Cell transfer method and cell transfer device

Abstract

A cell transfer method comprises: a supply step for transferring cells remaining in a pipe (first circulation flow path (50)) connected to a bioreactor (24) to the bioreactor (24) by supplying a culture medium to the bioreactor (24) via the pipe in a state in which the cells remain in the pipe (first circulation flow path (50)); an acquisition step for acquiring a predetermined physical quantity relating to the culture medium flowing into the bioreactor (24); and a stopping step for stopping the supply of the culture medium on the basis of the physical quantity reaching the predetermined threshold value.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell transfer method and a cell transfer apparatus for moving cells remaining in a pipe to a bioreactor. BACKGROUND

[0002] A culture apparatus capable of performing a cell culture process is disclosed in Japanese Patent Application Publication No. 2002-148258. SUMMARY

[0003] For example, there is a cell culture apparatus in which a pipe through which a cell suspension flows is connected to a bioreactor. In order to culture cells using this cell culture apparatus, it is preferable to fill the bioreactor with cells by moving cells remaining in the pipe to the bioreactor before culture. This process is called a cell pack process. In order to optimize a cell culture process, a technique that contributes to optimization of the cell pack process is desired.

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

[0005] (1) The cell transfer method of the first application includes: a supply process of transferring cells remaining in a pipe to a bioreactor by supplying a culture medium to the bioreactor via the pipe in a state in which cells remain in the pipe; a measurement process of measuring a predetermined physical quantity related to the culture medium flowing into the bioreactor; and a stop process of stopping the supply of the culture medium based on the physical quantity reaching a predetermined threshold value.

[0006] In the first application, in the stop process, the supply of the culture medium is stopped based on the physical quantity becoming equal to or less than the predetermined threshold value. According to the present application, it is possible to perform the cell pack process for only a necessary minimum time. According to the present application, it is possible to shorten the execution time of the cell pack process, and it is possible to shorten the execution time of the cell culture process. Therefore, according to the present application, it is possible to optimize the cell culture process. In addition, according to the present application, it is possible to reduce the amount of consumption of the culture medium by shortening the cell pack time.

[0007] (2) In the cell transfer method described in the above item (1), the pipe can have a first pipe connected to a first port of the bioreactor and a second pipe connected to a second port of the bioreactor, and in the supply process, the culture medium can be supplied to the bioreactor via the first pipe and the culture medium can be supplied to the bioreactor via the second pipe.

[0008] (3) In the cell transfer method described in the above item (2), in the stopping process, the supply of the culture medium can be stopped based on a case where the physical quantity in the first pipe reaches the threshold value and the physical quantity in the second pipe reaches the threshold value.

[0009] (4) In the cell transfer method described in the above item (2) or (3), in the supply process, the culture medium can be supplied to the first pipe and the second pipe, respectively, in such a manner that a difference between a time point at which the physical quantity in the first pipe reaches the threshold value and a time point at which the physical quantity in the second pipe reaches the threshold value becomes equal to or less than a prescribed difference.

[0010] According to the above configuration, it is possible to end the cell filling process in the shortest time.

[0011] (5) In the cell transfer method described in the above item (4), in the aforementioned acquisition process, the physical quantity can be acquired over time, and in the supply process, the supply of the culture medium to the first pipe and the second pipe can be adjusted based on a temporal change in the physical quantity.

[0012] (6) In the cell transfer method described in any one of the above items (1) to (5), the physical quantity can be acquired using an optical sensor.

[0013] (7) In the cell transfer method described in any one of the above items (1) to (6), the physical quantity can be at least one of an amount of light transmitted through the culture medium and an amount of light scattered in the culture medium.

[0014] (8) The cell transfer apparatus of the second application includes: a supply unit that transfers cells remaining in a pipe connected to a bioreactor to the bioreactor by supplying a culture medium to the bioreactor via the pipe in a state where the cells remain in the pipe; an acquisition unit that acquires a prescribed physical quantity related to the culture medium flowing into the bioreactor; and a stopping unit that stops the supply of the culture medium based on a case where the physical quantity reaches a prescribed threshold value.

