JP2011103882A - Cell culture apparatus, and method and program for culturing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture apparatus for preventing contamination and hardly intermingling with impurities. <P>SOLUTION: The cell culture apparatus includes: a culture module 10 divided into an outer chamber 10b and an inner chamber 10a; a culture solution vessel 4 which receives culture solution for supplying to the culture module 10; a cleaning liquid container 5 which receives cleaning liquid for washing the culture module 10; pumps P1 and P2 for supplying the culture solution and the cleaning liquid; and a control portion 30 for controlling the pumps P1 and P2. The cleaning liquid is supplied prior to culturing cells. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell culture device, a cell culture method, and a cell culture program. The present invention particularly relates to an apparatus for culturing adherent cells, a culture method, and a culture program.

  In recent years, attention has been focused on a technique for culturing cells in vitro and reconstructing tissues with the cultured cells. Therefore, in order to cultivate cells easily in large quantities, it is required to semi-automate or automate the cell culture in vitro and the recovery of the cells cultured thereby.

  For example, a culture apparatus that can circulate a culture solution in a cell culture vessel has been proposed (see Patent Document 1 and Patent Document 2). A culture apparatus capable of exchanging the culture medium in the cell culture vessel has also been proposed (see Patent Document 3). Furthermore, a culture apparatus capable of automatically replacing the culture solution in the cell culture container is disclosed (see Patent Document 4).

JP-A-5-260958 (claims, etc.) JP-A-5-276922 Japanese Patent Laid-Open No. Hei 8-172956 (claims, etc.) JP 2002-262856 A

  Here, in the cell culture devices of Patent Document 1 and Patent Document 2 described above, it is preferable to culture the cells in a state where there is little contamination with impurities that affect cell culture. Therefore, the cell culture apparatus may be sterilized before culturing the cells. For such sterilization, for example, the cell culture apparatus is sterilized by irradiation with gamma rays. However, when gamma rays are applied to the cell culture apparatus, some members constituting the cell culture apparatus may be eluted by gamma rays, and there is a problem that impurities such as the elution affect cells to be cultured.

  This invention is made | formed in view of this request, Comprising: The subject which this invention tends to solve is providing the cell culture apparatus which is hard to contaminate and is hard to mix an impurity.

  The cell culture device according to the present invention stores a culture module partitioned into an outer chamber and an inner chamber, a culture solution container for storing a culture solution to be supplied to the culture module, and a cleaning solution for cleaning the culture module. A cell culture apparatus having a cleaning liquid container, a pump for supplying the culture liquid and the cleaning liquid, and a controller for controlling the pump, wherein the cleaning liquid is supplied before culturing the cells. is there.

  In the cell culture device according to the present invention, the entire culture circuit including the culture module, the culture solution container, and the washing solution container is detachably attached to a control device having a pump and a control unit.

  In the cell culture device according to the present invention, the control unit may control the flow rate for supplying the culture solution and the flow rate for supplying the washing solution.

  Furthermore, in the cell culture device according to the present invention, at least one of the culture solution container and the washing solution container may be disposed on the antigravity direction side of the culture module.

  Furthermore, in the cell culture apparatus according to the present invention, a valve for opening and closing the flow path is provided in the flow path connecting the culture module and each part. The valve is preferably a multi-way cock having a tube projecting in a plurality of directions and a rotatable turning member for switching a fluid flow path, and a turning member by fixing the stopper portion of the multi-way cock. Is provided with a multi-way stopcock switching device. The multi-way cock switching device preferably includes a drive source for applying a rotational driving force to the rotational member, and one or more gripping columns connected to the rotational shaft of the drive source and gripping the rotational member. A clamp, and the clamp rotates the rotation member based on the rotation operation of the drive source controlled by the control unit. Further, the valve may be a system that does not allow entry of germs from the outside, for example, a pinch valve.

  In another embodiment of the present invention, the cell culture method includes a washing step for washing the culture module partitioned into the outer chamber and the inner chamber, a culture solution filling step for filling the culture module with the culture solution, and an outer chamber. A cell filling step of injecting the cells into the cell, a culture step of culturing the cells while supplying the culture solution to the inner chamber, and a recovery step of collecting the cells cultured in the culture module.

  In another embodiment of the present invention, the cell culture program includes a washing step for supplying a washing solution to the culture module partitioned into the outer chamber and the inner chamber, a culture solution filling step for supplying the culture solution to the culture module, A control unit includes a culture step of culturing cells while supplying a culture solution to the inner chamber, and a recovery step of supplying at least one of a release agent for peeling cells, the cleaning agent, and the culture solution to the culture module To be executed.

  ADVANTAGE OF THE INVENTION According to this invention, the cell culture apparatus which is hard to contaminate and is hard to mix an impurity can be provided.

It is a block diagram which shows the structure of the cell culture apparatus which concerns on this Embodiment. It is a block diagram which shows the structure of the cell culture circuit which the cell culture apparatus of FIG. 1 has. It is a perspective view which shows the external appearance of the culture module which the cell culture apparatus of FIG. 1 has. It is an expansion perspective view which shows the external appearance of the hollow fiber contained in the culture module of FIG. It is an expanded sectional view at the time of cut | disconnecting the culture module of FIG. 3 along the AA line. It is a perspective view which shows the external appearance of the control apparatus which the cell culture apparatus of FIG. 1 has. It is a block diagram which shows the structure of the control apparatus of FIG. It is a flowchart which shows the flow of the cell culture method which concerns on this Embodiment. It is a flowchart which shows the flow of step S101 of FIG. It is a block diagram which shows the state of the cell culture apparatus of FIG. 1 in step S201. It is a block diagram which shows the state of the cell culture apparatus of FIG. 1 in step S202. It is a block diagram which shows the state of the cell culture apparatus of FIG. 1 in step S203. It is a block diagram which shows the state of the cell culture apparatus 1 of FIG. 1 in step S204. FIG. 14 is a block diagram showing a state after FIG. 13 in the cell culture device of FIG. 1. It is a block diagram which shows the state of the cell apparatus of FIG. 1 in step S102 and step S104. It is a block diagram which shows the state of the cell apparatus of FIG. 1 in step S102. It is a block diagram which shows the state of the cell apparatus of FIG. 1 in step S105. It is a block diagram which shows the state of the cell apparatus of FIG. 1 after FIG. 17 among step S105. It is a disassembled perspective view which shows the structure of the multiway cock used for the cell culture apparatus which concerns on the modification of this Embodiment. It is a perspective view which shows the state which assembled the clamp and the stopper part among the multiway cocks of FIG. It is a top view which shows an example of the relationship between the rotation member when the multiway cock of FIG. 19 rotates with the rotation of a pulse motor, and the clamp rotates, and the flow path of a three-way cock, and shows the state at the time of zero angle. . It is a top view which shows an example of the relationship between the rotation member at the time of rotating the multiway cock shown in FIG. 19 90 degree | times counterclockwise from the state of FIG. 21, and the flow path of a stopper part. It is a top view which shows an example of the relationship between the rotation member at the time of rotating the multiway cock shown in FIG. 19 90 degree | times counterclockwise from the state of FIG. 22, and the flow path of a stopper part.

