Cell harvesting method

Abstract

When detaching cultured cells from the culture vessel using a detachment solution and recovering them, the goal is to minimize cell damage while keeping costs down. [Solution] The cell recovery method for recovering cells 13 comprises a culture medium transfer step, a detachment solution supply step, and a mixing step. After the cells 13 have been cultured, in the culture medium transfer step, the used culture medium 11 in the culture vessel 6 is transferred to the recovery container 5. After the culture medium transfer step, in the detachment solution supply step, a detachment solution 12A for detaching the cells 13 from the inner surface 9 of the culture vessel 6 is supplied to the culture vessel. After the detachment solution supply step, in the mixing step, the detachment solution 12A and the used culture medium 11 are mixed.

Description

Technical Field

[0001] The present invention relates to a cell collection method for culturing cells in a container filled with a liquid medium and collecting the cells adhering to the inner surface of the container.

Background Art

[0002] When cells are cultured in a culture container filled with a liquid medium, generally, the cells grow while adhering to the inner surface of the culture container. Therefore, when collecting the cultured cells, it is necessary to detach the cells from the inner surface of the culture container. For this purpose, first, generally, the used medium is discharged from the culture container. Thereafter, for example, as described in Non-Patent Document 1, the cells can be detached from the inner surface of the culture container by supplying a detachment solution to the culture container. Note that if the cells are immersed in the detachment solution for a long time, they may be damaged by the detachment solution. Therefore, by mixing the suspension in which the detachment solution and the cells are mixed with a fresh medium, the reaction by the detachment solution is stopped (that is, the detachment solution is inactivated).

Prior Art Documents

Non-Patent Documents

[0003] <9000017>

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In recent years, there has been interest in culturing large quantities of cells using large-scale facilities. When it becomes necessary to harvest large amounts of cells, the method of inactivating the detachment solution using fresh culture medium, as described above, would consume a large amount of fresh culture medium. Therefore, the cost could become extremely high.

[0005] The objective of the present invention is to suppress cell damage while minimizing cost increases when detaching cultured cells from the culture vessel using a detachment solution and recovering them. [Means for solving the problem]

[0006] 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 have adhered to the inner surface of the culture vessel, and is characterized by comprising: a culture medium transfer step of moving the used culture medium in the culture vessel to a predetermined storage area after culturing the cells; a detachment solution supply step of supplying a detachment solution to the culture vessel after the culture medium transfer step to detach the cells from the inner surface of the culture vessel; and a mixing step of mixing at least a portion of the detachment solution with the used culture medium after the detachment solution supply step.

[0007] If cells are immersed in the detachment solution for a long time, the cells may be damaged. In this invention, the detachment solution can be diluted at least with the 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 even without using fresh culture medium. Therefore, when detaching cultured cells from the culture vessel with the detachment solution and recovering them, it is possible to suppress cell damage while keeping costs down.

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

[0009] In the mixing step, the used culture medium may be transferred from the storage unit to the culture vessel. However, in this case, an additional step is required to transfer the suspension of the used culture medium and cells from the culture vessel to a recovery container in order to recover the suspension. In the present invention, the mixing step also serves as the step of transferring the cells to the recovery container (i.e., the recovery step). Therefore, the 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 unit has a predetermined first storage unit and a second storage unit separate from the first storage unit, the culture medium transfer step has 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 storage unit, and a second culture medium transfer 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 storage unit, the mixing step has a suspension in which the detachment solution and the cells are mixed, which is transferred from the culture vessel to the first storage unit, and after the mixing step, a return step of transferring the second used culture medium from the second storage unit to the culture vessel.

[0011] During the mixing process, when the suspension is moved to the first storage unit, some of the cultured cells may remain in the culture vessel. In this invention, some of the cells remaining in the culture vessel can be mixed with the second used culture medium returned to the culture vessel during the return process. This allows for the recovery of these cells together with the second used culture medium. Therefore, the cell recovery rate can be improved while keeping costs down.

