Automatic culture device, automatic culture system, and cell culture method

Abstract

To provide an automatic culture device, an automatic culture system, and a culture method. Provided are an automatic culture device, an automatic culture system, and a culture method capable of accurately conveying a cell suspension between any containers without passing through a drive unit of a pump mechanism, regardless of the type of a cell culture container, a container containing the cell suspension, and a cell collection container to be used, and capable of efficiently culturing cells by conveying the cell suspension between any containers without passing through the drive unit of the pump mechanism. Cell supply and recovery with reduced physical damage to cells can be achieved. This automatic culture device is provided with: a cell suspension storage container capable of storing a cell suspension; a cell processing container for processing the cell suspension conveyed from the cell suspension storage container; a flow path connecting at least the cell suspension storage container and the cell processing container; a first buffer space connected to the flow path and having a predetermined pressure resistance; and a pressure source for introducing the cell suspension from the cell suspension storage container into the first buffer space by bringing the interior of the first buffer space into negative pressure.

Description

TECHNICAL FIELD

[0001] The present application relates to an automatic culturing apparatus for culturing cells or tissues, an automatic culturing system, and a cell culturing method. BACKGROUND

[0002] In recent years, cell therapy for repairing or restoring a damaged or functionally reduced tissue by transplanting a cultured cell or tissue has attracted attention. So far, the manufacturing of a cell product has been performed by manual operation, but the manufacturing cost is high, and quality deviation due to the proficiency of an operator is a problem, and automation of the manufacturing is being promoted. On the other hand, the manufacturing scale of a cell product differs depending on whether autologous therapy using a patient's own cells or allogeneic therapy using a healthy person's cells is performed, and in addition, the process thereof involves many aspects such as selection, washing, gene introduction, proliferation, differentiation induction, and tissue formation of cells. Therefore, a plurality of devices dedicated to each process are sometimes used for the manufacturing, and in this case, a cell supply and recovery operation is performed in each device.

[0003] When a cell is supplied in each device, a transport mechanism (driving unit) such as a peristaltic pump is usually provided between a container that accommodates a cell suspension and a container of a transport destination, and is connected by a tube, respectively. Such a technique is described in, for example, Patent Literature 1.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT LITERATURE

[0006] Patent Literature 1: International Publication No. 2015 / 025425 SUMMARY

[0007] PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] In a case where a culture medium is transported using a transport mechanism having a driving unit such as a peristaltic pump, there is a concern that a cell is physically damaged when the cell passes through the driving unit. In FIG. 11 of Patent Literature 1, it is disclosed that a cell is transported without passing through a driving unit by supplying a gas to a container that accommodates a cell suspension and pressurizing. However, a cell culture container, a container in which a cell suspension is filled, and a cell recovery container are used by a user using various containers (for example, a synthetic resin container having flexibility called a bag), and therefore in a case where the pressure resistance of a container pressurized for the purpose of transporting a cell suspension is low, it is possible that an adverse situation occurs in the transportation.

[0009] An object of the present application is to provide an automatic culture device, an automatic culture system, and a cell culture method, which can accurately transfer regardless of the type of a cell culture container, a container filled with a cell suspension, and a cell recovery container, and which can achieve cell supply and recovery with reduced physical damage to cells by transferring a cell suspension between arbitrary containers without a driving section of a pump mechanism.

[0010] Method for solving the problem

[0011] The present application for achieving the above object is configured as follows.

[0012] An automatic culture device includes a cell suspension storage container that can store a cell suspension, a cell processing container that processes the cell suspension transferred from the cell suspension storage container, a flow path that links at least the cell suspension storage container and the cell processing container, a first buffer space that is connected to the flow path and has a predetermined pressure resistance, and a pressure source that introduces the cell suspension from the cell suspension storage container into the first buffer space by making the inside of the first buffer space negative.

[0013] In addition, an automatic culture system includes the above automatic culture device and a control device that controls each mechanism of the automatic culture device including the pressure source.

[0014] In addition, a cell culture method is a cell culture method of an automatic culture device that includes a cell suspension storage container that can store a cell suspension, a cell processing container that processes the cell suspension transferred from the cell suspension storage container, a flow path that links at least the cell suspension storage container and the cell processing container, a first buffer space that is connected to the flow path and has a predetermined pressure resistance, and a pressure source that can make the inside of the first buffer space negative. The cell culture method includes a step of providing a liquid containing cells to be cultured in the cell suspension storage container, a step of making the inside of the first buffer space negative by operating the pressure source and transferring the liquid containing cells provided in the cell suspension storage container to the inside of the first buffer space, and a step of making the inside of the first buffer space positive by operating the pressure source and transferring the liquid containing cells transferred to the first buffer space to the cell processing container.

