Cell harvesting method

Abstract

To strip off a cultured cell from a culture vessel using a stripper to collect it without causing cell injury or cost increase.SOLUTION: A cell collection method for collecting a cell 13 includes a medium movement step, a stripper feeding step, and a mixing step. After the cell 13 is cultured, in the medium movement step, a used medium 11 in a culture vessel 6 is moved to a collection vessel 5. After the medium movement step, in the stripper feeding step, a stripper 12A for stripping off the cell 13 from the inner surface 9 of the culture vessel 6 is supplied to the culture vessel. After the stripper feeding step, in the mixing step, the stripper 12A is mixed with the used medium 11.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a cell recovery method for recovering cells that have been cultured in a vessel containing a liquid medium and that have adhered to the inner surface of the vessel. [Background technology]

[0002] When cells are cultured in a culture vessel containing a liquid medium, the cells generally grow while attached to the inner surface of the culture vessel. Therefore, when recovering the cultured cells, the cells must be detached from the inner surface of the culture vessel. To do this, the used medium is generally first discharged from the culture vessel. Then, as described in Non-Patent Document 1, for example, a detachment solution is supplied to the culture vessel, thereby detaching the cells from the inner surface of the culture vessel. Note that if the cells are immersed in the detachment solution for a long period of time, they may be damaged by the detachment solution. Therefore, the reaction caused by the detachment solution is stopped (i.e., the detachment solution is inactivated) by mixing the suspension of the detachment solution and the cells with fresh medium. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Corning Incorporated, “Corning HYPERStack Cell Culture Vessel Closed System”, [online], 2016 (Heisei 28), Corning Incorporated, [Retrieved July 27, 2021], Internet <URL:https: / / www.corning.com / catalog / cls / documents / selection-guides / CLS-AN-364%20DL%20HYPERStack%20User%20Guide.pdf> Summary of the Invention [Problem to be solved by the invention]

[0004] In recent years, the cultivation of large amounts of cells using large-scale facilities has been considered. When it becomes necessary to recover a large amount of cells, the method of inactivating the detachment solution using fresh medium as described above will consume a large amount of fresh medium. This may result in a very high cost.

[0005] An object of the present invention is to suppress damage to cells and to suppress increases in costs when cultured cells are detached from a culture vessel using a detachment solution and recovered. [Means for solving the problem]

[0006] The cell recovery method of the first invention is a cell recovery method for recovering cells that have been cultured in a culture vessel containing a liquid culture medium and that have adhered to the inner surface of the culture vessel, and is characterized by comprising: a culture medium transfer step for transferring the spent culture medium in the culture vessel to a predetermined storage section after culturing the cells; a detachment liquid supply step for supplying a detachment liquid to the culture vessel after the culture medium transfer step for detaching the cells from the inner surface of the culture vessel; and a mixing step for mixing at least a portion of the detachment liquid with the spent culture medium after the detachment liquid supply step.

[0007] If cells are immersed in the detachment solution for a long period of time, the cells may be damaged. In the present invention, the detachment solution can be at least diluted with used culture medium used to culture the cells. Furthermore, if the used culture medium contains a component that inactivates the detachment solution, the detachment solution can also be inactivated. In this way, cell damage caused by the detachment solution can be suppressed without using fresh culture medium. Therefore, when cultured cells are detached from a culture vessel using the detachment solution and recovered, cell damage can be suppressed while preventing cost increases.

[0008] The cell recovery method of the second invention is characterized in that, in the first invention, the storage section has a recovery container for recovering the cells, and in the mixing process, a suspension containing the detachment solution and the cells is moved from the culture container to the recovery container.

[0009] In the mixing step, the spent medium may be transferred from the reservoir to the culture vessel. In this case, however, an additional step of transferring the suspension from the culture vessel to a collection vessel is required to recover the suspension of the spent medium and cells. In the present invention, the mixing step also serves as a step of transferring the cells to the collection vessel (i.e., a collection step). Therefore, an increase in the number of steps can be suppressed.