[0012] The cell recovery method of the fourth invention is characterized in that, in the first invention, between the detachment solution supply 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 discharge step, the vessel is left waiting for a predetermined time with a portion of the detachment solution remaining in the culture vessel, and in the mixing step, the used culture medium is moved from the storage unit to the culture vessel.

[0013] When the detachment solution is drained from the culture vessel before the cells have completely detached from the inner surface of the vessel, cells and a small amount of detachment solution generally remain in the vessel. By waiting for a predetermined time in this state, it is possible to completely detach the cells from the inner surface of the culture vessel while minimizing cell damage caused by the detachment solution. Furthermore, by moving the used culture medium to the culture vessel during the mixing process, the cells can be suspended in the used culture medium within the culture vessel. Therefore, even if the cells are susceptible to damage from the detachment solution, for example, the cells can be recovered while minimizing cell damage.

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

[0015] Generally, cells can become activated and denatured in warm environments. In this respect, the present invention makes it possible to maintain the temperature inside the recovery container lower than the temperature inside the culture vessel. Therefore, compared to cases where the temperature inside the recovery container is high, the denaturation of cells recovered in the recovery container is suppressed, and the cells can be stored for a longer period of time. [Brief explanation of the drawing]

[0016] [Figure 1] This is a schematic diagram showing a cell culture system for carrying out the cell retrieval method according to the first embodiment. [Figure 2]It is a flowchart showing the procedure of the cell recovery method. [Figure 3] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 4] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 5] It is a schematic diagram showing a cell culture system according to a modification of the first embodiment. [Figure 6] It is a flowchart showing the procedure of the cell recovery method. [Figure 7] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 8] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 9] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 10] It is a flowchart showing the procedure of the cell recovery method according to another modification. [Figure 11] It is a schematic diagram showing a cell culture system according to the second embodiment. [Figure 12] It is a flowchart showing the procedure of the cell recovery method. [Figure 13] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 14] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Figure 15] (a) and (b) are explanatory diagrams showing a part of the procedure of the cell recovery method. [Embodiments for Carrying Out the Invention]

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

[0018] (Outline of the cell culture system) The outline of the cell culture system 1 for carrying out the cell recovery method according to the first embodiment will be described with reference to Figure 1. Figure 1 is a schematic front view of the cell culture system 1. Note that Figure 1 shows the cell culture system 1 in the state immediately before the cell culture of the cells 13, which will be described later, is completed and the cells 13 are harvested (recovered).

[0019] As shown in Figure 1, the cell culture system 1 comprises a refrigerator 2 that constitutes a low-temperature area and an incubator 3 that constitutes a culture area. The refrigerator 2 is a device for storing various reagents while suppressing changes in the components of those reagents. The incubator 3 is a device for culturing cells 13, which will be described later. Examples of 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] Refrigerator 2 is configured to maintain the temperature of the low-temperature area at approximately 4°C. This allows for the storage of various reagents within Refrigerator 2 while suppressing changes in the components of the reagents. Refrigerator 2 may also be configured to allow the temperature of the low-temperature area to be adjusted to a temperature other than approximately 4°C. Refrigerator 2 has an openable and closable door (not shown). Refrigerator 2 contains at least a stripping solution storage container 4 and a recovery container 5 (storage unit of the present invention). The stripping solution storage container 4 is a container for storing the stripping solution 12, which will be described later. The recovery container 5 is a container for recovering cultured cells 13. In addition, Refrigerator 2 may also contain, for example, a container (not shown) for storing a liquid culture medium (not shown) used for culturing cells 13.

[0021] Incubator 3 is configured to maintain the temperature of the culture area at approximately 37°C, for example. This allows cells 13 to be cultured within incubator 3. Incubator 3 may also be configured to adjust the temperature of the culture area to a temperature other than approximately 37°C. Incubator 3 has a door (not shown) that can be opened and closed. Inside incubator 3 are at least a culture vessel 6 and a detachment buffer container 7. The culture vessel 6 is a container for culturing cells 13. When the cell culture is complete, the culture vessel 6 contains used liquid culture medium 11, in which the cells 13 have grown sufficiently. Generally, the cells 13 grow while attached to the inner surface 9 (more specifically, the bottom surface) of the culture vessel 6. The detachment 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. In addition, the incubator 3 may also contain, for example, a container (not shown) for warming culture medium (not shown). In other words, for example, the culture medium buffer container 21 (see Figure 7), which will be used in the modified example described later, may be contained inside the incubator 3. However, a detailed explanation will be omitted here.