  Next, preferred embodiments of the cell culture method, the cell culture apparatus, and the program thereof according to the present invention will be described. The embodiments of the cell culture device according to the present invention will be described first, and the embodiments of the cell culture method and the program thereof will be described together with the description of the operation of the device.

1. Cell Culture Device FIG. 1 is a block diagram showing a configuration of a cell culture device 1 according to the present embodiment. FIG. 2 is a block diagram showing a configuration of the cell culture circuit 2 included in the cell culture device 1 of FIG.

  The cell culture device 1 according to the present embodiment includes a cell culture circuit 2 and a control device 3. The cell culture circuit 2 is detachably attached to the control device 3.

  The cell culture circuit 2 is a circuit for cell culture. The cell culture circuit 2 mainly includes a culture solution container 4, a washing solution container 5, a recovery container 6, a release agent container 7, a gas exchanger 8, and a culture module 10. The culture solution container 4, the washing solution container 5, the recovery container 6 and the release agent container 7 may be used in exchange for the later-described waste solution container.

  The culture solution container 4 is a container for storing a culture solution such as a culture medium. As the culture solution stored in the culture solution container 4, various culture solutions suitable for cells to be cultured can be used.

  The cleaning liquid container 5 is a container that holds a cleaning liquid for cleaning the cell culture circuit 2. As the cleaning liquid retained by the cleaning liquid container 5, a general cleaning liquid can be used as long as it does not adversely affect the cells to be cultured. For example, an isotonic buffer such as phosphate buffered saline (PBS) or tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (Tris), or a sucrose solution having substantially the same osmotic pressure as the cells to be cultured. An isotonic solution such as saline is preferably used. Among these, those substantially free of calcium are preferable, and PBS is particularly preferable.

  The collection container 6 is a container for collecting the cells cultured in the culture module 10. The release agent container 7 is a container for storing a release agent used for removing cells adhered to a nonwoven fabric or the like and improving the recovery rate when the cells cultured in the culture module 10 are collected. Examples of the release agent to be put into the release agent container 7 include 0.5 mM to 10 mM EDTA, EGTA, DTPA, NTTA or TTHA bivalent metal chelating agents, trypsin or collagen proteolytic enzymes, integrins or cadherins, etc. Peptides that competitively inhibit the adhesion of these proteins are preferably used. When such a release agent is supplied to the culture module 10, an integrin or the like that attaches cells to the inside wall of the culture module 10 or a nonwoven fabric, or an adhesive factor such as cadherin that adheres cells can be cut. When the adhesion factor is cleaved, the adherent cells float and the cells can be easily collected.

  The culture liquid container 4, the cleaning liquid container 5, the recovery container 6 and the release agent container 7 hold each liquid in a bag-type container mainly made of a flexible resin, for example. In such a case, since the culture solution container 4, the washing solution container 5, the recovery container 6 and the release agent container 7 are bent when the amount of the solution held therein increases or decreases, air is supplied to the cell culture circuit 2. Hard to mix. Moreover, it is preferable that the culture solution container 4, the cleaning solution container 5, the recovery container 6, and the release agent container 7 are arranged vertically above the culture module 10 and the flow path is arranged on the lower side in the vertical direction. In such a case, even if air is mixed in the culture solution container 4, the cleaning solution container 5, the recovery container 6 or the release agent container 7, the air moves upward in the container, so that the cell culture circuit 2 It is difficult to enter and it is possible to prevent impurities mixed in via air from affecting the cells.

  The gas exchanger 8 is provided for discharging carbon dioxide or the like and injecting oxygen or the like into the cell culture circuit 2. The cells take in oxygen necessary for survival from the culture medium and discharge carbon dioxide. Since the gas exchanger 8 appropriately maintains the carbon dioxide concentration and the oxygen concentration in the culture solution, the pH of the culture solution is prevented from becoming high, so that the cell activity can be prevented from decreasing. The gas exchanger 8 has a gas exchange membrane, for example, and can transmit carbon dioxide and oxygen through the gas exchange membrane. Further, the gas exchanger 8 may have a carbon dioxide sensor and adjust the supply amount of carbon dioxide in response to a signal from the carbon dioxide sensor. Moreover, it is preferable to supply the gas exchanger 8 with a gas whose oxygen concentration and flow rate are adjusted so that the cell activity does not decrease due to a decrease in the oxygen concentration in the culture solution.

  FIG. 3 is a perspective view showing an appearance of the culture module 10 included in the cell culture circuit 2 according to the present embodiment. FIG. 4 is a perspective view showing a structure of a hollow fiber included in the culture module 10 of FIG. FIG. 5 is a cross-sectional view of the culture module 10 of FIG. Note that, in FIG. 5, in consideration of legibility, the scale of each member is changed and illustrated, and the number of hollow fibers 11 is reduced and the hollow fibers 11 are illustrated linearly. .

  The culture module 10 is a module for culturing cells. Any culture module 10 may be used as long as it is a known module. For example, the culture module 10 includes a hollow fiber 11 as shown in FIG. The hollow fiber 11 is a hollow fiber. The culture module 10 is divided into two chambers, an inner chamber 10a and an outer chamber 10b, by a hollow fiber 11. Here, the inner chamber 10 a refers to the hollow portion of the hollow fiber 11, and the outer chamber 10 b refers to the outer portion of the hollow fiber 11. The wall portion of the hollow fiber 11 allows a culture solution, a cleaning solution, a release agent, etc. (hereinafter, the culture solution, a cleaning solution, a release agent, etc. are collectively referred to as “culture solution”), but does not pass cells to be cultured. Fiber.

