liquid introduced from the second supply flow path 52 to the bioreactor 24. In addition, the second circulation flow path 54 introduces the liquid discharged from the bioreactor 24 to the bioreactor 24 again. The first end portion 54a of the second circulation flow path 54 is connected to the third port 44 of the bioreactor 24. The second end portion 54b of the second circulation flow path 54 is connected to the fourth port 46 of the bioreactor 24. The second circulation flow path 54 communicates with the space (the second region 38) between the plurality of hollow fiber membranes 32 and the housing 34. The second confluence portion 62 is provided in the second circulation flow path 54. In addition, the second branch portion 74 is provided in the portion of the second circulation flow path 54 between the second confluence portion 62 and the second end portion 54b.

[0048] The recovery flow path 56 introduces the cell suspension discharged from the bioreactor 24 to the cell recovery portion 18. The recovery flow path 56 branches from the first circulation flow path 50. The first end portion 56a of the recovery flow path 56 is connected to the recovery branch portion 64 of the first circulation flow path 50. The second end portion 56b of the recovery flow path 56 is connected to the medical bag of the cell recovery portion 18.

[0049] The waste liquid flow path 58 introduces the liquid of the first circulation flow path 50 and the second circulation flow path 54 to the waste liquid container 20. The waste liquid flow path 58 includes a first waste liquid flow path 66, a second waste liquid flow path 68, and a third waste liquid flow path 70. The first waste liquid flow path 66 branches from the first circulation flow path 50. A first end portion 66a of the first waste liquid flow path 66 is connected to the first branch portion 72 of the first circulation flow path 50. The second waste liquid flow path 68 branches from the second circulation flow path 54. A first end portion 68a of the second waste liquid flow path 68 is connected to the second branch portion 74 of the second circulation flow path 54. Second end portions 66b and 68b of the first and second waste liquid flow paths 66 and 68, respectively, are connected to a first end portion 70a of the third waste liquid flow path 70. A second end portion 70b of the third waste liquid flow path 70 is connected to the medical bag of the waste liquid container 20.

[0050] The sensor portion 28 is disposed at the first flow path 51a (measurement target portion). Note that the sensor portion 28 can also be disposed at the second flow path 51b. The sensor portion 28 detects a prescribed physical quantity related to the liquid (e.g., cell suspension) flowing in the first circulation flow path 50. The prescribed physical quantity is a physical quantity proportional to the number of cells in the liquid. For example, the sensor portion 28 includes a light source and one or more optical sensors (light receivers). The light source irradiates light to the liquid. The optical sensor receives transmitted light that has passed through the liquid. The optical sensor outputs an electrical signal corresponding to the amount of light received to the controller 112 (not shown). Figure 2 ). Note that the optical sensor can also receive scattered light (forward scattered light, side scattered light, back scattered light, etc.) instead of transmitted light. Alternatively, the sensor portion 28 can include a sensor (e.g., permittivity sensor) having two or more electrodes instead of the light source and the optical sensor.

[0051] The gas exchange portion 30 is disposed at a portion of the second circulation flow path 54 between the second confluence portion 62 and the third port 44. The gas exchange portion 30 supplies a prescribed component gas to the liquid (culture medium) flowing in the second circulation flow path 54. The gas used in the gas exchange portion 30 has a component close to that of air, for example. That is, the gas includes nitrogen, oxygen, and carbon dioxide.

[0052] [1-2 Support device 14]

[0053] The cell culture circuit 12 described above is detachable with respect to the support device 14. The support device 14 includes a case that supports the cell culture circuit 12. The support device 14 is a reusable member that can be used multiple times.

[0054] The support device 14 is provided with a plurality of pumps 76, a plurality of clamps 78, and a reactor driving section 80. The plurality of pumps 76, the plurality of clamps 78, and the reactor driving section 80 are each provided with an electric actuator. Note that the plurality of pumps 76, the plurality of clamps 78, and the reactor driving section 80 can also be each provided with a fluidic actuator.