[0015] According to the second application, the same effects as those of the first application can be obtained.

[0016] (9) In the cell transfer apparatus described in the above item (8), the pipe can include a first pipe connected to a first port of the bioreactor and a second pipe connected to a second port of the bioreactor, and the supply unit can supply the culture medium to the bioreactor via the first pipe and supply the culture medium to the bioreactor via the second pipe.

[0017] (10) The cell transfer device according to the above item (9), wherein the stop unit stops the supply of the culture medium based on a case where the physical quantity in the first pipe reaches the threshold value and the physical quantity in the second pipe reaches the threshold value.

[0018] (11) The cell transfer device according to the above item (9) or (10), wherein the supply unit supplies the culture medium to the first pipe and the second pipe in such a manner that a difference between a time point at which the physical quantity in the first pipe reaches the threshold value and a time point at which the physical quantity in the second pipe reaches the threshold value becomes equal to or less than a predetermined difference.

[0019] According to the configuration according to the above item (11), the same effects as those according to the above item (4) can be obtained.

[0020] (12) The cell transfer device according to the above item (11), wherein the acquisition unit acquires the physical quantity over time, and the supply unit adjusts the supply of the culture medium to the first pipe and the second pipe based on a temporal change in the physical quantity.

[0021] (13) The cell transfer device according to any one of the above items (8) to (12), wherein the physical quantity is acquired using an optical sensor.

[0022] (14) The cell transfer device according to any one of the above items (8) to (13), wherein the physical quantity is at least one of an amount of light transmitted through the culture medium and an amount of light scattered in the culture medium.

[0023] According to the present application, it is possible to optimize a cell culture process. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic view showing a cell culture device.

[0025] Figure 2 is a block diagram showing a culture control device (cell transfer device) provided in the cell culture device.

[0026] Figure 3 is a flowchart of a cell culture process.

[0027] Figure 4 is a schematic view showing a cell inputting process.

[0028] Figure 5 is a schematic view showing a cell stirring process.

[0029] Figure 6is a schematic view showing a cell filling process.

[0030] Figure 7 is a schematic view showing a cell culture process.

[0031] Figure 8 is a flowchart showing a cell filling process.

[0032] Figure 9 is a time chart showing a time change in the light receiving amount of the transmitted light detected by the sensor section. DETAILED DESCRIPTION

[0033] [1 Configuration of Cell Culture Device 10]

[0034] Figure 1 is a schematic view showing a cell culture device 10. 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-described cells.

[0035] [1-1 Cell Culture Circuit 12]

[0036] A liquid flows in the cell culture circuit 12. As the liquid, a cell suspension, a culture medium, a washing liquid, a peeling liquid, 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 in accordance with the cells to be cultured. As the culture medium, for example, MEM (Minimum Essential Media) can be used. The washing liquid is a solution for washing the inside of the cell culture circuit 12. As the washing liquid, 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 liquid is a solution for peeling cells from the bioreactor 24 of the cell culture circuit 12. As the peeling liquid, for example, a trypsin solution, an EDTA solution, and the like can be given. Note that the culture medium, the washing liquid, and the peeling liquid are not limited to the above-described liquids.

[0037] The cell culture circuit 12 is a disposable article that is replaced each time a cell culture process is performed. The cell culture circuit 12 is provided with the liquid supply section 16, the cell recovery section 18, the waste liquid accommodation section 20, and the culture main body 22.

[0038] The liquid supply section 16 is provided with a plurality of medical bags (not shown). In each of the medical bags, a liquid to be supplied to the culture main body 22 is filled. For example, a cell suspension is filled in a first medical bag. A culture medium is filled in a second medical bag. A washing liquid is filled in a third medical bag. A stripping liquid is filled in a fourth medical bag. Note that a plurality of kinds of culture media can be separately filled in the medical bags.

[0039] The cell recovery section 18 and the waste liquid storage section 20 are each provided with 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 a waste liquid generated by the culture main body 22.