[0022] At a minimum, the stripping solution storage container 4, the recovery container 5, the culture container 6, and the stripping solution buffer container 7 are connected to each other by a piping system 8 having, for example, multiple tubes. The liquid or suspension (a mixture of liquid and solid) used in the cell culture system 1 can be sent (moved) from any of the above-mentioned containers to any other container via the piping system 8. Part of the piping system 8, for example, is provided with multiple valves (not shown) and one or more pumps. Also, for example, the stripping solution buffer container 7 is connected to a pressurizing device (not shown). By operating the pump or pressurizing device with the multiple valves appropriately opened and closed, a predetermined liquid or suspension is sent to a predetermined container. The valves, pumps, and pressurizing device may be operated by an operator. Alternatively, a control device (not shown) that controls the valves, pumps, and pressurizing device may be provided. The control device may control the valves, pumps, and pressurizing device on behalf of the operator to move 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 necessary for the proliferation of the cells 13. These nutrients include, for example, inorganic salts, sugars, amino acids, and vitamins. As for the type of culture medium, for example, a serum medium containing animal-derived serum (for example, bovine serum) can be used, but is not limited to this. As the culture medium, for example, a xeno-free medium containing human-derived components but no non-human animal-derived components may be used. Alternatively, as the culture medium, for example, an animal-free medium that does not contain animal-derived components may be used. After the culture of the cells 13 is complete, the culture medium becomes used culture medium 11.

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

[0025] Here, a conventional procedure for harvesting (recovering) cultured cells 13 could be as follows: When the used culture 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, the 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, fresh culture medium (not shown) is placed in the recovery container 5 beforehand. At the appropriate time, a suspension of detachment solution 12 and cells 13 is sent to the recovery container 5. This mixes the suspension with the fresh culture medium. Generally, fresh culture medium contains substances that inactivate cell-detaching enzymes. For example, serum culture medium contains inorganic salts (calcium and magnesium, etc.). Also, for example, trypsin, a cell-detaching enzyme, is inactivated by calcium and magnesium. In this way, the culture medium inactivates the detachment solution 12. Therefore, the cells 13 are recovered while suppressing damage to the cells 13 by the detachment solution 12.

[0026] In recent years, there has been consideration of culturing large quantities of cells 13 using large-scale facilities. When it becomes necessary to recover a large quantity of cells 13, the method of inactivating the detachment solution 12 using fresh culture medium, as described above, would consume a large amount of fresh culture medium. This could lead to very high costs. Therefore, in order to suppress damage to the cells 13 while keeping costs down, in the first embodiment, the cells 13 are recovered by the following procedure.

[0027] The procedure for recovering cells 13 in cell culture system 1 will be explained with reference to the flowchart in Figure 2 and the schematic diagrams in Figures 3(a) to 4(b). For the sake of explanation, it will be assumed that an operator performs the work below. The operator operates the valves, pumps, and pressurizing devices (not shown) described above to transfer (move) various liquids or suspensions from one container to another. Alternatively, a control device (not shown) may control the valves, pumps, and pressurizing devices instead of the operator.

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

[0029] Next, the operator transfers the used culture medium 11 from the culture vessel 6 to the collection container 5 (S102. Culture medium transfer process. See Figure 3(b)). The temperature of the space where the collection container 5 is located (low temperature area) is maintained lower than the temperature of the space where the culture vessel 6 is located (culture area). Therefore, the used culture medium 11 is cooled in the refrigerator 2. The cells 13 remain in the culture vessel 6, attached 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 vessel 6 (S103. Detachment solution supply step). After waiting for a predetermined time, the cells 13 are detached from the inner surface 9 of the culture vessel 6 by the detachment solution 12A. As a result, the detachment solution 12A and the cells 13 mix to form a suspension 14 (see Figure 4(a)).