[0010] The cell recovery method of the third invention is characterized in that, in the first or second invention, the storage section has a predetermined first storage section and a second storage section separate from the first storage section, and the medium transfer process includes a first medium transfer process of transferring a first used medium, which is a part of the used medium, from the culture vessel to the first storage section, and a second medium transfer process of transferring a second used medium, which is a part of the used medium separate from the first used medium, from the culture vessel to the second storage section, in the mixing process, transferring a suspension in which the detachment solution and the cells are mixed from the culture vessel to the first storage section, and after the mixing process, a return process of transferring the second used medium from the second storage section to the culture vessel.

[0011] When the suspension is transferred to the first reservoir in the mixing step, some of the cultured cells may remain in the culture vessel. In the present invention, the remaining cells can be mixed with the second spent medium returned to the culture vessel in the returning step. This allows the remaining cells to be recovered together with the second spent medium. Therefore, the cell recovery rate can be improved while suppressing increases in costs.

[0012] The cell recovery method of the fourth invention is the same as that of the first invention, except that, between the detachment solution supplying step and the mixing step, the detachment solution is discharged from the culture vessel before the cells are completely detached from the inner surface of the culture vessel, and after the detachment solution discharging step, the culture vessel is left standing for a predetermined time with some of the detachment solution remaining in the culture vessel, and the spent culture medium is moved from the reservoir to the culture vessel in the mixing step.

[0013] When the detachment solution is discharged from the culture vessel before the cells are completely detached from the inner surface of the culture vessel, the cells and a small amount of detachment solution generally remain in the culture vessel. Waiting for a predetermined time in this state allows the cells to be completely detached from the inner surface of the culture vessel while minimizing cell damage caused by the detachment solution. Furthermore, by transferring the spent medium to the culture vessel in the mixing process, the cells can be suspended in the spent medium within the culture vessel. Therefore, even if the cells are susceptible to damage by the detachment solution, the cells can be recovered while minimizing cell damage.

[0014] The cell recovery method of the fifth invention is characterized in that, in any one of the first to fourth inventions, the storage section has a recovery container for recovering the cells, and the temperature of the space in which the recovery container is placed is maintained lower than the temperature of the space in which the culture vessel is placed.

[0015] Generally, cells may become activated and degenerate in warm places. In this regard, the present invention can maintain the temperature inside the collection container lower than the temperature inside the culture container. Therefore, compared to when the temperature inside the collection container is high, the degeneration of cells collected in the collection container is suppressed, and the cells can be preserved for a long time. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing a cell culture system for carrying out a cell recovery method according to a first embodiment. [Figure 2]1 is a flowchart showing the steps of a cell recovery method. [Figure 3] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 4] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 5] FIG. 10 is a schematic diagram showing a cell culture system according to a modified example of the first embodiment. [Figure 6] 1 is a flowchart showing the steps of a cell recovery method. [Figure 7] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 8] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 9] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 10] 10 is a flowchart showing the steps of a cell recovery method according to another modified example. [Figure 11] FIG. 10 is a schematic diagram showing a cell culture system according to a second embodiment. [Figure 12] 1 is a flowchart showing the steps of a cell recovery method. [Figure 13] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 14] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. [Figure 15] 1(a) and 1(b) are explanatory diagrams showing part of the procedure of the cell recovery method. DETAILED DESCRIPTION OF THE INVENTION

[0017] First Embodiment Next, a first embodiment of the present invention will be described. For the sake of convenience, the up-down direction on the paper surface of Fig. 1 is taken as the up-down direction (the vertical direction in which gravity acts).

[0018] (Outline of cell culture system) An outline of a cell culture system 1 for carrying out a cell recovery method according to a first embodiment will be described with reference to Fig. 1. Fig. 1 is a schematic front view showing the cell culture system 1. Fig. 1 shows the cell culture system 1 in a state where the culture of cells 13, which will be described later, has been completed and the cells 13 are about to be harvested (recovered).

[0019] As shown in FIG. 1, the cell culture system 1 includes a refrigerator 2 constituting a low-temperature area and an incubator 3 constituting a culture area. The refrigerator 2 is a device for storing various reagents while suppressing changes in the components of the reagents. The incubator 3 is a device for culturing cells 13, which will be described later. The types of cells 13 to be cultured include, but are not limited to, mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs).