[0015] Effects of the Invention

[0016] According to the present application, it is possible to provide an automatic culturing device, an automatic culturing system, and a cell culturing method, which can accurately perform transfer regardless of the type of cell culturing containers, containers filled with cell suspensions, and cell recovery containers, and which can achieve cell supply and recovery with reduced physical damage to cells by transferring cell suspensions between arbitrary containers without a driving section of a pump mechanism. The above and other objects, configurations, and effects will become apparent from the following description of the embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a view showing one configuration of a cell suspension supply and recovery mechanism of Example 1.

[0018] Figure 2 is a view showing a step example of cell suspension supply and recovery of Example 1.

[0019] Figure 3 is a view showing one configuration of a flow path circuit of a culturing device of Example 1.

[0020] Figure 4 is a view showing one configuration of a cell suspension supply and recovery mechanism of Example 2.

[0021] Figure 5 is a view showing one configuration of a cell suspension supply and recovery mechanism of Example 3.

[0022] Figure 6 is a view showing a step example of cell suspension supply and recovery of Example 3.

[0023] Figure 7 is a view showing one configuration of a cell suspension supply and recovery mechanism of Example 4.

[0024] Figure 8 is a view showing a step example of cell suspension supply and recovery of Example 4.

[0025] Figure 9 is a view showing a decrease in cell proliferation rate due to a peristaltic pump. DETAILED DESCRIPTION

[0026] Hereinafter, embodiments of the present application will be described using the drawings. The following description shows specific examples of the content of the present application, but the present application is not limited to these descriptions, and various modifications and corrections can be made by those skilled in the art within the scope of the technical idea disclosed in the present specification.

[0027] In addition, in all the drawings used to describe the present application, the same symbols are attached to parts having the same function, and the repeated description thereof is sometimes omitted.

[0028] Example 1

[0029] In this embodiment, using Figures 1-4 This illustrates an example of using a pressure-resistant container for cell supply and retrieval without a drive unit. Figure 1 This is an example of the configuration of the closed system flow path in Embodiment 1 of the present invention. Figure 2 express Figure 1 Example of steps in the flow path.

[0030] First, the cell processing device (also called "cell culture container" or "cell processing container") 1, which supplies cells, is connected to the flow path (represented by unsigned solid lines) using connector 3. Figure 2 In step S01), a container (also called a "cell suspension holding container"; in this embodiment, the aforementioned "bag") 7 containing the cell suspension (also called "suspension") is connected to the flow path. Figure 2 (S02). Next, open solenoid valves 8 and 16 ( Figure 2 S03), causing the peristaltic pump (also known as a "pressure source" or simply a "pump") 17 to rotate to the right ( Figure 2 The S04) thereby creates a negative pressure within the buffer space 13 (also known as the "first buffer space"), introducing the suspension from the container 7 into the buffer space 13. The buffer space 13 uses a container with sufficient pressure resistance, so the container will not deform due to the negative pressure, allowing the suspension to be introduced into the buffer space 13.

[0031] As a pressure-resistant container, containers made of high-strength synthetic resin or glass containers can be used. To allow external viewing of the introduced suspension, a transparent material is preferred, but containers made of metals such as stainless steel or aluminum can also be used. A transparent window can be provided in part of the container to allow viewing of the interior. The required pressure resistance varies depending on the elevation difference of the transport path, the diameter of the flow path, and its length; for example, it is approximately several hundred kPa when using a flow path with an inner diameter and outer diameter of several millimeters. Qualitatively, "predetermined pressure resistance" means that when the buffer space 13 is made negatively pressurized and the cell suspension in container 7 is introduced into the buffer space 13, if the buffer space 13 is flattened due to the negative pressure, the cell suspension cannot be adequately introduced into the buffer space 13. Therefore, it is "pressure resistance to the point that it will not be flattened even when the buffer space 13 is made negatively pressurized." For example, even if a "bag" made of flexible synthetic resin is made negatively pressurized, it will only be flattened and the suspension cannot be introduced into the bag. Therefore, "bags" do not belong to containers with predetermined pressure resistance.