[0040] The culture main body 22 is provided with a bioreactor 24, a flow path 26, sensor sections 28, 29, and a gas exchange section 30.

[0041] The bioreactor 24 is provided with 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 a length direction of the bioreactor 24. The hollow fiber membrane 32 is composed of, for example, a high molecular material. The hollow fiber membrane 32 has 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.

[0042] The bioreactor 24 is provided with 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 an outer peripheral surface of each of the hollow fiber membranes 32 and an 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.

[0043] The housing 34 is provided with 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. Thereby, 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. Thereby, the second port 42 communicates with the first region 36.

[0044] 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 is provided with 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.

[0045] The flow path 26 is composed of a plurality of tubes (piping) through which a liquid flows. Each tube 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.

[0046] The first supply flow path 48 introduces various liquids of the liquid supply section 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 section 16. The first upstream flow path 48a is connected to the medical bag of the liquid supply section 16. In addition, each first upstream flow path 48a is connected to the first downstream flow path 48b. The first downstream flow path 48b is connected to the first confluence section 60 in the first circulation flow path 50.

[0047] 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 section 60 is provided in the first circulation flow path 50. A portion of the first circulation flow path 50 divided by the first end portion 50a and the first confluence section 60 is referred to as a first flow path 51a (first piping). A portion of the first circulation flow path 50 divided by the second end portion 50b and the first confluence section 60 is referred to as a second flow path 51b (second piping). The recovery branch section 64 is provided in the second flow path 51b. In addition, the first branch section 72 is provided in a portion of the second flow path 51b between the recovery branch section 64 and the second end portion 50b.

[0048] The second supply flow path 52 introduces various liquids of the liquid supply section 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 section 16. The second upstream flow path 52a is connected to the medical bag of the liquid supply section 16. In addition, each second upstream flow path 52a is connected to the second downstream flow path 52b. The second downstream flow path 52b is connected to the second confluence section 62 in the second circulation flow path 54.

[0049] 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 (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.

[0050] 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.

[0051] 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 storage portion 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. The 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. The 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. The second end portion 66b of the first waste liquid flow path 66 and the second end portion 68b of the second waste liquid flow path 68 are respectively connected to the first end portion 70a of the third waste liquid flow path 70. The second end portion 70b of the third waste liquid flow path 70 is connected to the medical bag of the waste liquid storage portion 20.

[0052] The sensor portion 28 is provided in the first flow path 51a. On the other hand, the sensor portion 29 is provided in the second flow path 51b. The sensor portion 28 detects a prescribed physical quantity related to the liquid flowing in the first flow path 51a. The sensor portion 29 detects a prescribed physical quantity related to the liquid flowing in the second flow path 51b. The prescribed physical quantity is a physical quantity proportional to the number of cells in the liquid. For example, the sensor portions 28, 29 each include 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 Figure 2) output. Note that the optical sensor can also receive scattered light (front scattered light, side scattered light, back scattered light, etc.) instead of transmitted light. In addition, the sensor sections 28, 29 can each have a sensor (e.g., a permittivity sensor) having two or more electrodes instead of the light source and the optical sensor.

[0053] The gas exchange section 30 is provided in a portion of the second circulation flow path 54 between the second confluence section 62 and the third port 44. The gas exchange section 30 supplies a gas having a prescribed composition to the liquid (culture medium) flowing in the second circulation flow path 54. The gas used in the gas exchange section 30 has, for example, a composition close to that of air. That is, the gas includes nitrogen, oxygen, and carbon dioxide.

[0054] [1-2 Support device 14]

[0055] The cell culture circuit 12 described above is detachable with respect to the support device 14. The support device 14 has a cassette that supports the cell culture circuit 12. The support device 14 is a reusable member that can be used multiple times.

[0056] The support device 14 has a plurality of pumps 76, a plurality of clamps 78, and a reactor drive section 80. The plurality of pumps 76, the plurality of clamps 78, and the reactor drive section 80 each have an electric actuator. Note that the plurality of pumps 76, the plurality of clamps 78, and the reactor drive section 80 can each have a fluidic actuator.