[0020] The refrigerator 2 is configured to maintain the temperature of the low-temperature area at approximately 4°C. This allows various reagents to be stored in the refrigerator 2 while suppressing changes in their components. The refrigerator 2 may be configured to adjust the temperature of the low-temperature area to a temperature other than approximately 4°C. The refrigerator 2 has an openable and closable door (not shown). The refrigerator 2 contains at least a stripping liquid storage container 4 and a collection container 5 (a storage section of the present invention). The stripping liquid storage container 4 is a container for storing the stripping liquid 12 described below. The collection container 5 is a container for collecting the cultured cells 13. The refrigerator 2 may also contain other containers (not shown) for storing, for example, a liquid medium (not shown) used for culturing the cells 13.

[0021] The incubator 3 is configured to maintain the temperature of the culture area at, for example, approximately 37°C. This allows cells 13 to be cultured within the incubator 3. The incubator 3 may be configured to adjust the temperature of the culture area to a temperature other than approximately 37°C. The incubator 3 has an openable and closable door (not shown). The incubator 3 contains at least a culture vessel 6 and a detachment solution buffer container 7. The culture vessel 6 is a container for culturing the cells 13. After the cells 13 have been cultured, a liquid spent medium 11 is contained within the culture vessel 6, and the cells 13 have sufficiently proliferated within the spent medium 11. The cells 13 generally proliferate while attached to the inner surface 9 (more specifically, the bottom surface) of the culture vessel 6. The detachment solution buffer container 7 is a container for warming the detachment solution 12 used to detach the cultured cells 13 from the inner surface 9 of the culture vessel 6. The incubator 3 may also contain, for example, a container (not shown) for warming a culture medium (not shown). That is, for example, a culture medium buffer container 21 (see FIG. 7) used in a modified example described later may be housed inside the incubator 3. However, detailed description thereof will be omitted here.

[0022] At least the detachment solution storage container 4, the recovery container 5, the culture container 6, and the detachment solution buffer container 7 are connected to one another by, for example, a pipe 8 having multiple tubes. The liquid or suspension (a mixture of liquid and solid) used in the cell culture system 1 can be transferred (moved) from any one of the multiple containers to any other one of the multiple containers via the pipe 8. For example, multiple valves and one or more pumps (not shown) are provided along the pipe 8. For example, a pressure device (not shown) is connected to the detachment solution buffer container 7. By operating the pump or pressure device with the multiple valves appropriately opened and closed, a predetermined liquid or suspension is transferred to a predetermined container. The valves, pump, and pressure device may be operated by an operator. Alternatively, a control device (not shown) that controls the valves, pump, and pressure device may be provided. The control device may control the valves, pump, and pressure device on behalf of the operator to transfer the liquid or suspension.

[0023] Next, the culture medium will be described. In this embodiment, a liquid culture medium (culture solution) is used to culture the cells 13. The culture medium contains nutrients for the proliferation of the cells 13. The nutrients include, for example, inorganic salts, sugars, amino acids, vitamins, etc. The type of culture medium may be, for example, a serum medium containing animal-derived serum (for example, bovine serum), but is not limited to this. For example, a xeno-free medium containing human-derived components but not containing components derived from animals other than humans may be used as the culture medium. Alternatively, for example, an animal-free medium containing no animal-derived components may be used as the culture medium. After the culture of the cells 13 is completed, the culture medium becomes used culture medium 11.

[0024] Next, the detachment liquid 12 will be described. The detachment liquid 12 is a liquid containing a cell detachment enzyme for detaching cells 13 adhering to the inner surface 9 of the culture vessel 6 from the inner surface 9. The type of cell detachment enzyme that can be used is, for example, the well-known trypsin, but is not limited to this. The detachment liquid 12 may contain a cell detachment enzyme other than trypsin. Alternatively, the detachment liquid 12 does not necessarily have to contain a cell detachment enzyme, as long as it has the function of detaching cells 13 from the inner surface 9 of the culture vessel 6. For example, the detachment liquid 12 may contain ethylenediaminetetraacetic acid (EDTA).