[0055] Each pump 76 can impart a flow force to a liquid in the flow path 26 by extruding a tube that forms the flow path 26. Each pump 76 operates using electric power supplied from a pump driving circuit 114 Figure 2 ).

[0056] The plurality of pumps 76 includes a first supply pump 82, a first circulation pump 84, a second supply pump 86, and a second circulation pump 88. Each pump 76 functions as follows. Note that, as shown in Figure 1 , a state in which the cell culture circuit 12 is installed to the support device 14 is referred to as an "installed state".

[0057] In the installed state, the first supply pump 82 is fitted with the first downstream flow path 48b. The first supply pump 82 imparts a flow force to a liquid in the first supply flow path 48 in a direction from the liquid supply section 16 toward the first circulation flow path 50.

[0058] In the installed state, the first circulation pump 84 is fitted with the second flow path 51b of the first circulation flow path 50. The first circulation pump 84 imparts a flow force to a liquid in the first circulation flow path 50 in a direction from the second port 42 toward the first port 40. Note that the first circulation pump 84 can also impart a flow force to a liquid in the first circulation flow path 50 in a direction from the first port 40 toward the second port 42.

[0059] In the installed state, the second supply pump 86 is fitted with the second downstream flow path 52b. The second supply pump 86 imparts a flow force to a liquid in the second supply flow path 52 in a direction from the liquid supply section 16 toward the second circulation flow path 54.

[0060] In the installed state, the second circulation pump 88 is fitted with the second circulation flow path 54. The second circulation pump 88 imparts a flow force to a liquid in the second circulation flow path 54 in a direction from the fourth port 46 toward the third port 44. Note that the second circulation pump 88 can also impart a flow force to a liquid in the second circulation flow path 54 in a direction from the third port 44 toward the fourth port 46.

[0061] Each clamp 78 can close the flow path 26 by compressing a tube that forms the flow path 26 in the width direction. Each clamp 78 functions as an on-off valve. Each clamp 78 operates using electric power supplied from a clamp driving circuit 116 Figure 2 ).

[0062] The plurality of clamps 78 include a plurality of first supply clamps 90, a plurality of second supply clamps 92, a recovery clamp 94, a first waste liquid clamp 96, a second waste liquid clamp 98, and a third waste liquid clamp 100. Each clamp 78 functions as follows.

[0063] In the installed state, one first supply clamp 90 is fitted with one first upstream flow path 48a. In other words, each first upstream flow path 48a is supported by any one of the first supply clamps 90. The first supply clamps 90 open and close the first supply flow paths 48.

[0064] In the installed state, one second supply clamp 92 is fitted with one second upstream flow path 52a. In other words, each second upstream flow path 52a is supported by any one of the second supply clamps 92. The second supply clamps 92 open and close the second supply flow paths 52.

[0065] In the installed state, the recovery clamp 94 is fitted with the recovery flow path 56. The recovery clamp 94 opens and closes the recovery flow path 56. In the installed state, the first waste liquid clamp 96 is fitted with the first waste liquid flow path 66. The first waste liquid clamp 96 opens and closes the first waste liquid flow path 66. In the installed state, the second waste liquid clamp 98 is fitted with the second waste liquid flow path 68. The second waste liquid clamp 98 opens and closes the second waste liquid flow path 68. In the installed state, the third waste liquid clamp 100 is fitted with the third waste liquid flow path 70. The third waste liquid clamp 100 opens and closes the third waste liquid flow path 70.

[0066] The reactor drive section 80 supports the bioreactor 24. In addition, the reactor drive section 80 is capable of rotating the bioreactor 24 about an axis orthogonal to the length direction of the bioreactor 24 in one direction and the opposite direction. The reactor drive section 80 operates using electric power supplied from a reactor drive circuit 118 Figure 2 ).

[0067] [1-3 Culture Control Device (Agitator Device) 110]

[0068] Figure 2 is a block diagram of the culture control device 110 provided in the cell culture device 10. The culture control device 110 controls the plurality of pumps 76, the plurality of clamps 78, and the reactor drive section 80, respectively. Note that the culture control device 110 functions as an agitator device that agitates the cell suspension of the bioreactor 24.