[0057] Each pump 76 can impart a flow force to the liquid in the flow path 26 by pressing the tube that forms the flow path 26. Each pump 76 operates using electric power supplied from a pump drive circuit 114 Figure 2 ).

[0058] 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 Fig. 6, the state in which the cell culture circuit 12 is mounted to the support device 14 is referred to as an "installed state".

[0059] In the installed state, the first downstream flow path 48b is fitted to the first supply pump 82. The first supply pump 82 imparts a flow force to the liquid in the first supply flow path 48 in a direction from the liquid supply section 16 toward the first circulation flow path 50.

[0060] In the installed state, the second flow path 51b of the first circulation flow path 50 is fitted with the first circulation pump 84. The first circulation pump 84 imparts a flow force to the liquid in the first circulation flow path 50 in the 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 the liquid in the first circulation flow path 50 in the direction from the first port 40 toward the second port 42.

[0061] In the installed state, the second downstream flow path 52b is fitted with the second supply pump 86. The second supply pump 86 imparts a flow force to the liquid in the second supply flow path 52 in the direction from the liquid supply portion 16 toward the second circulation flow path 54.

[0062] In the installed state, the second circulation flow path 54 is fitted with the second circulation pump 88. The second circulation pump 88 imparts a flow force to the liquid in the second circulation flow path 54 in the 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 the liquid in the second circulation flow path 54 in the direction from the third port 44 toward the fourth port 46.

[0063] Each clamp 78 can close the flow path 26 by compressing the 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 drive circuit 116 Figure 2 ).

[0064] The plurality of clamps 78 includes 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.

[0065] In the installed state, one first upstream flow path 48a is fitted with one first supply clamp 90. In other words, each first upstream flow path 48a is supported by any one of the first supply clamps 90. The first supply clamp 90 opens and closes the first supply flow path 48.

[0066] In the installed state, one second upstream flow path 52a is fitted with one second supply clamp 92. In other words, each second upstream flow path 52a is supported by any one of the second supply clamps 92. The second supply clamp 92 opens and closes the second supply flow path 52.

[0067] 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.

[0068] 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 ).

[0069] [1-3 Culture Control Device (Cell Transfer Device) 110]

[0070] 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 a cell transfer device that transfers cells remaining in the first circulation flow path 50 to the bioreactor 24.

[0071] The culture control device 110 includes a sensor section 28, 29, 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 can be used. The controller 112 includes an arithmetic section 120 and a storage section 122.

[0072] The arithmetic section 120 can be constituted by a processor such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), or the like. That is, the arithmetic section 120 can be constituted by a processing circuit.

[0073] The arithmetic section 120 includes a supply control section 124 (supply section), an acquisition section 126, and a stop control section 128 (stop section). The supply control section 124, the acquisition section 126, and the stop control section 128 can be realized by executing a program stored in the storage section 122 by the arithmetic section 120, respectively.

[0074] Note that at least a part of the supply control section 124, the acquisition section 126, and the stop control section 128 can 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 supply control section 124, the acquisition section 126, and the stop control section 128 can be constituted by an electronic circuit including discrete devices.

[0075] The supply control section 124 performs various controls related to the supply of the liquid from the liquid supply section 16 to the bioreactor 24. 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 electric signals output from the sensor sections 28, 29. The stop control section 128 performs various controls related to the stop of the supply of the liquid from the liquid supply section 16 to the bioreactor 24.

[0076] The storage section 122 can be constituted by a not-illustrated volatile memory and a not-illustrated non-volatile memory. As the volatile memory, for example, a RAM (Random Access Memory) or the like can be given. This volatile memory can be used as a work memory of the processor, and temporarily stores data and the like required for processing or operation. As the non-volatile memory, for example, a ROM (Read Only Memory), a flash memory, or the like can be given. This non-volatile memory can be used as a storage memory for storing programs, tables, charts, and the like. At least a part of the storage section 122 can be provided to the processor, the integrated circuit, or the like described above.

[0077] 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. Each 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.