[0025] Here, the following procedure, for example, can be considered as a conventional procedure for harvesting (recovering) the cultured cells 13. That is, when the used medium 11 is discharged from the culture vessel 6, the cells 13 remain attached to the inner surface 9 of the culture vessel 6. Then, a detachment solution 12 is supplied to the culture vessel 6. Note that if the cells 13 are immersed in the detachment solution 12 for a long time, they may be damaged by the detachment solution 12. Therefore, for example, a fresh medium (not shown) is placed in the collection vessel 5 in advance. At an appropriate time, a suspension containing the detachment solution 12 and the cells 13 is sent to the collection vessel 5. This allows the suspension to be mixed with the fresh medium. Generally, a fresh medium contains a substance that inactivates a cell detachment enzyme. For example, a serum medium contains inorganic salts (such as calcium and magnesium). Furthermore, for example, trypsin, a cell detachment enzyme, is inactivated by calcium and magnesium. In this way, the detachment solution 12 is inactivated by the medium. Therefore, the cells 13 are recovered while suppressing damage to the cells 13 caused by the detachment solution 12.

[0026] In recent years, the cultivation of a large amount of cells 13 using large-scale facilities has been considered. When it becomes necessary to recover a large amount of cells 13, the method of inactivating the detachment solution 12 using a fresh medium as described above results in a large amount of fresh medium being consumed. This may result in a very high cost. Therefore, in order to suppress damage to the cells 13 while suppressing an increase in cost, in the first embodiment, the cells 13 are recovered in the following manner.

[0027] The steps of the cell recovery method for recovering cells 13 in the cell culture system 1 will be described with reference to the flowchart in FIG. 2 and the schematic diagrams in FIGS. 3(a) to 4(b). For ease of explanation, it is assumed below that the work is performed by an operator. The operator operates the above-mentioned valves, pumps, and pressure devices (not shown) to send (move) various liquids or suspensions from one container to another. Instead of the operator, a control device (not shown) may control the valves, pumps, and pressure devices.

[0028] First, after the culture of the cells 13 is completed (after the cells 13 in the culture vessel 6 have sufficiently proliferated), the operator transfers the detachment liquid 12 from the detachment liquid storage vessel 4 to the detachment liquid buffer vessel 7 (S101). For example, as shown in FIG. 3(a), a portion of the detachment liquid 12 (detachment liquid 12A) in the detachment liquid storage vessel 4 may be transferred to the detachment liquid buffer vessel 7. The detachment liquid 12A is warmed in the incubator 3. Another portion of the detachment liquid 12 (detachment liquid 12B) remains in the detachment liquid storage vessel 4.

[0029] Next, the operator transfers the used culture medium 11 from the culture vessel 6 to the collection vessel 5 (S102, culture medium transfer process; see FIG. 3(b)). The temperature of the space (low temperature area) in which the collection vessel 5 is located is maintained lower than the temperature of the space (culture area) in which the culture vessel 6 is located. Therefore, the used culture medium 11 is cooled in the refrigerator 2. The cells 13 remain in the culture vessel 6 while adhering to the inner surface 9.

[0030] Next, the operator sends the warmed detachment solution 12A from the detachment solution buffer container 7 to the culture container 6 (S103, detachment solution supply step). After that, by waiting for a predetermined time, the detachment solution 12A detaches the cells 13 from the inner surface 9 of the culture container 6. As a result, the detachment solution 12A and the cells 13 mix together to form a suspension 14 (see FIG. 4(a)).

[0031] Finally, the operator transfers the suspension 14 from the culture vessel 6 to the collection vessel 5 (S104, mixing step). This causes the suspension 14 (the detachment solution 12A and the cells 13) to mix with the spent culture medium 11, resulting in a suspension 15 (see FIG. 4(b)). Therefore, the detachment solution 12A is diluted by the spent culture medium 11. Furthermore, the inventors of the present application discovered the following fact: In the spent culture medium 11, most of the nutrients other than inorganic salts are absorbed by the cells 13 and almost none remain, but some inorganic salts remain in the spent culture medium 11. Therefore, the inorganic salts can inactivate the detachment solution 12A. This effectively prevents the spent culture medium 11 from damaging the cells 13 with the detachment solution 12A.