[0069] The culture control device 110 includes a sensor section 28, a controller 112, a pump drive circuit 114, a clamp drive circuit 116, and a reactor drive circuit 118. As the controller 112, for example, a computer is used. The controller 112 includes an arithmetic section 120 and a storage section 122.

[0070] The arithmetic unit 120 can be constituted by, for example, a processor such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), or the like. That is, the arithmetic unit 120 can be constituted by a processing circuit.

[0071] The arithmetic unit 120 is provided with a control section 124, an acquisition section 126, and a determination section 128. The control section 124, the acquisition section 126, and the determination section 128 can each be realized by executing a program stored in a storage section 122 by the arithmetic unit 120.

[0072] Note that at least a part of the control section 124, the acquisition section 126, and the determination section 128 can also be realized by an integrated circuit such as an ASIC (Application Specific Integrated Circuit), an FPGA (Field-Programmable Gate Array), or the like. In addition, at least a part of the control section 124, the acquisition section 126, and the determination section 128 can also be constituted by an electronic circuit including discrete devices.

[0073] The control section 124 outputs various action signals and controls the actions of the respective pumps 76, the actions of the respective clamps 78, and the action of the reactor driving section 80. The acquisition section 126 acquires the light-receiving amount of the transmitted light (or the light-receiving amount of the scattered light) over time on the basis of the electrical signals output from the sensor section 28. The determination section 128 performs various determinations.

[0074] The storage section 122 can be constituted by a not-shown volatile memory and a not-shown non-volatile memory. As the volatile memory, for example, a RAM (Random Access Memory) or the like can be cited. This volatile memory is used as a work memory of the processor and temporarily stores data and the like necessary for processing or arithmetic. As the non-volatile memory, for example, a ROM (Read Only Memory), a flash memory, or the like can be cited. This non-volatile memory is used as a storage memory for saving and stores programs, tables, charts, and the like. At least a part of the storage section 122 can also be provided to the above-described processor, integrated circuit, or the like.

[0075] The pump drive circuit 114 supplies electric power to the actuator of the pump 76 that is an operation target, in accordance with a pump operation signal output from the controller 112. The clamp drive circuit 116 supplies electric power to the actuator of the clamp 78 that is an operation target, in accordance with a clamp operation signal output from the controller 112. The reactor drive circuit 118 supplies electric power to the actuator of the reactor drive section 80, in accordance with a reactor operation signal output from the controller 112.

[0076] [2 Cell culture processing]

[0077] Figure 3 is a flowchart of the cell culture processing. The user uses the input device to perform a start operation of the cell culture processing. The arithmetic unit 120 starts the cell culture processing in accordance with the start operation Figure 3 the cell culture processing.

[0078] In step S1, the arithmetic unit 120 performs a cell charging process. The control unit 124 outputs a pump operation signal corresponding to the cell charging process to the pump drive circuit 114. In addition, the control unit 124 outputs a clamp operation signal corresponding to the cell charging process to the clamp drive circuit 116. As a result, the cell culture device 10 becomes the state shown in Figure 4 The cell suspension filled in the medical bag of the liquid supply section 16 flows in the first supply flow path 48 and the first circulation flow path 50 to be supplied to the bioreactor 24. Thus, the cells are charged to the first region 36 (the inside of the hollow fiber membrane 32) of the bioreactor 24. The control unit 124 stops the cell charging process at a prescribed timing. When step S1 ends, the processing moves to step S2.