[0031] Finally, the operator transfers the suspension 14 from the culture vessel 6 to the collection vessel 5 (S104, mixing step). This mixes the suspension 14 (exfoliation solution 12A and cells 13) with the used culture medium 11 to form suspension 15 (see Figure 4(b)). Thus, the exfoliation solution 12A is diluted by the used culture medium 11. Furthermore, the inventors of this application have found the following fact: In the used culture medium 11, most of the nutrients other than inorganic salts are absorbed by the cells 13 and hardly remain, but inorganic salts remain in the used culture medium 11 to some extent. Therefore, these inorganic salts can inactivate the exfoliation solution 12A. As a result, the used culture medium 11 can effectively suppress damage to the cells 13 by the exfoliation solution 12A.

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

[0033] As described above, the detachment solution 12A can be diluted at least by the used culture medium 11 used to culture the cells 13. Furthermore, if the used culture medium 11 contains a component that inactivates the detachment solution 12A, the detachment solution 12A can also be inactivated. In this way, cell damage caused by the detachment solution 12A can be suppressed even without using fresh culture medium. Therefore, when detaching and recovering cultured cells 13 from the culture vessel 6 with the detachment solution 12A, it is possible to suppress damage to the cells 13 while keeping costs down.

[0034] Furthermore, during the mixing process, the suspension 14, which is a mixture of the detachment solution 12A and the cells 13, is transferred from the culture vessel 6 to the collection container 5. In other words, the mixing process also serves as the process of transferring the cells 13 to the collection container 5 (i.e., the collection process). Therefore, compared to the case where the used culture medium 11 is transferred from the collection container 5 to the culture vessel 6 during the mixing process, the increase in the number of steps can be suppressed.

[0035] Furthermore, the internal temperature of the recovery container 5 housed in the refrigerator 2 can be maintained lower than the internal temperature of the culture vessel 6 housed in the incubator 3. Therefore, compared to the case where the internal temperature of the recovery container 5 is high, the deterioration of the cells 13 recovered in the recovery container 5 is suppressed, and the cells 13 can be stored for a longer period of time.

[0036] Next, a modified example of the first embodiment described above will be explained. However, components having the same configuration as the first embodiment will be denoted by the same reference numerals and their descriptions will be omitted as appropriate.

[0037] (1) In the first embodiment, all used culture medium 11 is sent from the culture vessel 6 to the recovery container 5 during the culture medium transfer process. However, this is not the only embodiment. A specific example will be given below. For example, as shown in Figure 5, the cell culture system 1a may include a culture medium buffer container 21 (second storage unit of the present invention) in addition to the detachment solution storage container 4, recovery container 5 (first storage unit of the present invention), culture vessel 6 and detachment solution buffer container 7 described above. The culture medium buffer container 21 is housed, for example, in an incubator 3. The detachment solution storage container 4, recovery container 5, culture vessel 6, detachment solution buffer container 7 and culture medium buffer container 21 are connected to each other by piping 8a.