[0032] The weight of the introduced suspension is measured by measuring the weight of the buffer space using a weight gauge 14. Figure 2S05), the pump 17 is stopped at the moment when the desired weight is reached Figure 2 S06), the electromagnetic valves 8, 16 are closed Figure 2 S07).

[0033] Next, the electromagnetic valves 6, 16 are opened Figure 2 S08), the pump 17 is rotated to the left Figure 2 S09), thereby making the buffer space 13 positive, and the suspension is delivered to the cell processing device 1. The weight of the buffer space is measured by the weight meter 14 Figure 2 S10), the pump 17 is stopped at the moment when the desired weight is reached Figure 2 S11), the electromagnetic valves are closed Figure 2 S12).

[0034] After the cell processing (e.g., cell culture, etc.) Figure 2 S13), the electromagnetic valves 5, 15 are opened Figure 2 S14), the pump 17 is rotated to the right Figure 2 S15), thereby making the buffer space 11 (also referred to as "second buffer space") negative, and the suspension after the processing is introduced from the cell processing device 1 to the buffer space 11. The weight of the buffer space is measured by the weight meter 12 Figure 2 S16), the pump 17 is stopped at the moment when the desired weight is reached Figure 2 S17), the electromagnetic valves 5 are closed and the electromagnetic valves 9 are opened Figure 2 S18).

[0035] Next, the buffer space 11 is made positive by rotating the pump 17 to the left Figure 2 S19), and the suspension is delivered to the cell recovery container (also referred to as "recovery container". In this embodiment, a bag is used) 10. The weight of the buffer space is measured by the weight meter 12 Figure 2 S20), the pump 17 is stopped at the moment when the desired weight is reached Figure 2 S21), the electromagnetic valves are closed Figure 2 S22).

[0036] After that, the recovery container 10 is cut off from the flow path and recovered Figure 2two buffer spaces are used because if the cell suspension before treatment is mixed with the cell suspension after treatment, there is a concern that the cells before treatment (at the time of seeding) will remain and die. Depending on the kind of cells to be treated, the contents of the treatment, even if mixed, there is no effect, in which case, it is possible to deliver only in the first buffer space from the container 7 containing the cell suspension to the cell treatment device 1, and to recover the cell suspension after the treatment to the cell recovery container 10. By being configured in this way, it is possible to realize miniaturization and low cost of the automatic culture device.

[0037] In addition, in Figure 2 , the buffer space 13 is provided in the flow path between the connecting portion of the container 7 containing the cell suspension and the peristaltic pump 17, but can also be provided in the flow path between the cell treatment device 1 and the connecting portion of the container 7 containing the cell suspension. In this case, compared to the configuration of Figure 2 , it is possible to shorten the flow path length, and thus it is possible to realize miniaturization and low cost of the device. Figure 2 is an example when the cell supply and recovery mechanism shown in Figure 2 is incorporated into the flow path capable of automatic culture (the illustration of the step example in the flow path of Figure 2 is omitted).

[0038] First, the electromagnetic valves 8, 16, 26 are opened, the pump 17 is rotated to the right, whereby the inside of the buffer space 13 becomes negative pressure, and the cell suspension is introduced from the container 7 to the buffer space 13. After the desired amount is introduced, the pump is stopped and all the valves are closed. Next, the electromagnetic valves 6, 16, 26 are opened, the pump 17 is rotated to the left, whereby the inside of the buffer space 13 becomes positive pressure, and the cell suspension is delivered from the buffer space 13 to the cell treatment container 1, and the pump is stopped, and all the electromagnetic valves are closed.