[0078] [2 Cell culture processing]

[0079] Figure 3 is a flowchart of the cell culture processing. The user performs a start operation of the cell culture processing using the input device. The operation section 120 starts the cell culture processing shown in Figure 3 Fig. 1, in accordance with the start operation.

[0080] In step S1, the arithmetic unit 120 performs a cell feeding process. The supply control unit 124 outputs a pump operation signal corresponding to the cell feeding process to the pump drive circuit 114. In addition, the supply control unit 124 outputs a gripper operation signal corresponding to the cell feeding process to the gripper drive circuit 116. Thus, the cell culture device 10 becomes the state shown in FIG. 1. The cell suspension liquid filled in the medical bag of the liquid supply portion 16 flows in the first supply flow path 48 and the first circulation flow path 50 and is supplied to the bioreactor 24. Thus, the cells are fed to the first region 36 (the inside of the hollow fiber membrane 32) of the bioreactor 24. The stop control unit 128 stops the cell feeding process at a prescribed time point. When step S1 ends, the process shifts to step S2. Figure 4

[0081] In step S2, the arithmetic unit 120 performs a cell stirring process. The supply control unit 124 outputs a pump operation signal corresponding to the cell stirring process to the pump drive circuit 114. In addition, the supply control unit 124 outputs a gripper operation signal corresponding to the cell stirring process to the gripper drive circuit 116. Thus, the cell culture device 10 becomes the state shown in FIG. 2. The cell suspension liquid circulates in the circulation path 130 formed by the bioreactor 24 and the first circulation flow path 50. Thus, the cell suspension liquid is stirred, and the cells are diffused in the cell suspension liquid. A state in which the cells are uniformly diffused in the cell suspension liquid is referred to as a uniform diffusion state. In the uniform diffusion state, nutrients are equally distributed to the cells. Note that the supply control unit 124 can also output a reactor operation signal for causing the reactor drive unit 80 to operate to the reactor drive circuit 118. The reactor drive unit 80 causes the bioreactor 24 to alternately rotate in one direction and the opposite direction in accordance with the reactor operation signal. Thus, it is possible to make the cell suspension liquid become the uniform diffusion state more quickly. The stop control unit 128 stops the cell stirring process at a time point at which a prescribed stirring time has elapsed from the start of the cell stirring process. When step S2 ends, the process shifts to step S3. Figure 5

[0082] In step S3, the arithmetic unit 120 performs a cell filling process. The supply control unit 124 outputs a pump operation signal corresponding to the cell filling process to the pump drive circuit 114. In addition, the supply control unit 124 outputs a gripper operation signal corresponding to the cell filling process to the gripper drive circuit 116. Thus, the cell culture device 10 becomes the state shown in FIG. 3. The cell suspension liquid filled in the medical bag of the liquid supply portion 16 flows in the second supply flow path 52 and the second circulation flow path 54 and is supplied to the bioreactor 24. Thus, the cells are filled in the second region 38 (the outside of the hollow fiber membrane 32) of the bioreactor 24. The stop control unit 128 stops the cell filling process at a prescribed time point. When step S3 ends, the process shifts to step S4. Figure 6 ​​The state shown. Details of the cell filling process will be described later. The medium filled in the medical bag of the liquid supply part 16 flows in the first supply flow path 48 and the first flow path 51a and is supplied to the bioreactor 24. In addition, the medium filled in the medical bag of the liquid supply part 16 flows in the first supply flow path 48 and the second flow path 51b and is supplied to the bioreactor 24. Thereby, the cells remaining in the first circulation flow path 50 move to the first region 36 (the inside of the hollow fiber membrane 32) of the bioreactor 24. In addition, the medium circulates in the circulation path 132 formed by the bioreactor 24 and the second circulation flow path 54. The remaining portion of the medium is discharged to the waste liquid container 20. When step S3 ends, the process shifts to step S4.