[0079] In step S2, the arithmetic unit 120 performs a cell stirring process. The control unit 124 outputs a pump operation signal corresponding to the cell stirring process to the pump drive circuit 114. In addition, the control unit 124 outputs a clamp operation signal corresponding to the cell stirring process to the clamp drive circuit 116. As a result, the cell culture device 10 becomes the state shown in Figure 5 The details of the cell stirring process will be described later. In the cell stirring process, the cell suspension is stirred so that the cells are diffused in the cell suspension. A state in which the cells are uniformly diffused in the cell suspension is called a uniform diffusion state. In the uniform diffusion state, nutrients are equally distributed to the cells. Thus, by forming the uniform diffusion state, it is possible to accelerate the growth speed of the cells. In addition, by forming the uniform diffusion state, it is possible to increase the survival rate of the cells. When step S2 ends, the processing moves to step S3.

[0080] In step S3, the calculation unit 120 performs the cell filling process. The control unit 124 outputs a pump operation signal corresponding to the cell filling process to the pump drive circuit 114. In addition, the control unit 124 outputs a clamp operation signal corresponding to the cell filling process to the clamp drive circuit 116. Thus, the cell culture apparatus 10 becomes Figure 6 The state shown. The culture medium in the medical bag filled in the liquid supply unit 16 flows through the first supply flow path 48 and the first flow path 51a and is supplied to the bioreactor 24. In addition, the culture medium in the medical bag filled in the liquid supply unit 16 flows through the first supply flow path 48 and the second flow path 51b and is supplied to the bioreactor 24. As a result, the cells remaining in the first circulation flow path 50 move to the first area 36 (inside the hollow fiber membrane 32) of the bioreactor 24. Preferably, the amount of culture medium supplied from the first flow path 51a to the bioreactor 24 is equal to the amount of culture medium supplied from the second flow path 51b to the bioreactor 24. In addition, the culture medium circulates in the circulation path 132 formed by the bioreactor 24 and the second circulation flow path 54. Excess culture medium is discharged to the waste liquid storage unit 20. The control unit 124 stops the cell filling process at a time point when a predetermined packaging time has passed since the start of the cell filling process or when the supply amount of culture medium has reached a predetermined amount. When step S3 is completed, the process moves to step S4.

[0081] In step S4, the calculation unit 120 performs the cell culture process. Here, the control unit 124 outputs the pump operation signal corresponding to the cell culture process to the pump drive circuit 114. In addition, the control unit 124 outputs the clamp operation signal corresponding to the cell culture process to the clamp drive circuit 116. Thus, the cell culture device 10 becomes Figure 7 The state shown. The culture medium filled in the medical bag of the liquid supply unit 16 flows through the first supply flow path 48 and the second flow path 51b and is supplied to the first region 36 of the bioreactor 24 (inside the hollow fiber membrane 32). Furthermore, the culture medium circulates in the circulation path 130 formed by the bioreactor 24 and the first circulation flow path 50. This provides nutrients to the cells. Furthermore, the culture medium circulates in the circulation path 132 formed by the bioreactor 24 and the second circulation flow path 54. It should be noted that the remaining culture medium is discharged to the waste liquid storage unit 20.

[0082] In step S5, the determination unit 128 determines whether a predetermined culture time (predetermined time) has elapsed since the start of the cell culture step. The storage unit 122 pre-stores the predetermined time. If the predetermined time has elapsed (yes in step S5), the process proceeds to step S6. On the other hand, if the predetermined time has not elapsed (no in step S5), the cell culture step continues in step S4.

[0083] When moving from step S5 to step S6, the determination unit 128 determines Figure 3 Whether the termination condition of the series of cell culture processes shown is met. The storage unit 122 pre-stores a calibration curve representing the relationship between the amount of light received by the transmitted light (or scattered light) of the cell suspension and the number of cells. In addition, the storage unit 122 pre-stores a threshold value as the termination condition. The determination unit 128 estimates the number of cells contained in the cell suspension based on the amount of light received by the sensor unit 28 and the calibration curve. When the estimated number of cells is above the threshold, the determination unit 128 determines that the termination condition is met. It should be noted that the storage unit 122 may also store a calibration curve representing the relationship between the amount of light received by the transmitted light (or scattered light) of the cell suspension and the cell concentration. When the estimated number of cells is above the threshold (yes in step S6), the series of cell culture processes ends. On the other hand, when the estimated number of cells is less than the threshold (no in step S6), the process returns to the cell stirring process of step S2.