[0032] Thereafter, the suspension 15 is separated into the cells 13 and other substances using, for example, a centrifuge (not shown) (concentration step), and the cells 13 can be harvested.

[0033] As described above, the detachment solution 12A can be at least diluted with the used medium 11 used to culture the cells 13. Furthermore, if the used medium 11 contains a component that inactivates the detachment solution 12A, the detachment solution 12A can also be inactivated. In this way, damage to the cells caused by the detachment solution 12A can be suppressed without using a fresh medium. Therefore, when the cultured cells 13 are detached from the culture vessel 6 using the detachment solution 12A and recovered, damage to the cells 13 can be suppressed while preventing an increase in costs.

[0034] Furthermore, in the mixing step, the suspension 14 in which the detachment solution 12A and the cells 13 are mixed is moved from the culture vessel 6 to the collection vessel 5. That is, the mixing step also serves as a step of moving the cells 13 to the collection vessel 5 (i.e., a collection step). Therefore, compared to the case in which the used culture medium 11 is moved from the collection vessel 5 to the culture vessel 6 in the mixing step, an increase in the number of steps can be suppressed.

[0035] Furthermore, the temperature inside the collection container 5 housed in the refrigerator 2 can be maintained lower than the temperature inside the culture container 6 housed in the incubator 3. Therefore, compared to when the temperature inside the collection container 5 is high, deterioration of the cells 13 collected in the collection container 5 can be suppressed, and the cells 13 can be preserved for a long period of time.

[0036] Next, a modified example of the first embodiment will be described, with the same reference numerals being used to designate components similar to those in the first embodiment, and the description thereof will be omitted where appropriate.

[0037] (1) In the first embodiment, all of the used culture medium 11 is transferred from the culture vessel 6 to the collection vessel 5 in the culture medium transfer step. However, this is not limited to this. A specific description will be given below. For example, as shown in FIG. 5, a cell culture system 1a may include a culture medium buffer container 21 (a second storage container of the present invention) in addition to the above-mentioned detachment solution storage container 4, collection container 5 (a first storage container of the present invention), culture vessel 6, and detachment solution buffer container 7. The culture medium buffer container 21 is housed, for example, in an incubator 3. The detachment solution storage container 4, collection container 5, culture vessel 6, detachment solution buffer container 7, and culture medium buffer container 21 are connected to one another by piping 8a.

[0038] The steps of the cell recovery method in the cell culture system 1a will be described with reference to the flowchart in FIG. 6 and the schematic diagrams in FIGS. 7(a) to 9(b). First, after the culture of the cells 13 is completed, the operator transfers the detachment solution 12A from the detachment solution storage container 4 to the detachment solution buffer container 7 (S201, see FIG. 7(a)). Next, as part of the medium transfer step, the operator transfers a portion of the used medium 11 (used medium 11A, the first used medium of the present invention) from the culture container 6 to the recovery container 5 (S202, the first medium transfer step, see FIG. 7(b)). Furthermore, as part of the medium transfer step, the operator transfers the remaining used medium 11B (the second used medium of the present invention) from the culture container 6 to the culture medium buffer container 21 (S203, the second medium transfer step, see FIG. 7(b)). The used medium 11B is a portion of the used medium 11 that is separate from the used medium 11A. Note that the order of S202 and S203 may be reversed. After that, the operator sends the warmed detachment solution 12A from the detachment solution buffer container 7 to the culture container 6 (S204, detachment solution supplying step). After that, by waiting for a predetermined time, the detachment solution 12A causes the cells 13 to detach from the inner surface 9 of the culture container 6. As a result, the detachment solution 12A and the cells 13 mix to form a suspension solution 22 (see FIG. 8(a)). After that, the operator sends the suspension solution 22 from the culture container 6 to the collection container 5 (S205, mixing step). As a result, the suspension solution 22 (detachment solution 12A and cells 13) and the used culture medium 11A mix to form a suspension solution 23 (see FIG. 8(b)). Here, it is most desirable for all cells 13 in suspension 22 to flow out of culture vessel 6 in step S205. However, in reality, some cells 13 may not flow out and remain in culture vessel 6 (see cells 13A in Figures 8(a) and 8(b)). Therefore, next, the operator transfers used medium 11B from culture medium buffer container 21 to culture vessel 6 (S206, return step). This mixes cells 13A and used medium 11B to form suspension 24 (see Figure 9(a)), and cells 13A contained in suspension 24 can be recovered. Finally, the operator transfers suspension 24 from culture vessel 6 to recovery container 5 (S207). This mixes suspension 23 and suspension 24 to form suspension 25 (see Figure 9(b)).In this way, some of the cells 13A remaining in the culture vessel 6 can be mixed with the used medium 11B returned to the culture vessel 6 in the returning step. This allows the cells 13A to be recovered together with the used medium 11B. Therefore, the recovery rate of the cells 13 can be improved while suppressing an increase in costs.