  As shown in FIG. 4, the culture module 10 has an inlet 14 for supplying a culture solution or the like to the inner chamber 10a of the hollow fiber 11, and an outlet 15 for discharging the culture solution or the like from the inner chamber 10a. . In addition, the culture module 10 discharges cells, culture solution, and the like from the supply port 16 for supplying cells, culture solution, and the like to the outer chamber 10b from a direction substantially perpendicular to the extending direction of the hollow fiber 11, and the outer chamber 10b. And a discharge port 17 for the purpose. The culture module 10 arrange | positions a nonwoven fabric as a support | carrier in the outer chamber 10b, for example.

  When the hollow fiber 11 is used in the culture module 10, a laminated body in which a large number of hollow fibers 11 and a nonwoven fabric are overlapped is wound into a rod shape. Subsequently, both ends thereof are bonded with an adhesive so that they do not fall apart. Finally, it is put into a cylindrical housing provided with the supply port 16 and the discharge port 17, and a cap provided with an inlet 14 and a cap provided with an outlet 15 are attached to the opening of the cylindrical housing. . In addition, when collecting cultured cells, in order to prevent the cells from being caught on the nonwoven fabric and reducing the recovery rate, for example, a nonwoven fabric shorter than the hollow fiber 11 is overlapped, and a space where no nonwoven fabric is present on the outlet 15 side is provided. Form. With this structure, the cultured cells are not caught on the nonwoven fabric before the outlet 15, so that the cell recovery rate can be improved.

  When the cell culture circuit 2 is assembled in advance and sterilized as a single unit, it is preferable because contamination inside the circuit of the cell culture circuit 2 can be avoided. Further, the cyst culture circuit 2 is more preferably sterilized in a state in which a fluid such as distilled water or physiological saline is filled therein. When the cell culture circuit 2 is sterilized in a state where fluid such as distilled water or physiological saline is filled in the cell culture circuit 2, when the cell culture circuit 2 is mounted on the control device 3 during transportation or the like. In the cell culture circuit 2, valves V1, V2, V3, V4, V5, V6, and V7 described later in the cell culture circuit 2 (hereinafter collectively referred to as all valves) In this case, it is preferable that a portion attached to the pump P1 and the pump P2 is sandwiched by a detachable disposable clip to block fluid flow in the cell culture circuit 2. The disposable clip is removed at an appropriate step such as after the cell culture circuit 2 is set in the control device 3 and the power switch is turned on or after the valve V is first fully closed.

  FIG. 6 is a perspective view showing an appearance of the control device 3 included in the cell culture device 1 according to the present embodiment. FIG. 7 is a block diagram illustrating a configuration of the control device 3. In the drawings other than FIG. 6, the illustration of the cover 33 is omitted.

  The control device 3 is a device for arranging the cell culture circuit 2 and controlling each part so that cells can be cultured in the cell culture circuit 2. It has a control unit 30, a gas exchanger holder 31, a culture module holder 32, a cover 33, a heater 34, a temperature sensor 35, pumps P1 and P2, and a valve V.

  The control unit 30 is means for controlling each unit of the control device 3. The control unit 30 acquires information from the temperature sensor 35, temperature adjusting means 30a for driving the heater 34, valve opening / closing means 30b for opening / closing the valve V, pump driving means 30c for driving the pumps P1, P2, and It functions as an input / output means 30d for obtaining an instruction input by the user from the input / output unit 39 and displaying an operation status on the input / output unit 39. The control unit 30 includes, for example, a CPU, a ROM, a RAM, a timer, and the like. The ROM and RAM of the control unit 30 store a program executed by the control unit 30, temporarily store data to be processed, or function as a work memory of the control unit 30. The control unit 30 controls each unit by a program stored in the ROM or RAM. The control unit 30 may process information from the temperature sensor 35 and the pumps P1 and P2, or may count the time using a timer. The control unit 30 controls the heater 34, the pumps P1 and P2, and the valve V. The control unit 30 may be connected to an input / output unit 39 configured by a touch panel or the like, and the user instructs the control unit 30 from the input / output unit 39 or confirms the current status. May be.

  The gas exchanger holder 31 is a fixture for holding the gas exchanger 8. The culture module holder 32 is a fixture for holding the culture module 10. The gas exchanger holder 31 and the culture module holder 32 may be anything as long as the gas exchanger holder 31 and the culture module holder 32 can be fixed.

  The cover 33 is a member that covers the culture module holder 32 and other parts in order to protect the culture module holder 32 and to keep the temperature constant. In the present embodiment, the gas exchanger holder 31, the culture module holder 32, the heater 34, and the temperature sensor 35 are housed inside the cover 33.

  The heater 34 is provided to keep the temperature of the cell culture circuit 2 constant. This is to prevent the temperature of the culture solution from decreasing due to the decrease in the ambient temperature of the cell culture circuit 2 and the decrease in the activity of the cells in culture. As the heater 34, for example, a so-called warm air heater that maintains the temperature inside the cover 33 at a constant temperature by warm air can be employed. It is sufficient that at least the culture module holder 32, the heater 34, and the temperature sensor 35 are accommodated in the cover 33, and more preferably, the cell culture circuit 2 other than the culture module holder 32, the heater 34, and the temperature sensor 35. Is also stored. In particular, since the culture solution container 4 is also kept warm, the temperature of the supplied culture solution can be kept constant.

  The pump P1 and the pump P2 are drive units for supplying a culture solution or the like from each unit to the culture module 10. Any pump may be used as the pump P1 and the pump P2, but a so-called roller pump can be suitably used when a tube having high elasticity is used as the flow path. A roller pump is a pump that crushes one point of an elastic tube with a roller, moves the roller as it is, and moves while extruding liquid inside the tube. When the roller moves, when the crushed portion returns to its original shape by the restoring force of the tube, the next liquid can be sucked by the vacuum generated inside the tube.

  In the present embodiment, the pump P1 is provided in a flow path for supplying the culture solution from the culture solution container 4 to the inner chamber of the culture module 10 via the gas exchanger 8. Therefore, by driving the pump P1, the culture solution is supplied from the culture solution container 4 to the culture module 10 through the gas exchanger 8. Further, the pump P <b> 2 is provided on the culture module 10 side of the cleaning solution container 5 and the release agent container 7 in the flow path continuing from the cleaning solution container 5 and the release agent container 7 to the outer chamber of the culture module 10. Moreover, it is preferable that the pump P2 can adjust the discharge amount per unit time. The pump P2 is preferably one that can set the discharge amount per unit time to be larger than the discharge amount of the pump P1. This is because when the cell culture circuit 2 is washed with the washing liquid, the larger the flow rate, the more efficiently the impurities attached to the channel wall can be removed.