[0084] when Figure 3 When the cell culture process shown is completed, the control unit 124 supplies the stripping solution to the bioreactor 24 to transfer the cells inside the bioreactor 24 to the cell recovery unit 18 .

[0085] [3. Cell stirring process]

[0086] Figure 8 This is a flow chart of the cell stirring process. Figure 3 After step S1 shown, or when a negative determination is made in step S6, a cell stirring step described below is performed.

[0087] In step S11 (cell stirring process), the control unit 124 stirs the cell suspension inside the bioreactor 24. The control unit 124 outputs a pump operation signal for operating the first circulation pump 84 to the pump drive circuit 114. The first circulation pump 84 operates according to the pump operation signal. Figure 5 As shown, the cell suspension circulates in the circulation path 130 formed by the bioreactor 24 and the first circulation flow path 50. Thus, the cell suspension is stirred. Furthermore, the control unit 124 may output a reactor actuation signal to the reactor drive circuit 118 for activating the reactor drive unit 80. The reactor drive unit 80 rotates the bioreactor 24 alternately in one direction and in the opposite direction based on the reactor actuation signal. This allows the cell suspension to be more uniformly diffused more quickly. It should be noted that the process of step S11 continues until the process moves to step S14.

[0088] In step S12 (acquisition process), the acquisition section 126 acquires the light-receiving amount of the transmitted light based on the electrical signal output by the sensor section 28. The acquisition section 126 can also acquire the light-receiving amount of the scattered light instead of the light-receiving amount of the transmitted light. The acquisition section 126 acquires the light-receiving amount of the transmitted light at every prescribed sampling time.

[0089] In step S13 (determination process), the determination section 128 compares the degree of variation of the light-receiving amount of the transmitted light with a threshold value of the degree of variation. For example, the determination section 128 calculates the standard deviation of the light-receiving amount of the transmitted light as the degree of variation. The storage section 122 stores in advance the threshold value of the degree of variation (threshold value of the standard deviation). As the threshold value, an upper limit value of the allowable degree of variation (standard deviation) is set. The determination section 128 calculates the standard deviation of the light-receiving amount of the transmitted light based on the light-receiving amount of the transmitted light of the most recent prescribed number of times. The determination section 128 determines that the cells have reached the uniform diffusion state in the cell suspension based on the fact that the standard deviation is smaller than the threshold value. In the case where the standard deviation is smaller than the threshold value (Yes in step S13), the processing moves to step S14. On the other hand, in the case where the standard deviation is equal to or larger than the threshold value (No in step S13), the processing returns to step Sll. In this case, the processing of step Sll is continued.

[0090] When moving from step S13 to step S14, the control section 124 stops the stirring of the cell suspension. That is, the control section 124 stops the first circulating pump 84. In addition, the control section 124 stops the rotation of the bioreactor 24. When step S14 ends, the processing moves to Figure 3 The cell filling process (step S3) is shown in FIG. 6.

[0091] [4Effects of the Present Embodiment]

[0092] Figure 9A 、 Figure 9B is a time chart showing the time variation of the light-receiving amount of the transmitted light detected by the sensor section 28. In Figure 9A is shown the time variation of the light-receiving amount of the transmitted light in the cell culture processing without using the culture control device 110. In Figure 9B is shown the time variation of the light-receiving amount of the transmitted light in the cell culture processing with using the culture control device 110. In Figure 9A is shown the cell culture processing, the time Tl from the time point tl to the time point t2 is the execution time of the cell stirring process. In Figure 9A is shown the cell culture processing, the time T2 from the time point t2 to the time point t3 is the execution time of the cell filling process. In Figure 9A is shown the cell culture processing, the time T3 from the time point t3 to the time point t4 is the execution time of the cell culture process.