[0039] In the culture, a desired gas such as 5% CO2 gas is supplied to the cell processing container 1 from the gas cylinder 30 through the regulator 29, the mass flow controller (also referred to as "mass flow meter") 28 at a desired flow rate. In this embodiment, the supplied gas is humidified using the humidifying bottle 32 in a manner that the medium in the container is not evaporated. At the time of medium replacement, first, the electromagnetic valves 21, 22 are opened, and the pump 17 is rotated to the right, whereby the culture supernatant is transported to the drain container (also referred to as "drain recovery container") 23. After the culture supernatant is drained, the pump is stopped, and all the electromagnetic valves are closed. Next, the electromagnetic valve 24 is opened, and the pump 20 is rotated to the left, whereby the medium is supplied from the medium bottle (also referred to as "medium or the like reagent supply bottle") 25 to the cell processing container 1. After the desired amount of medium is supplied, the pump is stopped, and all the electromagnetic valves are closed. At the time of cell recovery at the end of the culture, the electromagnetic valves 5, 15, 26 are opened, and the pump 17 is rotated to the right, whereby the buffer space 11 is made negative, and the cell suspension is introduced from the cell processing container 1 to the buffer space 11. After all the suspension is recovered, the pump is stopped, and all the electromagnetic valves are closed. Next, the electromagnetic valves 9, 15, 26 are opened, and the pump 17 is rotated to the left, whereby the buffer space 11 is made positive, and the cell suspension is transported from the buffer space 11 to the cell recovery container 10. After the transportation is completed, the cell recovery container 10 is aseptically separated from the flow path, and transferred to another operation.

[0040] Example 2

[0041] In Example 1, a container was used as the buffer space, but as shown in Figure 1 , the buffer space can also be provided as a tube having a certain pressure resistance. By being provided as a tube, an exhaust filter or the like connected to the bottle is not needed, and the flow path configuration can be simplified.

[0042] As such a tube, for example, a tube obtained by winding a rubber tube of silicon, a thermoplastic elastomer or the like having sufficient strength into a coil shape can be used. The number of turns of the coil shape is preferably adjusted according to the amount of the suspension to be transported. The flow path diameter is preferably a system that can be controlled by the pump and the electromagnetic valves used and has a certain pressure resistance. Alternatively, a metal tube can also be considered. Although the introduction cost is higher than that of a rubber tube, in the case where the tube length is long, the tube can be reused by sterilization treatment, and the cost can be suppressed by repeated use.

[0043] Example 3

[0044] In this embodiment, a tube is used as the buffer space. Figure 1 , Figure 3Let's take an example of setting a buffer space for both cell supply and recycling. In a closed system flow path, no contaminants will enter from the outside, so the buffer space used during cell supply can be reused during recycling. However, as mentioned above, depending on the type of cells being treated and the processing procedures, if the cell suspension before treatment is mixed with the cell suspension after treatment, there is a concern that residual cells from before treatment (at seeding) may die.

[0045] Therefore, in this embodiment, a flow path and a buffer space are provided for cleaning the cell suspension before delivery treatment with a washing solution such as physiological saline (PBS). This is an example of a closed system flow path configuration. Figure 1 and as an example of its steps Figure 3 This is used to represent this embodiment.

[0046] The flow path is set in the same manner as in Example 1. Figure 4 S101), in connecting the container 7 containing cells to the flow path ( Figure 5 After S102), first, open solenoid valves 8, 16, and 24. Figure 7 S103), causing the peristaltic pump 17 to rotate to the right ( Figure 5 (S104), thereby creating a negative pressure within the buffer space 13, introducing the suspension from the container 7 into the buffer space 13. The weight of the buffer space is measured using a weighing scale 14. Figure 6 S105), stops pump 17 when the desired weight is reached. Figure 6 S106), close solenoid valves 8, 16, and 24. Figure 6 (S107). Next, open solenoid valves 6, 16, and 24 ( Figure 6 S108), causing pump 17 to rotate to the left ( Figure 6 (S109), thereby creating positive pressure in the buffer space 13 and supplying the suspension to the cell processing device 1. The weight of the buffer space is measured using a weighing scale 14. Figure 6 S110), stops pump 17 when the desired weight is reached. Figure 6 S111), close solenoid valves 6, 16, and 24. Figure 6 (S112).

[0047] Next, open solenoid valves 15, 22, and 26. Figure 6 S113), causing pump 17 to rotate to the left ( Figure 6 (S114), thereby supplying cleaning fluid from cleaning fluid bottle 31 to buffer space 13.

[0048] Measure the weight of the buffer space using a weight scale 14. Figure 6 S115), when the desired weight is reached, closes solenoid valve 22 and opens solenoid valve 24. Figure 6S116), after the cleaning fluid in the flow path is completely squeezed into the buffer space 13 by maintaining the left rotation of pump 17 for a predetermined time, pump 17 is stopped. Figure 6 S117), close solenoid valves 15, 22, and 26. Figure 6 (S118).