  The valve V is used for opening and closing the flow path. As the valve V, any method may be used to open and close the flow path as long as it is a method that does not cause contamination by germs or the like from the outside. For example, a pinch cock can be used as the valve V. The valve V1 is at the outlet of the release agent container 7, the valve V2 is at the outlet of the cleaning liquid container 6, the valve V3 is at the flow path from the pump P2 to the gas exchanger, and the valve V4 is between the pump P2 and the outer chamber. The valve V5 is between the collection container 6 and the outer chamber, the valve V6 is between the collection container 6 and the outlet 15 of the inner chamber 10a, and the valve V7 is between the culture medium container 4 and the outlet 15 of the inner chamber 10a. , Respectively.

  By configuring the cell culture apparatus 1 as described above, the channel and each part can be washed with a washing solution before the cells are cultured. Therefore, even when the cell culture circuit 2 is sterilized by irradiating the cell culture circuit 2 with a gamma ray, after the part of the members constituting the cell culture circuit 2 is eluted by the gamma ray, the cell culture circuit 2 is washed with the washing liquid. Impurities can be removed for cleaning.

  Moreover, by using the cell culture device 1 as described above, the cell culture circuit 2 can be detached from the control device 3 as it is. Therefore, when cells are cultured to some extent, the cell culture circuit 2 can be removed as it is and stored frozen. In particular, when the culture solution container 4, the washing solution container 5, the recovery container 6, the release agent container 7, and the like are bag-like containers, they can be stored frozen after being removed. Further, when the cell culture circuit 2 is thawed and the cell culture is started again, it is preferable because impurities are not mixed.

  Moreover, since the culture temperature can be kept constant by using the cell culture apparatus 1 as described above, the cell culture efficiency is improved. In the cell culture device 1, unlike the conventional case, not only the culture solution is kept at a constant temperature, but the culture module 10 can be kept warm, or a part or all of the cell culture circuit 2 including the culture module 10 can be kept warm. It is possible to prevent the temperature of the culture solution from changing during the transfer of the culture solution due to the outside air temperature. In particular, when a warm air heater is used as the heater 34, the temperature inside the cover 33 is uniform as compared with the case of hot water or electric heating.

2. Cell Culture Method Next, the cell culture method according to the embodiment of the present invention will be described. The cell culture program according to this embodiment is a program that causes the control unit 30 of the cell culture device 1 to execute each step in the cell culture method according to the embodiment of the present invention. Hereinafter, the cell culture method and program according to the present embodiment will be described together with the operation of the cell culture apparatus 1.

  FIG. 8 is a flowchart showing the flow of the cell culture method according to the embodiment of the present invention.

  When the user turns on the power after setting the circuit 2 to the control device 3, the control unit 30 fully closes all the valves V. Subsequently, when the user gives a start instruction to the control unit 30 via the input / output unit 39, the control unit 30 starts cleaning each flow path and the culture module 10 with a cleaning solution (step S101: cleaning step).

  FIG. 9 is a flowchart for explaining the flow of processing in step S101 of FIG. FIG. 10 is a block diagram showing the state of the cell culture device 1 in step S201. 10 to 18, the flow path through which the cleaning liquid passes is shown in black, the opened valve V is outlined, and the closed valve V is shown in black.

  Step S101 includes four steps of steps S201 to S204 in FIG. First, the controller 30 opens the valves V2, V3, and V6, closes the valves V1, V4, V5, and V7, stops the pump P1, and drives the pump P2 to clean the inner chamber 10a. Control (step S201). As a result, the cleaning liquid supplied from the cleaning liquid container 5 passes through the inner chamber 10 a of the culture module 10 and is discharged into a waste liquid container placed in place of the collection container 6.

  FIG. 11 is a block diagram showing the state of the cell culture device 1 in step S202.

  Next, in step S202, the controller 30 opens the valves V2, V3, and V5 and closes the valves V1, V4, V6, and V7 in order to clean the membrane wall of the hollow fiber 11, and the pump P1. Is stopped and the pump P2 is driven. With this cleaning, the cleaning liquid supplied from the inlet 14 to the inner chamber is closed by the valves V6 and V7 connected to the inner chamber 10a, so that the cleaning liquid supplied to the inner chamber 10a is pressurized and the hollow fiber 11 Permeates through the membrane wall. Through this step, the membrane wall of the hollow fiber 11 is washed, and the washing liquid is discharged into a waste liquid container placed in place of the collection container 6.

  FIG. 12 is a block diagram showing the state of the cell culture device 1 in step S203.

  Next, in step S203, the control unit 30 opens the valves V2, V4, and V5 and closes the valves V1, V3, V6, and V7, as shown in FIG. 12, to clean the outer chamber 10b. The pump P1 is stopped and the pump P2 is driven. By such cleaning, the cleaning liquid passes through the outer chamber 10b of the culture module 10 and cleans the outer chamber 10b when passing. The used cleaning liquid is discharged into a waste liquid container. At that time, the nonwoven fabric arranged in the outer chamber is also washed at the same time.

  13 and 14 are block diagrams showing the state of the cell culture device 1 in step S204.

  Finally, in step S204, the control unit 30 passes the flow path from the outlet 15 of the inner chamber 10a to the culture medium container 4, and the flow path from the culture liquid container 4 to the inlet 14 of the inner chamber 10a via the pump P1. Wash. First, the control unit 30 opens the valves V2, V3, and V7 and cleans the valves V1, V4, and V5 as shown in FIG. 13 in order to wash the flow path from the outlet 15 of the inner chamber 10a to the culture solution container 4. And the valve V6 are closed, the pump P1 is stopped, and the pump P2 is driven. By such cleaning, the cleaning liquid is discharged through the inner chamber 10a of the culture module 10 to a waste liquid container attached instead of the culture liquid container 4.

  Subsequently, the controller 30 opens the valves V2, V3, and V7 as shown in FIG. 14 in order to wash the flow path that reaches from the culture solution container 4 to the inlet 14 of the inner chamber 10a via the pump P1. The valves V1, V4, V5, and V6 are closed, the pump P2 is driven, and the pump P1 is driven in the reverse direction. At this time, it is preferable that the flow rate of the pump P1 and the flow rate of the pump P2 are the same as or smaller than the flow rate of the pump P2. When the flow rate of the pump P1 is the same as or smaller than the flow rate of the pump P2 and the valve V7 is opened as a vent, the pressure inside the cell culture circuit 2 can be properly maintained. Can be prevented from bursting or crushing. As a result, the cleaning liquid cleans the inside of the cell culture circuit 2, and the excess cleaning liquid is discharged to the waste liquid container attached instead of the culture liquid container 4 via the inner chamber 10a and the valve V7. By appropriately setting the flow rate of the pump P1 and the flow rate of the pump P2, the cleaning process in the state shown in FIG. 13 and FIG. 14 in step S204 can be made into one cleaning process. The flow path from the outlet 15 of the inner chamber 10a to the culture medium container 4 and the flow path from the culture medium container 4 to the inlet 14 of the inner chamber 10a via the pump P1 can be washed simultaneously.

