JP6003070B2 - Cell recovery method - Google Patents

Description

  The present invention relates to a method for recovering cells cultured using a culture vessel, and more particularly to a cell recovery method for improving cell recovery efficiency.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
In such a large-scale culture of cells, generally, the cells are cultured using a culture vessel. In cell culture using this culture container, it is necessary to collect cells from the culture container after culture.
When recovering cultured cells (especially suspension cells), conventionally, a method of recovering the entire cell suspension from the culture container and separating the cells by centrifugation has been performed. However, in this method, when cells are cultured in a large amount and there are a large amount of cell suspension, it is necessary to perform centrifugation several times, which is time consuming and troublesome.

  On the other hand, as a technique related to the cell recovery method, for example, a recovery method used in the cell culture method described in Patent Document 1 can be exemplified. In this recovery method, first, the spent medium supernatant is discharged from the culture vessel, and then the cell suspension containing the cultured cells is recovered, thereby making it possible to obtain a concentrated cell suspension. ing.

International Publication No. 2007/052716 Pamphlet

However, this method has a problem that a relatively large number of cells remain in the culture vessel and cannot be recovered, and the recovery efficiency cannot be said to be sufficient.
That is, according to this recovery method, a concentrated cell suspension can be recovered, but even if the cell suspension is recovered, cells that remain attached to the wall surface in the culture vessel remain. Exists. In addition, when the culture vessel is made of a flexible material, if the cell suspension becomes small, the inside of the culture vessel tends to be blocked, and a relatively large amount of the cell suspension remains in the culture vessel. There was also a problem that it remained inside.

  In order to solve such problems, after collecting the cell suspension, a new washing solution (physiological saline or phosphate buffer) is added to the culture vessel and stirred, and the cells remaining in the culture vessel are used as the washing solution. A method of suspending and collecting again is also conceivable. However, the method for recovering cells remaining in the culture vessel using the washing solution as described above requires a new washing solution and a washing solution storage container for storing the washing solution, resulting in an extra cost. .

  The present invention is considered in view of the above circumstances, in collecting the cells cultured using the culture vessel, to prevent the culture vessel from clogging, without using a new washing culture solution or washing solution, An object of the present invention is to provide a cell recovery method capable of improving the cell recovery efficiency.

  In order to achieve the above object, the cell recovery method of the present invention comprises at least a culture container for culturing cells, a cell recovery container for recovering cells after culture, and a waste liquid container for recovering a culture medium after culture. , A method for recovering cells cultured using a cell culture kit connected by a conduit, wherein a supernatant discharge step of transferring a cultured culture supernatant from a culture vessel to a waste vessel, the bottom area of the culture vessel And reducing the concentration of the concentrated cell suspension to the cell collection port side of the culture vessel, and the reduction and recovery step of transferring the collected cell suspension from the culture vessel to the cell recovery vessel There is a way.

  According to the present invention, when recovering cells cultured using a culture container, it is possible to prevent clogging of the culture container and improve cell recovery efficiency without using a new culture medium or washing solution for washing. It becomes possible.

It is a figure which shows the kit for cell cultures used in the cell collection | recovery method of embodiment of this invention. It is a figure which shows the reduction | restoration collection procedure in the cell collection | recovery method of 1st embodiment of this invention. It is a figure which shows the washing | cleaning collection procedure in the cell collection method of 2nd embodiment of this invention. It is a figure which shows the stationary procedure in the cell collection | recovery method of 3rd embodiment of this invention. It is a figure which shows the experimental result by the cell collection | recovery method of embodiment of this invention.

Hereinafter, a preferred embodiment of the cell recovery method of the present invention will be described with reference to the drawings. First, a cell culture kit that can be suitably used in the cell recovery method of the present embodiment will be described with reference to FIG. This cell culture kit includes a culture container 10, a medium storage container 20, a cell injection container 30, a cell collection container 40, a sampling container 50, and a tube 60. The medium storage container 20 is also used as a waste liquid container.
The cell collection method of this embodiment is a method used when cells are collected from the culture container 10 in the cell culture kit to the cell collection container 40 after cell culture is performed using such a cell culture kit. It is.