[0039] (2) In the above-described embodiments, the suspension (suspension 14 or suspension 22) of the detachment solution 12A and the cells 13 is sent from the culture vessel 6 to the collection vessel 5 in the mixing step. However, this is not limited to this. The operator may send the used culture medium 11 from the collection vessel 5 to the culture vessel 6 in the mixing step. By such an operation, the detachment solution 12A can be diluted and inactivated. In this case, in order to collect the cells 13, it is necessary to finally send the suspension (not shown) containing the cells 13 from the culture vessel 6 to the collection vessel 5, which increases the number of steps.

[0040] (3) As another modification of the configuration shown in FIG. 5, in the cell culture system 1a, a cell recovery method may be performed in the following procedure similar to the procedure shown in the first embodiment (S101 to S104 described above). Hereinafter, a description will be given with reference to the flowchart in FIG. 10. After performing step S101, the operator may perform, for example, a step (S102A) of transferring all of the used culture medium 11 from the culture vessel 6 to the culture medium buffer container 21, instead of S102 (the step of transferring the used culture medium 11 from the culture vessel 6 to the collection container 5). In this case, the culture medium buffer container 21 corresponds to the reservoir of the present invention. Furthermore, after performing step S103, the operator may perform a step (S104A, corresponding to the mixing step of the present invention) of returning the used culture medium 11 from the culture medium buffer container 21 to the culture vessel 6, instead of S104 (the step of transferring the suspension liquid 14 from the culture vessel 6 to the collection container 5). Thereafter, the operator may execute a step (S105A) of transferring all of the suspension (not shown) in the culture container 6 to the collection container 5.

[0041] Alternatively, in this modification, instead of step S104A, the operator may perform a step of transferring the suspension 14 from the culture vessel 6 to the culture medium buffer vessel 21 (corresponding to a mixing step of the present invention). Thereafter, instead of step S105A, the operator may perform a step of transferring all of the suspension (not shown) in the culture medium buffer vessel 21 to the collection vessel 5.

[0042] Second Embodiment Next, a second embodiment of the present invention will be described, with the same reference numerals being used to designate components having the same configuration as those in the first embodiment, and the description thereof will be omitted where appropriate.

[0043] In the first embodiment, in the mixing step, all of the stripping solution 12A sent to the culture vessel 6 is mixed with the used medium 11. In the second embodiment, in the mixing step described below, a portion of the stripping solution 12A is mixed with the used medium 11. That is, considering both the first and second embodiments, at least a portion of the stripping solution 12A is mixed with the used medium 11 in the mixing step.

[0044] For example, the above-mentioned iPSCs are generally more susceptible to damage by the detachment solution 12 than the above-mentioned MSCs. For this reason, the cell recovery method described below is particularly effective when recovering iPSCs. As shown in FIG. 11 , the cell culture system 1b includes the above-mentioned detachment solution storage container 4, recovery container 5, culture container 6, detachment solution buffer container 7, and medium buffer container 21, as well as a drainage container 32 disposed in a room temperature area 31. The detachment solution storage container 4, recovery container 5, culture container 6, detachment solution buffer container 7, medium buffer container 21, and drainage container 32 are connected to one another by piping 8b.