  By completing step S101 (including steps S201 to S204), the channel and each part can be washed with a washing solution before the cells are cultured. By washing the cell culture circuit 2, impurities affecting the cells to be cultured are removed, so that the cells can be cultured efficiently.

  In order to prevent the cleaning liquid in the cleaning liquid container 5 from being interrupted during the step S101, a liquid shorting sensor (not shown) may be attached between the outlet of the cleaning liquid container 5 and the valve V2. For example, when the control unit 30 receives a detection signal from a liquid shortage sensor (not shown), the control unit 30 automatically closes the valve V2, stops at least one of the pump P1 and the pump P2, and runs out of liquid. A warning means such as a buzzer or a lamp to notify or a display on the screen of the input / output unit 39 notifies the user that the cleaning liquid has run out. The user instructs the control unit 30 from the input / output unit 39 to start again after replenishing the cleaning solution container 5 with a new cleaning solution or replacing the cleaning solution container 5 with a new one. When the control unit 30 receives such an instruction, the control unit 30 drives at least one of the pump P1 and the pump P2, and restarts step S101.

  When culturing adhesive cells, it is preferable that step S101 further includes a step of washing the flow path from the cleaning solution container 5 to the release agent container 7. For example, when the cleaning liquid container 5 is provided at a position higher in the vertical direction than the release agent container 7, the control unit 30 opens the valves V1 and V2, and closes the valves V3, V4, V5, V6, and V7. When the pumps P1 and P2 are stopped, the cleaning liquid cleans the flow path from the cleaning liquid container 5 to the release agent container 7 by a drop (water head difference), and a waste liquid container (not shown) attached instead of the release agent container 7 is shown. Are discharged. Further, when the release agent has a stable composition and the release agent does not change during cell culture, it is preferable to connect the release agent container 7 to the cell culture circuit 2 before step S101. In such a case, the control unit 30 opens the valves V1, V4, and V5, closes the valves V2, V3, V6, and V7 and drives the pump P2 before step S101. With this control, the release agent can clean the flow path from the release agent container 7 to the waste liquid container disposed in place of the recovery container 6 and the circuit from the release agent container 7 to the pump P2. After such a process, once all the valves are closed and the pumps P1 and P2 are stopped, the cleaning in step S101 may be started. Note that this step is not necessary when culturing planktonic cells because the release agent container 7 is not used.

  When the user confirms the end of step 101 and instructs the control unit 30 to start the culture solution filling step, the control unit 30 fills the inner chamber 10a and the outer chamber 10b with the culture solution ( Step S102: culture solution filling step).

  15 and 16 are block diagrams showing the state of the cell culture device 1 in step S102.

  In step S102, the control unit 30 first fills the inner chamber 10a of the culture module 10 with the culture solution, and then fills the outer chamber 10b with the culture solution. As shown in FIG. 15, the controller 30 opens the valve V7 and closes the valves V1, V2, V3, V4, V5 and V6 to fill the inner chamber 10a of the culture module 10 with the culture solution, Pump P1 is driven and pump P2 is stopped. The culture solution in the culture solution container 4 is supplied to the inner chamber 10a through the pump P1 and the gas exchanger 8. The culture fluid circulates from the outlet 15 to the culture fluid container 4 through the inner chamber 10a of the culture module 10.

  Subsequently, the control unit 30 opens the valve V5 and fills the valves V1, V2, V3, V4, V6 and V7 as shown in FIG. 16 in order to fill the outer chamber 10b of the culture module 10 with the culture solution. The pump P1 is driven and the pump P2 is stopped. Since the culture solution in the culture solution container 4 is pressurized and supplied to the inner chamber 10a of the culture module 10, the culture solution permeates through the membrane wall of the hollow fiber 11 and permeates into the outer chamber 10b. Is supplied with culture medium. The excess culture solution is discharged to a waste solution container arranged instead of the collection container 6.

  When the user confirms the completion of step S102 and instructs the control unit 30 to start the cell filling step from the input / output unit 39, the control unit 30 fills the outer chamber 10b of the culture module 10 with cells (step S103). : Cell filling step).

  In step S103, the control unit 30 closes the valves V1, V2, V3, V6, and V7, opens the valves V4 and V5, and stops the pumps P1 and P2. Thereafter, a cell injection syringe is automatically or manually inserted into a cell filling port (not shown) provided between the valve V4 and the culture module 10, and the cells in the syringe are injected into the outer chamber 10b of the incubator. By step S103, the outer chamber 10b of the culture module 10 is filled with cells.

  When the user confirms the completion of step S103 and instructs the control unit 30 from the input / output unit 39 to start the culture solution circulation step, the control unit 30 starts supplying the culture solution to the inner chamber 10a (step). S104: culture medium circulation step).

  In step S104, the culture solution is circulated and supplied from the culture solution container 4 to the inner chamber 10a of the culture module 10. Specifically, as shown in FIG. 15, the control unit 30 opens the valve V7, closes the valves V1, V2, V3, V4, V5, and V6, drives the pump P1, and stops the pump P2. . In step S104, the culture medium circulating in the inner chamber 10a is injected into the outer chamber in order to permeate the membrane wall of the hollow fiber 11 by the pressure difference or diffusion action between the membranes and replace the culture solution in the outer chamber 10b. The cultured cells can be obtained as nutrients in the outer chamber 10b. In step S104, by changing the rotation speed of the pump P1, the circulation amount of the culture solution can be increased or decreased to maintain the optimum culture state. Step S104 is usually performed for 1 to 3 weeks depending on the type of cells and culture medium.

  In addition, if the oxygen concentration in the culture solution decreases during the circulation of the culture solution, the cell activity decreases, so it is preferable to replace the culture solution in the culture solution container 4 with a fresh culture solution. When the culture medium is exchanged, the control unit 30 stops all the valves V and the pump P1 when the user presses the stop key. When the user presses the start switch of the culture process again after completing the replacement of the culture medium in the culture medium container 4 or presses the recovery switch, the process returns to the process before pressing the stop key, The control unit 30 reopens the culture process by opening the valve V7 and driving the pump P1. When the culture is completed, the process proceeds to the next recovery step. The time for exchanging the culture solution can also be determined by measuring the pH of the culture solution.