  Note that the cell culture kit used in the cell collection method of the present embodiment is not limited to such a configuration. For example, a kit excluding the sampling container 50 or a waste liquid container separately from the medium storage container It is also possible to use a suitably modified one, such as one provided, a plurality provided with a plurality of medium storage containers 20.

The culture container 10 is a container for putting a cell to be cultured and a culture solution to culture the cells in large quantities. The culture vessel 10 is formed into a bag shape (back shape) using a flexible material.
The culture vessel 10 is provided with a port for injecting and collecting a culture solution and cells. The number of ports is not particularly limited, but those having 1 to 3 ports are preferably used. FIG. 1 shows a one-port cell culture kit, through which a culture solution or a cell suspension can be transferred while maintaining a closed system with other containers. Yes.
Note that suspension cells can be suitably used as the cells to be cultured. As the culture solution, various general media can be used.

The medium storage container 20 is a container for storing a culture solution (medium) for culturing cells in order to transfer it to the culture container 10. The culture medium storage container 20 can be used as a waste liquid container after culturing. That is, prior to recovering cells from the culture container 10 to the cell recovery container 40, the supernatant of the culture solution is transferred to the medium storage container 20 as a waste liquid container, and then the concentrated cell suspension is cultured. It becomes possible to send from the container 10 to the cell collection container 40.
In the cell culture kit of this embodiment, two or more medium storage containers 20 can be provided. Thereby, the same culture solution can be stored twice or more, and the culture solution can be supplied to the culture vessel 10 in various variations by storing different culture solutions for each.

  The cell injection container 30 is a container containing cells and a culture solution that are necessary at the start of culture. By transferring the cells and the culture solution in the cell injection container 30 to the culture container 10, cell culture is started. The cell injection container 30 is provided with at least one port, through which cells and a culture solution are transferred.

  The cell collection container 40 is a container for collecting a cell suspension containing a part of the culture solution and the cultured cells from the culture container 10 after completion of the culture. The cell collection container 40 is provided with at least one port, and a cell suspension containing a part of the culture solution and the cultured cells is transferred from the culture container 10 to the cell collection container 40 through this port. Is done.

The sampling container 50 is a container for performing sampling by transferring a part of the cell suspension in the culture container 10 during or after the culturing of the cells. The cell suspension as a sample to be transferred to the sampling container 50 includes cultured cells and a culture solution. The sampling container 50 includes at least one port to which a conduit (tube) connected to the culture container 10 is connected, and a cell suspension is sent from the culture container 10 to the sampling container 50.
The tube 60 is a conduit connecting each container in the cell culture kit.

  Next, the structure of the cell culture apparatus in which the cell collection method of this embodiment is performed will be described with reference to FIG. In the figure, a cell culture apparatus is schematically shown. In this cell culture apparatus, the culture vessel 10 in the above-described cell culture kit is arranged, and cells are cultured. As shown in FIG. 2, the cell culture apparatus includes a culture container 10, a loading table 11, a fixing member 12, a stirring member 13, a partition member 14, and a tube 60.

The loading table 11 is a flat table on which the culture vessel 10 is placed on the upper surface, and further, the stirring member 13 and the partition member 14 are disposed on the upper surface of the culture vessel 10.
The fixing member 12 is a member for fixing the culture vessel 10 to the loading table 11. In FIG. 2, the fixing member 12 is shown as a clamp. However, the fixing member 12 may be configured as a stopper member such as a pin provided with holes in the four corners of the culture vessel 10 for fixing through the holes.

  The stirring member 13 stirs the cell suspension in the culture container 10 by applying an external force to the culture container 10. Thereby, the cells in the culture vessel 10 can be dispersed in the cell suspension and can be easily transferred to the cell collection vessel 40. In addition, the cells attached to the inner surface of the culture container 10 can be dispersed in the cell suspension and can be easily transferred to the cell collection container 40. The stirring of the cell suspension in the culture vessel 10 by the stirring member 13 can be performed at an appropriate timing such as before the transfer of the cell suspension.