[0038] The procedure for cell retrieval in the cell culture system 1a will be explained with reference to the flowchart in Figure 6 and the schematic diagrams in Figures 7(a) to 9(b). First, after the cell culture 13 is complete, the operator sends the detachment solution 12A from the detachment solution storage container 4 to the detachment solution buffer container 7 (S201. See Figure 7(a)). Next, as part of the culture medium transfer process, the operator sends a portion of the used culture medium 11 (used culture medium 11A. First used culture medium of the present invention) from the culture container 6 to the retrieval container 5 (S202. First culture medium transfer process. See Figure 7(b)). Furthermore, as part of the culture medium transfer process, the operator sends the remaining used culture medium 11B (second used culture medium of the present invention) from the culture container 6 to the culture medium buffer container 21 (S203. Second culture medium transfer process. See Figure 7(b)). Used culture medium 11B is a portion of the used culture medium 11 that is different from used culture medium 11A. Note that the order of steps S202 and S203 may be reversed. The operator then sends the warmed detachment solution 12A from the detachment solution buffer container 7 to the culture vessel 6 (S204. Detachment solution supply step). After waiting for a predetermined time, the cells 13 are detached from the inner surface 9 of the culture vessel 6 by the detachment solution 12A. As a result, the detachment solution 12A and the cells 13 mix to form a suspension 22 (see Figure 8(a)). The operator then sends the suspension 22 from the culture vessel 6 to the collection container 5 (S205. Mixing step). As a result, the suspension 22 (detachment solution 12A and cells 13) and the used culture medium 11A are mixed to form a suspension 23 (see Figure 8(b)). Here, in step S205, it is most preferable that all cells 13 in the suspension 22 flow out of the culture vessel 6, but in reality, some cells 13 may remain in the culture vessel 6 without flowing out (see cells 13A in Figures 8(a) and (b)). Therefore, the operator then sends the used culture medium 11B from the culture medium buffer container 21 to the culture vessel 6 (S206, return step). This mixes the cells 13A with the used culture medium 11B to form a suspension 24 (see Figure 9(a)), making it possible to recover the cells 13A contained in the suspension 24. Finally, the operator sends the suspension 24 from the culture vessel 6 to the recovery container 5 (S207). This mixes the suspension 23 and the suspension 24 to form a suspension 25 (see Figure 9(b)).As described above, some of the cells 13A remaining in the culture vessel 6 can be mixed with the used culture medium 11B returned to the culture vessel 6 in the return process. This allows the cells 13A to be recovered together with the used culture medium 11B. Therefore, the recovery rate of cells 13 can be improved while keeping costs down.

[0039] (2) In the embodiments described above, in the mixing step, a suspension (suspension 14 or suspension 22) of the detachment solution 12A and cells 13 is sent from the culture vessel 6 to the collection vessel 5. However, this is not limited to this. The operator may also send the used culture medium 11 from the collection vessel 5 to the culture vessel 6 in the mixing step. This operation can dilute and inactivate the detachment solution 12A. In this case, however, in order to recover the cells 13, it is necessary to send the suspension (not shown) containing the cells 13 from the culture vessel 6 to the collection vessel 5, so the number of steps increases.

[0040] (3) As another modification of the configuration shown in Figure 5, the cell recovery method may be carried out in the cell culture system 1a by the following procedure, which is similar to the procedure shown in the first embodiment (S101 to S104 described above). The following will be explained with reference to the flowchart in Figure 10. After performing the step in S101, the operator may, instead of S102 (the step of sending the used culture medium 11 from the culture vessel 6 to the recovery container 5), perform, for example, the step of sending all of the used culture medium 11 from the culture vessel 6 to the culture medium buffer container 21 (S102A). In this case, the culture medium buffer container 21 corresponds to the storage unit of the present invention. Furthermore, after performing the step in S103, the operator may, instead of S104 (the step of sending the suspension 14 from the culture vessel 6 to the recovery container 5), perform the step of returning the used culture medium 11 from the culture medium buffer container 21 to the culture vessel 6 (S104A, which corresponds to the mixing step of the present invention). Subsequently, the operator may perform the step (S105A) of transferring all of the suspension (not shown) in the culture vessel 6 to the recovery vessel 5.

[0041] Alternatively, in the modified example, the operator may, instead of step S104A, perform the step of transferring the suspension 14 from the culture vessel 6 to the culture medium buffer container 21 (corresponding to the mixing step of the present invention). Subsequently, the operator may, instead of step S105A, perform the step of transferring all of the suspension (not shown) in the culture medium buffer container 21 to the recovery container 5.

[0042] <Second Embodiment> Next, a second embodiment of the present invention will be described. However, components having the same configuration as in the first embodiment will be denoted by the same reference numerals and their descriptions will be omitted as appropriate.

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

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

[0045] The procedure for cell retrieval in cell culture system 1b will be explained with reference to the flowchart in Figure 12 and the schematic diagrams in Figures 13(a) to 15(b).