  When the user completes step S104 and instructs the control unit 30 from the input / output unit 39 to start the cell recovery step, the control unit 30 starts supplying the release agent (step S105: recovery step).

  17 and 18 are block diagrams showing the state of the cell culture device 1 in step S105.

  In step S105, the control unit 30 first fills the outer chamber 10b with the release agent and maintains the state for a certain period of time, then supplies the culture solution to the inner chamber 10a and the outer chamber 10b and is expelled from the discharge port 17. The collected cells are collected in the collection container 6. First, as shown in FIG. 17, the control unit 30 opens the valves V1, V4, and V5, closes the valves V2, V3, V6, and V7, stops the pump P1, and drives the pump P2. In step S105, the release agent is supplied from the release agent container 7 to the outer chamber 10b of the culture module 10, and fills the outer chamber 10b. The release agent that has overflowed the outer chamber 10 b is collected in the collection container 6 from the discharge port 17. In the case of culturing floating cells, the cells do not adhere to a carrier such as a non-woven fabric, and thus a release agent may not be used.

  Next, the control unit 30 receives the flow rate detection signal from the pump P2 or the signal from the timer, closes all the valves V, stops the pumps P1 and P2, and fills the outer chamber 10b with the release agent. The state is maintained, and the process waits until the release agent decomposes the protein that attaches the cells to the inner wall and the nonwoven fabric, and causes the cells to release. The waiting time is determined by the type of cells and release agent, but is usually 3 to 8 minutes.

  Subsequently, the control unit 30 receiving the signal from the timer opens the valves V3, V4 and V5, closes the valves V1, V2, V6 and V7 and drives the pump P1 as shown in FIG. Stop P2. Then, the culture solution is supplied from the culture solution container 4 to both the inner chamber 10a and the outer chamber 10b of the culture module 10, and the cells are expelled from the outlet 17 of the outer chamber 10b. The expelled cells are collected in the collection container 6. By supplying the culture solution to the inner chamber 10 a of the culture module 10, the pressure in the inner chamber 10 a and the outer chamber 10 b becomes equal, so that the cultured cells are expelled from the discharge port 17, and the cells are stored in the collection container 6. Can be recovered.

  In step S105, the cultured cells may be caught by the nonwoven fabric and the cell recovery rate may be reduced. As a method for collecting the cells caught on the nonwoven fabric, the culture module 10 may be swung, or the culture solution supplied to the inner chamber 10a of the culture module 10 may be intermittently pressurized. For example, a pressure sensor (not shown) is provided in front of the inlet 14. In response to a signal from the pressure sensor (not shown), the control unit 30 opens the valve V5 when the pressure in the flow path near the inlet 14 exceeds the set pressure, and closes the valve V5 when the pressure falls below the set pressure. Thus, the cells caught on the nonwoven fabric can be removed by intermittently changing the pressure of the culture solution supplied to the inner chamber 10a of the culture module 10.

  Furthermore, step S105 may include a step of expelling cells floating on the membrane wall surface from the outer chamber 10b. In order to expel cells floating on the surface of the membrane wall from the outer chamber 10b, the control unit 30 receiving the signal from the timer opens the valve V5 and closes the valves V1, V2, V3, V4, V6 and V7. Then, the pump P1 is driven and the pump P2 is stopped. Then, the culture solution is supplied from the culture solution container 4 to the inner chamber 10 a of the culture module 10. Since the culture solution pressurized and supplied to the inner chamber 10a of the culture module 10 passes through the membrane wall of the hollow fiber 11 and moves to the outer chamber 10b, the cultured cells floating on the membrane wall surface can be peeled off from the membrane wall. it can. The cells peeled off from the membrane wall are expelled from the outer chamber 10 b and collected in the collection container 6 from the discharge port 17.

  When the user confirms the completion of step S105 and instructs the control unit 30 to start disposal from the input / output unit 39, all the valves V are opened. The user can remove the cell culture circuit 2 from the cell culture apparatus 1 after confirming that all the valves V are opened. At this time, with the control unit 30 stopping the pumps P1 and P2 and closing all the valves V, the user can attach and detach the circuit from the valves V5 and V6 to the cell collection container 6 before starting disposal. It is preferable to close with a clip or the like to prevent any possible contamination of the collection container.

  Note that the collected cells may be washed with a washing solution such as PBS after step S105. In order to prevent release agents such as proteolytic enzymes from degrading and destroying cell surface markers, channels and receptors other than adhesion factors, and adversely affecting cultured cells, the collected release agent is replaced with a washing solution such as PBS. This is because it is preferable. For example, a drainage channel is connected to the lower part of the collection container 6, and a filter containing a porous membrane that prevents the permeation of cells is provided in the drainage channel. By the drainage channel, the culture solution and the release agent collected together with the cells are permeated and discarded. Cells in solution accumulate on the surface of the filter. The cells that have accumulated in the filter may be washed with PBS or the like.

  By using the cell culture apparatus 1 described above, even a beginner can easily perform cell culture. The user prepares a culture solution, a release agent, and a cleaning agent in each container, and stores the cells in a syringe. Then, the user simply instructs the control unit 30 to start the washing step S101, the culture solution filling step S102, the cell filling step S103, the culture step S104, and the recovery step S105 (for example, just pressing the start switch). This is because each process is automatically performed. In addition, since all steps are performed in a closed system, it is difficult for germs to enter the cell culture circuit 2. Furthermore, since a washing step is included before the cells are injected, impurities harmful to the cells can be removed in advance.

3. Modifications The embodiments of the cell culture device 1 and the cell culture method of the present invention have been described above, but the present invention is not limited to the above-described embodiments and can be implemented with various modifications. For example, in the above-described embodiment, the release agent is not essential. Moreover, you may use a washing | cleaning liquid as a peeling agent. For example, when culturing floating cells, the release agent may not be used, and the release agent container 7 is not essential.

  In the above-described embodiment, the cell culture circuit 2 is detachably attached to the control device 3, but is not limited to such a form. The cell culture circuit 2 may be in a form that cannot be removed from the control device 3. Moreover, in the above-mentioned embodiment, although the gas exchanger 8 was provided in the cell culture circuit 2, the gas exchanger 8 is not essential. In addition, part of the gas exchanger 8 may be provided in the cell culture circuit 2 and the other part may be provided on the control device 3 side. For example, a tube mainly composed of a gas permeable membrane (the medium flows inside) is provided in the cell culture circuit 2, and a portion for supplying a gas such as carbon dioxide or air is provided on the control device 3 side. It may be done. In such a case, it is preferable that a portion of the gas exchanger 8 provided on the control device 3 side and a portion provided on the cell culture circuit 2 side can be detached.