[0049] Next, open solenoid valves 16, 21, and 24. Figure 6 S119), causing pump 17 to rotate to the left ( Figure 6 (S120), thereby creating positive pressure in the buffer space 13 and delivering cleaning fluid to the drain recovery container 23. The weight of the buffer space 13 is measured using a weighing gauge 14. Figure 6 S120), stops pump 17 when the desired weight is reached. Figure 6 S121), close solenoid valves 16, 21, 24 ( Figure 6 S122). Cell treatment is performed in culture processing device 1 ( Figure 6 After S123), open solenoid valves 5, 16, and 24. Figure 6 S124), causing pump 17 to rotate to the right ( Figure 6 (S125), thereby creating a negative pressure within the buffer space 13, introducing the suspension from the cell treatment device 1 into the buffer space 13. The weight of the buffer space is measured using a weighing scale 14. Figure 6 S126), stops pump 17 when the desired weight is reached. Figure 6 S127), close solenoid valves 5, 16, and 24. Figure 6 (S128).

[0050] Next, open solenoid valves 9, 16, and 24. Figure 6 S129), causing pump 17 to rotate to the left ( Figure 6 (S130), thereby creating positive pressure in the buffer space 13 and delivering the suspension to the cell recovery container 10. The weight of the buffer space 13 is measured using a weighing scale 14. Figure 6 S131), stop pump 17 when the desired weight is reached. Figure 6 S132), close solenoid valves 9, 16, and 24. Figure 7 (S133). Afterwards, the recycling container 10 is disconnected from the flow path and the contents are recycled. Figure 8 (S134).

[0051] Example 4

[0052] In this embodiment, using Figure 7 , Figure 8 This example illustrates how to measure the weight of a cell recycling container during cell recycling. Figure 7 This is an example of the configuration of a closed system flow path in Embodiment 4 of the present invention.Figure 7 indicates Figure 8 the steps in the flow path. If the weight of the recovery container 10 is measured at the time of cell recovery, the accurate recovery amount can be confirmed. In addition, in Figure 2 the cells are recovered into a single recovery container, but can be equally distributed by connecting a plurality of recovery containers.

[0053] In the case where a bag is used as the recovery container 10, space saving can be achieved by using a sling scale, but the center of gravity changes due to the change in shape of the suspension, and thus accurate weight measurement is not possible.

[0054] In Figure 8 , the steps S01 to S18 are exactly the same as in Figure 8 , and thus the description is omitted.

[0055] The point different from the steps of Example 1 is that, at the time of transporting the suspension from the buffer space 11 to the recovery container 10, the pump 20 is rotated to the left (S219 of Figure 8 ) to simultaneously supply gas to the recovery container 10, as a result of which the shape of the recovery container 10 can be kept constant and the weight can be accurately measured. The pressure in the recovery container 10 can also be measured, and feedback is applied to the operation of the pump 20 to keep the pressure constant.

[0056] The weights of the buffer space 11 and the recovery container 10 are measured (S220 of Figure 8 ), and at the time when the weight gauge 12 of the buffer space 11 is below the specified value and the weight gauge 33 of the recovery container 10 is above the specified value, the pump 17 and the pump 20 are stopped (S21 and S221 of Figure 8 ), the electromagnetic valves 9 and 15 are closed (S22 of Figure 9 ), and the cell recovery container 10 is recovered from the flow path (S23 of ​ ).

[0057] <Notes>

[0058] ​ is a graph in which iPS cells (201B7 strain) are used to evaluate the physical damage to cells by the peristaltic pump. Cells that were left standing after peeling, cells that passed through the peristaltic pump 10 times, and cells that were transported 10 times using the method described in Patent Literature 1, which does not pass through the pump, were re-seeded and cultured for 7 days, and the proliferation rate at that time was evaluated. The proliferation rate was significantly reduced when passing through the peristaltic pump. As described above, when transporting the cell suspension as described in the present embodiment, by being configured not to pass through the driving section, cell supply and recovery with reduced physical damage to cells can be achieved.

[0059] Explanation of symbols

[0060] 1: cell processing device, 2: exhaust filter, 3: connector of cell processing device and flow path, 4: branch connector, 5 / 6 / 8 / 9 / 15 / 16 / 21 / 22 / 24 / 26: solenoid valve, 7: container containing cell suspension, 10: cell recovery container, 11 / 13: buffer space, 12 / 14: gravimetric meter, 17 / 20: drive unit of peristaltic pump, 23: drain recovery container, 25: reagent supply bottle of culture medium, 27: pressure sensor, 28: mass flow meter, 29: regulator, 30: gas cylinder, 31: cleaning liquid bottle, 32: humidification bottle, 33: hanging scale.