[0046] First, after the cell culture 13 is complete, the operator sends the detachment solution 12A from the detachment solution storage container 4 to the detachment solution buffer container 7 (S301; see Figure 13(a)). Next, the operator sends the used culture medium 11 from the culture vessel 6 to the culture medium buffer container 21 (S302; culture medium transfer step; see Figure 13(b)). In the second embodiment, the culture medium buffer container 21 corresponds to the storage unit of the present invention. Next, the operator sends the warmed detachment solution 12A from the detachment solution buffer container 7 to the culture vessel 6 (S303; detachment solution supply step; see Figure 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 drain container 32 before the cells 13 have completely detached from the inner surface 9 of the culture vessel 6 (S304. Detachment solution discharge step. See Figure 14(b)). At this time, since the cells 13 are attached to the inner surface 9 of the culture vessel 6, they remain in the culture vessel 6 without being discharged with the detachment solution 12A. Also, a portion of the detachment solution 12A remains in the culture vessel 6, for example, attached to the cells 13. After that, the operator waits for a predetermined time (S305. Waiting step). This allows the cells 13 to be completely detached from the inner surface 9 of the culture vessel 6 by the small amount of detachment solution 12A remaining in the culture vessel 6. In this method, damage to the cells 13 by the detachment solution 12A can be minimized compared to the case where 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 vessel 6 (S306. Mixing step). Thus, a stripping solution discharge step and a waiting step are provided between the stripping solution supply step and the mixing step.

[0048] In the mixing step, the used culture medium 11 is transferred to the culture vessel 6, allowing the cells 13 to be suspended in the used culture medium 11 within the culture vessel 6. This mixes the used culture medium 11 and the cells 13 to form a suspension 33 (see Figure 15(a)). Additionally, some of the detachment solution 12A remaining in the culture vessel 6 is mixed with the used culture medium 11, thus diluting and inactivating the detachment solution 12A. Finally, the operator transfers the suspension 33 from the culture vessel 6 to the recovery container 5 (S307; see Figure 15(b)). In this way, even if the cells 13 are susceptible to damage by the detachment solution 12A, for example, the cells 13 can be recovered while minimizing damage to the cells 13.

[0049] Next, a modified example of the second embodiment described above will be explained. However, components having the same configuration as the second embodiment will be denoted by the same reference numerals and their descriptions will be omitted as appropriate.

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

[0051] Next, a modified example common to the first and second embodiments described above will be explained. However, components having the same configuration as those in the first or second embodiment will be denoted by the same reference numerals and their descriptions 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 heated when the cells 13 are collected. However, this is not limited to this. For example, the detachment solution 12 may be preheated during the culture of the cells 13 (at a time before the collection of the cells 13 is started).

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

[0054] (3) The used culture medium 11 does not necessarily have to contain components that inactivate the detachment solution 12. That is, the used culture medium 11 may be used simply to dilute at least a portion of the detachment solution 12 that was used to detach the cells 13 from the inner surface 9 of the culture vessel 6. Even in such cases, damage to the cells 13 by the detachment solution 12 can be suppressed without using fresh culture medium. [Explanation of Symbols]

[0055] 5. Recovery container (storage section, first storage section) 6 Culture vessel 9. Inner self 11. Used culture media 11A Used culture medium (First used culture medium) 11B Used culture medium (second used culture medium) 12 Stripping solution 13 cells 21 Culture medium buffer container (second storage section)

Claims

[Claim 1] A cell recovery method for recovering cells that have been cultured in a culture vessel containing a liquid culture medium and have adhered to the inner surface of the culture vessel, After culturing the cells, a medium transfer step is performed to move the used culture medium in the culture vessel to a predetermined storage area. After the culture medium transfer step, a detachment solution supply step is performed to supply a detachment solution to the culture vessel in order to detach the cells from the inner surface of the culture vessel, A cell recovery method characterized by comprising a mixing step of mixing at least a portion of the detachment solution with the used culture medium after the detachment solution supply step.

Images