  Moreover, in the above-mentioned embodiment, although the example of the culture module 10 using the hollow fiber 11 was shown, it is not restricted to such a form. For example, the culture module 10 may be a module using a flat membrane without using the hollow fiber 11.

  Further, in the above-described embodiment, the culture solution container 4, the cleaning solution container 5, and the release agent container 7 are arranged vertically above the culture module 10, but the recovery container 6 is further arranged than the culture module 10. It is preferable to install the waste liquid container at a lower position than the collection container 6. It is more preferable to provide an openable and closable flow path between the collection container 6 and the waste liquid container and between the culture module 10 and the collection container 6. In such a case, all the waste liquid is discharged via the culture module 10 and the recovery container 6 by opening the flow path between the recovery container 6 and the waste liquid container and the flow path between the culture module 10 and the recovery container 6. Since it can be collected in a container, step S101 can be performed without replacing the waste liquid container in place of the collection container 6. Thereafter, if the flow path between the recovery container 6 and the waste liquid container is closed, the risk of impurities or germs entering the cell culture circuit 2 due to the exchange between the recovery container 6 and the waste liquid container can be reduced. Furthermore, the opening and closing of each flow path is facilitated by using auxiliary clamps or forceps that are detachable from the outside. Moreover, since the culture solution container 4, the cleaning solution container 5 and the release agent container 7 are arranged vertically above the culture module 10, the release agent, the cleaning solution, and the culture solution can be allowed to flow with a head (water head). The inside of the cell culture circuit 2 can be cleaned and replaced, and finally all the liquid used for cleaning is collected in a waste container.

  In the above-described embodiment, the valve V that can switch between closing and opening of one flow path is used. However, the present invention is not limited to such a form. For example, the multiway cock 40 can be used as a valve. FIG. 19 is an exploded perspective view showing an example of the structure of the multiway cock 40.

  The multiway stopcock 40 mainly includes, for example, a pulse motor 41, a partition plate 43, a clamp 50, and a plug portion 60. The pulse motor 41 has a rotating shaft 42 extending upward. The pulse motor 41 can rotate the rotating shaft 42 by an angle corresponding to a predetermined number of pulses. The control unit 30 may function as a control unit of the pulse motor 41, and the control unit 30 can rotate the rotation shaft 42 around the major axis of the rotation shaft 42 according to each step.

The partition plate 43 located on the top of the pulse motor 41 has a hole 44, and the rotating shaft 42 of the pulse motor 41 extends from the hole 44. Further, switches 45 and 46 are disposed on the partition plate 43.

  FIG. 20 is a perspective view of the multi-way cock 40 in which the clamp 50 and the stopper 60 are combined.

  The clamp 50 has a substantially circular pedestal 51, and a recess 52 is provided at the center of the back side of the pedestal 51. The rotating shaft 42 of the pulse motor 41 is fitted in the recess 52. Therefore, when the rotating shaft 42 rotates, the clamp 50 also rotates integrally with the rotating shaft 42. The clamp 50 further includes a single pressing member 53 that extends from the center of the pedestal portion 51 toward the radially outer side of the clamp 50. The length of the pressing member 53 is a length that reaches the switches 45 and 46 when the clamp 50 rotates. In addition, four gripping pillars 54 are erected on the upper surface of the pedestal 51.

  In addition, a columnar portion 55 shorter than the gripping column 54 is provided so as to be surrounded by the four gripping columns 54. The gripping column preferably has an outer periphery made of a material having high flexibility and a high coefficient of friction such as rubber. For example, a screw is used as a core, and the screw is connected to the side surface of the rotating member 61. It is preferably configured by covering a cylindrical body having a thickness that can sufficiently fill the gap. A gripping column having a multilayer structure in which rubber is coated on the outer surface of a cylinder made of a resin having higher hardness than rubber (for example, fluororesin) may be used. Since the core is made of metal and the outer periphery of the core is made of a material (for example, resin) having a hardness lower than that of the core material, each of the gripping columns 54 can grip the rotating member 61 strongly, and the rotating member. It becomes difficult to slip on the surface of 61. Since the rotation member 61 is detachable by the gripping column 54, a commercially available three-way stopcock can be used as the plug portion 60 regardless of the size or shape.

  The gripping column 54 grips the substantially cross-shaped rotating member 61. Further, the core body 62 has a T-shaped flow path 63 that opens in three directions that are perpendicular to each other on a plane when viewed from above. The plug portion 60 has a substantially cross-shaped rotating member 61 and a core body 62 extending upward in the approximate center. The core body 62 and the flow path 63 are fixed so as not to rotate. On the other hand, inside the core body 62, a cylinder having a T-shaped channel fixed to the substantially cross-shaped rotating member 61 is joined to the core body 62 in a fluid-tight manner through a seal. Has been. In this example, the rotation of the rotating member 61 rotates the cylindrical T-shaped channel fixed to the rotating member 61, and the T-shaped channel of the channel 63 extending in three directions from the core body 62. When they match, it circulates in that direction. When the rotating member 61 is at a position inclined by about 45 degrees with respect to the flow path 63 extending in the three external directions from the core body 62, the flow path is fully closed.

  21 to 23 show the direction of the rotating member 61 according to the rotational movement of the clamp 50, the cylindrical T-shaped flow channel fixed to the rotating member 61 when viewed from the upper surface, and three directions from the core body 62 to the outside. It is a top view explaining the distribution | circulation state with the flow path 63 extended in a T shape. In FIGS. 21 to 23, the portion indicated by the broken line in the drawing on the right side is a T-shaped flow path 63 in the cylinder fixed to the rotating member 61, and the long axis direction of the rotating member 61 and the left side thereof. And has a T-shaped flow path opened in the wall. In addition, “tube 1”, “tube 2”, and “tube 3” in the figure flow when the end of the flow path 63 faces the direction of “tube 1”, “tube 2”, or “tube 3”. A pipe connected to the path 63 is shown.