[0083] In step S4, the operation part 120 performs a cell culture process. Here, the supply control part 124 outputs a pump operation signal corresponding to the cell culture process to the pump drive circuit 114. In addition, the supply control part 124 outputs a clamp operation signal corresponding to the cell culture process to the clamp drive circuit 116. As a result, the cell culture device 10 becomes Figure 7 the state shown. The medium filled in the medical bag of the liquid supply part 16 flows in the first supply flow path 48 and the second flow path 51b and is supplied to the first region 36 (the inside of the hollow fiber membrane 32) of the bioreactor 24. In addition, the medium circulates in the circulation path 130 formed by the bioreactor 24 and the first circulation flow path 50. Thereby, nutrients are supplied to the cells. In addition, the medium circulates in the circulation path 132 formed by the bioreactor 24 and the second circulation flow path 54. Note that the remaining portion of the medium is discharged to the waste liquid container 20.

[0084] In step S5, the stop control part 128 determines whether or not a predetermined culture time (predetermined time) has elapsed since the start of the cell culture process. The storage part 122 stores the predetermined time in advance. In the case where the predetermined time has elapsed (step S5: Yes), the process shifts to step S6. On the other hand, in the case where the predetermined time has not elapsed (step S5: No), the cell culture process of step S4 is continued.

[0085] When the process shifts from step S5 to step S6, the stop control part 128 determines Figure 3The end condition of the series of cell culture processes is determined. The storage section 122 stores in advance a calibration curve indicating the relationship between the light receiving amount of the transmitted light (or scattered light) of the cell suspension and the number of cells. In addition, the storage section 122 stores in advance a threshold value as the end condition. The stop control section 128 estimates the number of cells contained in the cell suspension based on the light receiving amount of the sensor section 28 and the calibration curve. In the case where the estimated number of cells is equal to or more than the threshold value, the stop control section 128 determines that the end condition is satisfied. Note that the storage section 122 can store a calibration curve indicating the relationship between the light receiving amount of the transmitted light (or scattered light) of the cell suspension and the cell concentration. In the case where the estimated number of cells is equal to or more than the threshold value (step S6: Yes), the series of cell culture processes ends. On the other hand, in the case where the estimated number of cells is less than the threshold value (step S6: No), the process returns to the cell stirring process of step S2.

[0086] When Figure 3 When the cell culture process ends, the supply control section 124 supplies the stripping solution to the bioreactor 24 to transfer the cells in the inside of the bioreactor 24 to the cell recovery section 18.

[0087] [3 Cell filling process]

[0088] Figure 8 is a flowchart of the cell filling process. In Figure 3 After step S2 shown above, the cell filling process described below is performed.

[0089] In step Sll (supply process), the supply control section 124 controls the first supply pump 82 and the first circulation pump 84 to supply the culture medium to the bioreactor 24 via the first supply flow path 48 and the first flow path 51a. In addition, the supply control section 124 controls the first supply pump 82 and the first circulation pump 84 to supply the culture medium to the bioreactor 24 via the first supply flow path 48 and the second flow path 51b. The cells remaining in the first flow path 51a are moved to the bioreactor 24 together with the culture medium. The cells remaining in the second flow path 51b are moved to the bioreactor 24 together with the culture medium. Thus, the cell concentration in the first flow path 51a and the cell concentration in the second flow path 51b are each reduced.

[0090] Here, the time point at which the cell concentration in the first flow path 51a becomes below the prescribed concentration threshold is referred to as a first time point. Also, the time point at which the cell concentration in the second flow path 51b becomes below the prescribed concentration threshold is referred to as a second time point. The supply control section 124 preferably controls the first supply pump 82 and the first circulation pump 84 in such a manner that the difference between the first time point and the second time point becomes below a prescribed difference. Thereby, it is possible to end the cell filling process in the shortest time. For example, it is preferable that the ratio of the flow rate of the medium supplied from the first supply flow path 48 to the first flow path 51a to the flow rate of the medium supplied from the first supply flow path 48 to the second flow path 51b and the ratio of the volume of the first flow path 51a to the volume of the second flow path 51b coincide. The storage section 122 stores in advance the target flow rate of the first supply pump 82 and the target flow rate of the first circulation pump 84. The supply control section 124 controls the first supply pump 82 and the first circulation pump 84 respectively in such a manner that each of the target flow rates stored in the storage section 122 is attained. Note that it is not necessary that the difference between the first time point and the second time point becomes below the prescribed difference. That is, the difference between the first time point and the second time point can be greater than the prescribed difference.