[0045] The steps of the cell recovery method in the cell culture system 1b will be described with reference to the flowchart in FIG. 12 and the schematic diagrams in FIGS. 13(a) to 15(b).

[0046] First, after the culture of the cells 13 is completed, the operator transfers the detachment solution 12A from the detachment solution storage container 4 to the detachment solution buffer container 7 (S301, see FIG. 13(a)). Next, the operator transfers the used culture medium 11 from the culture vessel 6 to the culture medium buffer container 21 (S302, culture medium transfer step, see FIG. 13(b)). In the second embodiment, the culture medium buffer container 21 corresponds to the reservoir of the present invention. Next, the operator transfers the warmed detachment solution 12A from the detachment solution buffer container 7 to the culture vessel 6 (S303, detachment solution supply step, see FIG. 14(a)).

[0047] Then, after waiting for a certain period of time, the operator discharges the detachment solution 12A from the culture vessel 6 and sends it to the drainage container 32 before the cells 13 are completely detached from the inner surface 9 of the culture vessel 6 (S304, detachment solution discharge step; see FIG. 14(b)). At this time, since the cells 13 are attached to the inner surface 9 of the culture vessel 6, they are not discharged together with the detachment solution 12A but remain in the culture vessel 6. Furthermore, a portion of the detachment solution 12A remains in the culture vessel 6, for example, adhering to the cells 13. Thereafter, the operator waits for a predetermined period of time (S305, waiting step). As a result, the small amount of detachment solution 12A remaining in the culture vessel 6 allows the cells 13 to be completely detached from the inner surface 9 of the culture vessel 6. In this method, damage to the cells 13 caused by the detachment solution 12A can be minimized compared to when the cells 13 are immersed in the detachment solution 12A until they are completely detached from the inner surface 9 of the culture vessel 6. Next, the operator transfers the used culture medium 11 from the culture medium buffer container 21 to the culture container 6 (S306, mixing step). In this way, the stripping liquid discharge step and the standby step are provided between the stripping liquid supply step and the mixing step.

[0048] In the mixing step, the spent medium 11 is transferred to the culture vessel 6, whereby the cells 13 can be suspended in the spent medium 11 within the culture vessel 6. As a result, the spent medium 11 and the cells 13 are mixed together to form a suspension 33 (see FIG. 15(a)). Furthermore, a portion of the detachment solution 12A remaining in the culture vessel 6 is mixed with the spent medium 11, so that the portion of the detachment solution 12A can be diluted and inactivated by the spent medium 11. Finally, the operator transfers the suspension 33 from the culture vessel 6 to the collection vessel 5 (S307; see FIG. 15(b)). In this manner, even if the cells 13 are susceptible to damage by the detachment solution 12A, the cells 13 can be collected while minimizing damage to the cells 13.

[0049] Next, a modified example of the second embodiment will be described, in which the same reference numerals will be used to designate components having the same configuration as the second embodiment, and the description thereof will be omitted as appropriate.

[0050] (1) In the second embodiment, all of the spent medium 11 is sent from the medium buffer container 21 to the culture vessel 6 in the mixing step. However, this is not limited to this. For example, a portion of the spent medium 11 may be sent from the medium buffer container 21 to the culture vessel 6 in the mixing step, and then a suspension (not shown) of the portion of the spent medium 11 and the cells 13 may be sent from the culture vessel 6 to the collection vessel 5. Then, the remaining spent medium 11 may be sent from the medium buffer container 21 to the culture vessel 6. Then, a suspension (not shown) of the remaining spent medium 11 and some of the cells 13 that may remain in the culture vessel 6 may be sent from the culture vessel 6 to the collection vessel 5.

[0051] Next, a description will be given of modifications common to the first and second embodiments. However, components having the same configuration as those in the first or second embodiment will be given the same reference numerals and descriptions thereof will be omitted as appropriate.