  In the position shown in FIG. 21, the flow path 63 allows the pipe 2 and the pipe 3 to flow, and the pipe 1 is in a closed state. When the clamp 50 rotates 90 degrees in the opposite direction to the timepiece hand from the state shown in FIG. 21, the state shown in FIG. 22 is obtained. In the state shown in FIG. 22, the flow path 63 allows the pipe 1 and the pipe 3 to circulate. Further, when the clamp 50 further rotates 90 degrees counterclockwise from the state shown in FIG. 22, the state shown in FIG. 23 is obtained, and the flow path 63 causes the tube 1 and the tube 2 to circulate.

  When the three-way cock as described above is used, the three-way cock that is originally used manually can be rotated electrically to switch the opening and closing of the flow path 63. For example, the tube 1 is connected to the culture module 10, the tube 2 is connected to the detergent container 5, and the tube 3 is connected to the culture solution container 4. When the flow path 63 connecting the tube 1 and the tube 3 needs to be accurately opened, the pulse motor 41 must be rotated by the number of pulses for rotating the clamp 50 exactly 90 degrees. For this reason, the clamp 50, which is uncertain whether or not the angle is zero, is rotated in the direction opposite to the clock hand by several to several tens of pulses, and the clamp 50 is pressed until the pressing member 53 pushes the switch 45 from that position. Rotate in the same direction as the clock hands. The position of the clamp 50 when the switch 45 is pushed and the pulse motor 41 is stopped is surely a position of zero angle. From this state, the clamp 50 is rotated by the number of pulses corresponding to an angle of 90 degrees. As a result, the state of the clamp 50 shown in FIG. 21 is exactly 90 degrees rotated, and the flow path 63 connecting the tube 1 and the tube 3 is completely open. In addition, when the accuracy of the flow rate is not required, the above-described method may not be adopted.

  In the above-described embodiment, each step is executed when the control unit 30 receives a start instruction for each step from the input / output unit 39. However, the present invention is not limited to this mode. For example, the control unit 30 may automatically start the next step after confirming that a predetermined step has been completed or after a predetermined time has elapsed.

  Moreover, although the program which operates the control part 30 of the cell culture apparatus 1 which concerns on the said embodiment is memorize | stored in RAM, ROM, etc. which the control part 30 has, these programs are memorize | stored before shipment of these apparatuses. Even those that have been stored may be stored after the shipment of these devices. Some of these programs may be stored after the shipment of these devices. As described above, the program stored in the memory of the control unit of the cell culture device 1 after shipping is a program installed on a computer-readable recording medium such as a CD-ROM or a semiconductor memory. Alternatively, it may be downloaded from a server device or the like via a transmission medium such as the Internet.

  In addition, part or all of the programs and various data stored in the memory of the cell culture device 1 according to the above-described embodiment are attached to and detached from an external storage device other than a computer, such as an HDD, or the cell culture device 1. For example, it may be stored in a memory card or CD-ROM.

  Moreover, the function of each part which comprises the cell culture apparatus 1 which concerns on the said embodiment may implement | achieve all or one part by software, or may implement | achieve at least one part by hardware. For example, all or a part of the processing in the control unit 30 may be realized on a computer by one or a plurality of programs, and at least a part thereof may be realized by hardware.

  Further, a computer program for operating as the whole or a part of the cell culture device 1 in the above embodiment is stored and distributed in a computer-readable recording medium such as a memory card or a CD, and is distributed to another computer, For example, it may be installed in a mobile phone, an audio device, an electronic watch, etc., and operated as the cell culture device 1, or a part or all of the steps performed by the cell culture device 1 may be executed. Furthermore, the program may be stored in a disk device or the like included in a server device on the Internet, and may be downloaded onto a computer serving as the control unit 30 of the cell culture device 1 by being superimposed on a carrier wave, for example.

  The present invention can be applied to a cell culture apparatus for culturing cells.

DESCRIPTION OF SYMBOLS 1 ... Cell culture apparatus 2 ... Cell culture circuit 3 ... Control apparatus 4 ... Culture solution container (a part of cell culture circuit)
5 ... Cleaning solution container (part of cell culture circuit)
10 ... Culture module (part of cell culture circuit)
30 ... Control unit (part of control device)
40 ... multi-way stopcock 41 ... pulse motor (drive source)
42 ... rotating shaft 50 ... clamp 54 ... gripping column 60 ... plug part 61 ... rotating member 63 ... flow path P1, P2 ... pump

Claims (7)

A culture module partitioned into an outer chamber and an inner chamber;
A culture solution container for storing a culture solution to be supplied to the culture module;
A cleaning liquid container for storing a cleaning liquid for cleaning the culture module;
A pump for supplying the culture solution and the washing solution;
A control unit for controlling the pump,
The cell culture apparatus, wherein the washing liquid is supplied at least before culturing the cells. The cell culture device according to claim 1,
The entire culture circuit including the culture module, the culture solution container, and the washing solution container,
A cell culture device, wherein the cell culture device is detachably attached to a control device having the pump and the control unit. The cell culture device according to claim 1 or 2, wherein
The control unit changes the flow rate when supplying the culture solution and the flow rate when supplying the washing solution. The cell culture device according to any one of claims 1 to 3,
At least one of the culture solution container and the washing solution container is disposed on the antigravity direction side of the culture module. The cell culture device according to any one of claims 1 to 4,
Furthermore, the flow path connecting the culture module and each part is provided with a valve,
The valve is attached to a plug portion including a tube projecting in a plurality of directions and a rotatable rotating member for switching a fluid flow path, thereby fixing the plug portion to fix the rotating member. A multi-way cock switching device that rotates,
A drive source for applying a rotational driving force to the rotational member;
A clamp that is connected to a rotation shaft of the drive source and includes one or more gripping columns that grip the rotating member;
The control unit controls the rotation operation of the drive source,
The cell culture apparatus according to claim 1, wherein the clamp rotates the rotation member based on a rotation operation of the drive source controlled by the control unit. A washing step of washing the culture module partitioned into the outer chamber and the inner chamber before filling the culture solution;
A culture solution filling step of filling the culture solution into the culture module;
A cell filling step of injecting cells into the outer chamber;
A culture step of culturing cells while supplying the culture solution to the inner chamber;
And a recovery step of recovering cells cultured in the culture module. By input from the input means,
A washing step of supplying a washing solution to the culture module partitioned into the outer chamber and the inner chamber before filling the culture solution;
A culture solution filling step for supplying the culture solution to the culture module;
A culture step of culturing cells while supplying the culture solution to the inner chamber;
A recovery step of supplying at least one of a release agent for peeling cells, the cleaning agent and the culture solution to the culture module;
A program for cell culture characterized by causing a control unit to execute.

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