Claims

1. An automated cultivation device, characterized in that, have: A cell suspension storage container that can hold cell suspensions; A cell processing container for processing a cell suspension delivered from the cell suspension receiving container; A flow path that connects at least the cell suspension receiving container to the cell processing container; A first buffer space, which is connected to the flow path and has a predetermined pressure resistance; and A pressure source for introducing the cell suspension from the cell suspension receiving container into the first buffer space by creating a negative pressure within the first buffer space.

2. The automatic cultivation device according to claim 1, characterized in that, The pressure source has the function of delivering the cell suspension in the first buffer space to the cell processing container by making the first buffer space positive pressure.

3. The automatic cultivation device according to claim 1, characterized in that, A recovery container for recovering processed cells is connected to the flow path. The automated culture device includes a cleaning mechanism for rinsing untreated cells remaining in the flow path and / or the first buffer space before transferring the treated cells in the cell processing container to the recovery container.

4. The automatic cultivation device according to claim 1, characterized in that, The flow path is connected to a recovery container for recovering treated cells and a second buffer space with predetermined pressure resistance. The pressure source has the function of transferring the cell suspension containing the treated cells from the cell treatment container into the second buffer space by creating a negative pressure within the second buffer space.

5. The automatic cultivation device according to claim 4, characterized in that, The pressure source has the function of conveying the cell suspension containing the treated cells in the second buffer space to the recovery container by making the second buffer space positive.

6. The automatic cultivation device according to claim 5, characterized in that, At least one of the cell suspension collection container, the cell processing container, and the recycling container is composed of a bag.

7. The automatic cultivation device according to claim 1, characterized in that, The buffer space is made of coil-shaped tubes.

8. The automatic cultivation device according to claim 5, characterized in that, The recycling container is made of a bag. The automated culture device has a gas supply mechanism that supplies gas to the bag without deflated when the cell suspension containing the treated cells in the second buffer space is transported to the bag.

9. An automated cultivation system, characterized in that, have: The automated culture apparatus according to any one of claims 1 to 8; and A control device that controls the various mechanisms of the automated culture apparatus, which includes the pressure source.

10. A cell culture method, which is a cell culture method using an automated culture device, characterized in that, The automated culture device includes: A cell suspension storage container that can hold cell suspensions; A cell processing container for processing a cell suspension delivered from the cell suspension receiving container; A flow path that connects at least the cell suspension receiving container to the cell processing container; A first buffer space, which is connected to the flow path and has a predetermined pressure resistance; as well as A pressure source that can create negative pressure within the first buffer space. The cell culture method includes: The step of setting up a liquid containing the cells to be cultured in the cell suspension container; The step of transferring cell-containing liquid from the cell suspension receiving container into the first buffer space by activating the pressure source to create a negative pressure within the first buffer space; and The step of transferring the cell-containing liquid transferred to the first buffer space to the cell processing container by activating the pressure source to create positive pressure within the first buffer space.

11. The cell culture method according to claim 10, characterized in that, The flow path is connected to a bag for recovering treated cells and a second buffer space with predetermined pressure resistance. The cell culture method includes: The step of transferring liquid containing treated cells from the cell treatment container into the second buffer space by activating the pressure source to create a negative pressure within the second buffer space; and The step of transferring the liquid containing the treated cells, which is transferred to the second buffer space, to a recycling container for recovering the treated cells by activating the pressure source to create a positive pressure in the second buffer space.

12. The cell culture method according to claim 10, characterized in that, The flow path is connected to a recovery container for recovering processed cells and a second buffer space. The cell culture method includes the following steps: By activating the pressure source to create a negative pressure within the second buffer space, the cell suspension containing the treated cells within the cell treatment container is transferred to the second buffer space.

13. The cell culture method according to claim 12, characterized in that, Includes the following steps: By activating the pressure source to create positive pressure within the second buffer space, the cell suspension containing the treated cells within the second buffer space is transported to the recovery container.

14. The cell culture method according to claim 13, characterized in that, The recycling container is made of a bag. The cell culture method includes the following steps: When the cell suspension containing the treated cells in the second buffer space is conveyed to the bag, gas is supplied to the bag in a manner that does not cause the bag to collapse.

Images