[0052] (1) In the first and second embodiments, the detachment solution 12A is sent to the detachment solution buffer container 7 and warmed when the cells 13 are collected. However, this is not limited to this. For example, the detachment solution 12A may be warmed in advance while the cells 13 are being cultured (before the collection of the cells 13 is started).

[0053] (2) In the first and second embodiments, the collection container 5 is accommodated in the refrigerator 2. However, this is not limited to this. For example, if the cells 13 sent to the collection container 5 are quickly harvested through a concentration process, the collection container 5 may be placed in a different location. In this case, the collection container 5 may be accommodated in the incubator 3, for example. Alternatively, the collection container 5 may be placed in a space outside the refrigerator 2 and the incubator 3 (for example, the room temperature area 31).

[0054] (3) The used medium 11 does not necessarily have to contain a component for inactivating the detachment solution 12. That is, the used medium 11 may be used simply to dilute at least a portion of the detachment solution 12 used to detach the cells 13 from the inner surface 9 of the culture vessel 6. Even in this case, damage to the cells 13 caused by the detachment solution 12 can be suppressed without using fresh medium. [Explanation of symbols]

[0055] 5. Collection container (storage section, first storage section) 6 Culture vessel 9. Interior 11 Spent medium 11A Spent medium (first spent medium) 11B Spent medium (second spent medium) 12 Stripping solution 13 cells 21 Medium buffer container (second reservoir)

Claims

1. A cell recovery method for recovering cells cultured in a culture vessel containing a liquid medium and housed in an incubator that constitutes a culture area for culturing cells, the cells adhering to the inner surface of the culture vessel being recovered, comprising: a medium transfer step of transferring used medium in the culture vessel to a predetermined reservoir after culturing the cells; a detachment solution supplying step of supplying a detachment solution to the culture vessel for detaching the cells from the inner surface of the culture vessel after the medium transfer step; a mixing step of mixing at least a portion of the stripping solution with the used culture medium after the stripping solution supply step, the storage unit has a collection container for collecting the cells, which is accommodated in a refrigerator constituting a low-temperature area that is at a lower temperature than the culture area, A cell recovery method, characterized in that the used culture medium transferred from the culture vessel to the recovery vessel in the culture medium transfer step is cooled in the low-temperature area.

2. A cell recovery method for recovering cells cultured in a culture vessel containing a liquid medium and adhering to the inner surface of the culture vessel, comprising: a medium transfer step of transferring used medium in the culture vessel to a predetermined reservoir after culturing the cells; a detachment solution supplying step of supplying a detachment solution to the culture vessel for detaching the cells from the inner surface of the culture vessel after the medium transfer step; a mixing step of mixing at least a portion of the stripping solution with the used culture medium after the stripping solution supply step, The storage section has a predetermined first storage section and a second storage section separate from the first storage section, The medium transfer step includes: a first culture medium transfer step of transferring a first used culture medium, which is a part of the used culture medium, from the culture vessel to the first reservoir; a second culture medium transferring step of transferring a second used culture medium, which is a part of the used culture medium separate from the first used culture medium, from the culture vessel to the second reservoir; In the mixing step, a suspension in which the detachment solution and the cells are mixed is moved from the culture vessel to the first reservoir; A cell recovery method comprising, after the mixing step, a return step of transferring the second used culture medium from the second reservoir to the culture vessel.

3. the storage unit has a collection container for collecting the cells, 3. The cell recovery method according to claim 2, wherein the temperature of the space in which the recovery container is placed is maintained lower than the temperature of the space in which the culture container is placed.

4. the storage unit has a collection container for collecting the cells, 4. The cell recovery method according to claim 1, wherein in the mixing step, a suspension in which the detachment solution and the cells are mixed is moved from the culture vessel to the recovery vessel.

5. Between the stripping solution supplying step and the mixing step, a detachment solution discharge step of discharging the detachment solution from the culture vessel before the cells are completely detached from the inner surface of the culture vessel; a waiting step of waiting for a predetermined time in a state where a portion of the detachment solution remains in the culture vessel after the detachment solution discharging step, 2. The cell recovery method according to claim 1, wherein the spent culture medium is transferred from the reservoir to the culture vessel in the mixing step.

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