[0091] In step S12 (acquisition process), the acquisition section 126 acquires the light receiving amount of each of the transmitted lights based on the electric signals output by the sensor sections 28, 29 respectively. The acquisition section 126 can acquire the light receiving amount of the scattered light instead of the transmitted light. The acquisition section 126 acquires the light receiving amount of each of the transmitted lights at every prescribed sampling time.

[0092] In step S13, the stop control section 128 calculates the cell concentration of the medium in the first flow path 51a based on the light receiving amount of the transmitted light (or the scattered light) detected by the sensor section 28 and the calibration curve. Similarly, the stop control section 128 calculates the cell concentration of the medium in the second flow path 51b based on the light receiving amount of the transmitted light (or the scattered light) detected by the sensor section 29 and the calibration curve. Further, the stop control section 128 compares each of the calculated cell concentrations with a prescribed concentration threshold. The calibration curve indicates the relationship between the light receiving amount and the cell concentration. As the concentration threshold, the upper limit value of the allowable cell concentration is set. The calibration curve and the concentration threshold are stored in the storage section 122 in advance respectively. In the case where each of the cell concentrations is below the concentration threshold (step S13: YES), the process shifts to step S14. On the other hand, in the case where at least one of the cell concentrations is greater than the concentration threshold (step S13: NO), the process returns to step Sll. In this case, the process of step Sll is continued.

[0093] When the process shifts from step S13 to step S14 (stop process), the stop control section 128 stops the supply of the medium to the bioreactor 24. That is, the stop control section 128 stops the first supply pump 82 and the first circulation pump 84. When step S14 ends, the process shifts toFigure 3 The cell culture process (step S4) shown.

[0094] Note that in step S13, the stop control section 128 converts the light reception amount into a cell concentration, and performs stop determination based on a comparison result of the cell concentration and the concentration threshold value. Alternatively, the stop control section 128 can perform stop determination based on a comparison result of the light reception amount and the light threshold value. The light reception amount is correlated with the cell concentration. As the light reception amount of the transmitted light increases, the cell concentration decreases. On the other hand, as the light reception amount of the scattered light increases, the cell concentration increases.

[0095] [4Effects of the Present Embodiment]

[0096] Figure 9 is a time chart showing a time variation of the light reception amount of the transmitted light detected by the sensor section 28. Note that a time variation of the light reception amount of the transmitted light detected by the sensor section 29 is also the same as Figure 9 As shown in Figure 9 , the light reception amount of the transmitted light gradually increases from the time point tl at which the cell filling process is started. That is, by performing the cell filling process, the cells remaining in the first flow path 51a are reduced. Then, the turbidity of the culture medium in the first flow path 51a is reduced, and the light reception amount of the transmitted light increases. The amount of increase per unit time of the light reception amount gradually increases with the passage of time. The amount of increase per unit time of the light reception amount gradually decreases after increasing to a certain degree, and approaches zero at the time point t2. The amount of increase per unit time of the light reception amount approaching zero indicates that most of the cells in the first flow path 51a have moved to the bioreactor 24.

[0097] In the present embodiment, the stop control section 128 stops the supply of the culture medium based on the case where the cell concentration (physical quantity) becomes below the concentration threshold value (predetermined threshold value). According to the present embodiment, it is possible to perform the cell filling process for only a necessary minimum time. According to the present embodiment, it is possible to shorten the execution time of the cell filling process, and it is possible to shorten Figure 3 the execution time of the cell culture process shown. Therefore, according to the present embodiment, it is possible to optimize the cell culture process. In addition, according to the present embodiment, by shortening the cell filling time, it is possible to reduce the consumption amount of the culture medium.