[0093] InFigure 9A In the cell stirring process shown, in the stage before stirring (before time point t1), most of the cells are located inside the bioreactor 24. Therefore, the culture medium flowing in the first circulation channel 50 contains few cells. Therefore, the turbidity of the liquid flowing in the first circulation channel 50 is low, and the amount of light received by the sensor unit 28 is large. When stirring is started at time point t1, the cells inside the bioreactor 24 flow in the first circulation channel 50. As a result, parts with high cell concentration and parts with low cell concentration appear alternately in the liquid flowing through the first circulation channel 50. As a result, the amount of light received by the sensor unit 28 changes over time. When stirring is continued, the amount of light received by the sensor unit 28 gradually converges to a roughly fixed value c. The fact that the amount of light received by the sensor unit 28 converges to a fixed value c means that the cells are evenly diffused in the liquid (cell suspension). Figure 9B The cell agitation step shown has already converged to the fixed value c at time point t2' earlier than time point t2. That is, the cell agitation step from time point t2' to time point t2 is unnecessary.

[0094] In this embodiment, the determination unit 128 compares the standard deviation (degree of variation) with a threshold value (predetermined degree). Figure 8 Step S13). Thus, the determination unit 128 can determine that a uniform diffusion state has been achieved. According to this embodiment, the cell stirring process can be executed for only the minimum necessary time. According to this embodiment, the execution time of the cell stirring process (time T1′) can be shortened, and the time for executing the cell stirring process can be shortened. Figure 3 Therefore, according to this embodiment, it is possible to contribute to the optimization of the cell culture process.

[0095] It should be noted that the present invention is not limited to the above disclosure, and various configurations can be adopted without departing from the gist of the present invention.

Claims

1. A stirring method comprising: a cell agitation step of agitating the cell suspension by circulating the cell suspension in a circulation circuit including the bioreactor; an acquisition step of acquiring, over time, a predetermined physical quantity related to the cell suspension at the measurement target site within the circulation path; as well as The determination step determines that the cells have been uniformly diffused in the cell suspension, that is, have reached a uniform diffusion state, based on the fact that the degree of variation in the physical quantity is smaller than a predetermined degree.

2. The stirring method according to claim 1, wherein The degree of variation of the physical quantity is grasped based on the standard deviations of the plurality of physical quantities within a predetermined unit time.

3. The stirring method according to claim 1, wherein The physical quantity is acquired using an optical sensor.

4. The stirring method according to claim 3, wherein The physical quantity is at least one of the amount of transmitted light transmitted through the cell suspension and the amount of scattered light scattered in the cell suspension.

5. The stirring method according to claim 1, wherein In the cell stirring step, if it is determined in the determination step that the uniform diffusion state has been achieved, stirring of the cell suspension is stopped.

6. The stirring method according to any one of claims 1 to 5, wherein In the cell stirring step, the bioreactor is rotated using an actuator to stir the cell suspension.

7. A stirring device, comprising: a control unit that stirs the cell suspension by circulating the cell suspension in a circulation circuit including the bioreactor; an acquiring unit that acquires, over time, a predetermined physical quantity related to the cell suspension at the measurement target site within the circulation path; and The determination unit determines that the cells are uniformly diffused in the cell suspension, that is, the uniform diffusion state is reached based on the fact that the degree of variation in the physical quantity is smaller than a predetermined degree.

8. The stirring device according to claim 7, wherein The degree of variation of the physical quantity is grasped based on a plurality of standard deviations of the physical quantities within a predetermined unit time.

9. The stirring device according to claim 7, wherein: The physical quantity is acquired using an optical sensor.

10. The stirring device according to claim 9, wherein The physical quantity is at least one of the amount of transmitted light transmitted through the cell suspension and the amount of scattered light scattered in the cell suspension.

11. The stirring device according to claim 7, wherein The control unit stops stirring the cell suspension when the determination unit determines that the uniform diffusion state has been reached.

12. The stirring device according to any one of claims 7 to 11, wherein The control unit rotates the bioreactor using an actuator, thereby stirring the cell suspension.

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