[0098] [5Other Embodiments]

[0099] Either of the sensor sections 28, 29 can be provided in the cell culture circuit 12 alone. In this case, in step Sll, the supply control section 124 controls the first supply pump 82 and the first circulation pump 84 in such a manner that the first time point at which the cell concentration in the first flow path 51a becomes equal to or lower than the prescribed concentration threshold value and the second time point at which the cell concentration in the second flow path 51b becomes equal to or lower than the prescribed concentration threshold value are substantially the same.

[0100] Note that the present application is not limited to the above-described disclosure, and various configurations can be employed without departing from the gist of the present application.

Claims

1. A cell transfer method comprising: a supply step of supplying a culture medium to the bioreactor via the pipe connected to the bioreactor while cells remain in the pipe, thereby transferring the cells remaining in the pipe to the bioreactor; an acquisition step of acquiring a predetermined physical quantity related to the culture medium flowing into the bioreactor; and The stopping step stops the supply of the culture medium when the physical quantity reaches a predetermined threshold value.

2. The cell transfer method according to claim 1, wherein The piping includes a first piping connected to a first port of the bioreactor and a second piping connected to a second port of the bioreactor. In the supplying step, the culture medium is supplied to the bioreactor via the first pipe, and the culture medium is supplied to the bioreactor via the second pipe.

3. The cell transfer method according to claim 2, wherein In the stopping step, supply of the culture medium is stopped based on the fact that the physical quantity in the first pipe reaches the threshold value and the physical quantity in the second pipe reaches the threshold value.

4. The cell transfer method according to claim 2, wherein In the supplying step, the culture medium is supplied to the first and second pipes respectively so that the difference between the time point when the physical quantity in the first pipe reaches the threshold value and the time point when the physical quantity in the second pipe reaches the threshold value becomes equal to or smaller than a predetermined difference.

5. The cell transfer method according to claim 4, wherein In the acquisition step, the physical quantity is acquired over time. In the supply step, the supply of the culture medium to the first pipe and the second pipe is adjusted based on the temporal change of the physical quantity.

6. The cell transfer method according to any one of claims 1 to 5, wherein The physical quantity is acquired using an optical sensor.

7. The cell transfer method according to claim 6, wherein The physical quantity is at least one of the amount of transmitted light passing through the culture medium and the amount of scattered light scattered in the culture medium.

8. A cell transfer device comprising: a supply unit that supplies a culture medium to the bioreactor via the pipe connected to the bioreactor while cells remain in the pipe, thereby transferring the cells remaining in the pipe to the bioreactor; an acquisition unit that acquires a predetermined physical quantity related to the culture medium flowing into the bioreactor; and A stopping unit stops the supply of the culture medium when the physical quantity reaches a predetermined threshold value.

9. The cell transfer device according to claim 8, wherein The piping includes a first piping connected to a first port of the bioreactor and a second piping connected to a second port of the bioreactor. The supply unit supplies the culture medium to the bioreactor via the first pipe, and supplies the culture medium to the bioreactor via the second pipe.

10. The cell transfer device according to claim 9, wherein The stopping unit stops the supply of the culture medium based on the fact that the physical quantity in the first pipe reaches the threshold value and the physical quantity in the second pipe reaches the threshold value.

11. The cell transfer device according to claim 9, wherein The supply unit supplies the culture medium to each of the first and second pipes so that a difference between a time point when the physical quantity in the first pipe reaches the threshold value and a time point when the physical quantity in the second pipe reaches the threshold value becomes equal to or smaller than a predetermined difference.

12. The cell transfer device according to claim 11, wherein The acquisition unit acquires the physical quantity over time. The supply unit adjusts the supply of the culture medium to each of the first pipe and the second pipe based on the temporal change of the physical quantity.

13. The cell transfer device according to any one of claims 8 to 12, wherein The physical quantity is acquired using an optical sensor.

14. The cell transfer device according to claim 13, wherein The physical quantity is at least one of the amount of transmitted light passing through the culture medium and the amount of scattered light scattered in the culture medium.

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