As the stirring member 13, for example, a roller can be used. The cell suspension can be stirred by pressing the upper surface of the culture vessel 10 to a predetermined depth by the stirring member 13 and moving the stirring member 13 in parallel with the loading table 11.
Although not shown, the stirring member 13 can be placed on the culture vessel 10 by a support stand or the like. And this support stand can be moved to the up-down direction and a horizontal direction with a drive device, and the stirring by the stirring member 13 can be performed.

The partition member 14 presses the culture container 10 from the end of the culture container 10 until the upper surface and the lower surface of the culture container 10 are in close contact with each other, and moves in the horizontal direction with respect to the loading table 11, thereby It is a member that can increase or decrease the area.
Since the culture container 10 is made of a flexible material, when the bottom area of the culture container 10 is reduced by the partition member 14, the cell suspension is pushed upward in the culture container 10, and the thickness of the culture container 10 is slightly increased. Increased state.

  In the shrinking step described later, it is possible to prevent the culture vessel 10 from being blocked by reducing the bottom area of the culture vessel 10 and increasing the thickness of the cell suspension in the culture vessel 10. As a result, the cell suspension recovered from the culture container 10 to the cell recovery container 40 increases, and as a result, the cell recovery efficiency can be improved.

Moreover, in the stationary process mentioned later, after leaving still in the state which reduced the bottom area of the culture container 10 and made the liquid thickness of the cell suspension liquid in the culture container 10 increase, The culture supernatant of the suspension can be transferred to a waste container.
When the culture supernatant is transferred to the waste liquid container in this way, the culture container after discharging the supernatant is compared with the case where the culture supernatant is transferred to the waste liquid container without reducing the bottom area of the culture container 10. The cell concentration of the cell suspension remaining at 10 can be increased.
This is because the amount of the supernatant with respect to the bottom area in the culture vessel 10 can be increased, and the discharge amount of the culture supernatant can be increased. As a result, the cell concentration of the cell suspension collected in the cell collection container 40 increases, and the amount of cell suspension to be collected can be reduced.

  The partition member 14 can be configured by a roller or the like, similarly to the stirring member 13. FIG. 2 shows a partition member 14 that sandwiches the culture vessel 10 from above and below by two rollers. However, the present invention is not limited to this, as long as the bottom area of the culture vessel 10 can be reduced. good. The partition member 14 can be driven in the same manner as the stirring member 13.

  In FIG. 2, the tube 60 connects the culture container 10, a cell collection container, a waste liquid container, and the like. In the example of the figure, the tube 60 is divided into A, B, and C, A is connected to the cell collection container, and C is connected to the waste liquid container. Moreover, B can be used as what connects with a sampling container, for example.

[First embodiment]
Next, a first embodiment of the cell recovery method of the present invention will be described with reference to FIG. In the present specification, the cell recovery method including the following reduction process and reduction recovery process may be referred to as reduction recovery.

(1) Supernatant discharge step First, after the culture vessel 10 is stationary and the cells are allowed to settle downward, the supernatant of the cell suspension is transferred from the culture vessel 10 to the waste liquid vessel.
As a result, the cell suspension in the culture vessel 10 can be concentrated, and the cell concentration can be increased. The culture supernatant can be discharged by operating a tube pump in the tube 60. In addition, the transfer of the cell suspension and the return of the culture supernatant described below can also be performed by operating the tube pump in the tube 60.

(2) Reduction process Next, the partition member 14 is used to reduce the bottom area of the culture vessel 10. Specifically, the bottom area of the culture vessel 10 can be reduced by moving the partition member 14 on the culture vessel 10 while bringing the front and back surfaces into close contact from the end of the culture vessel 10 with the partition member 14. . Thereby, the cell suspension can be collected near the cell collection port or the cell collection port side in the culture container 10.

(3) Reduced recovery step Further, the cell suspension is transferred from the culture vessel 10 to the cell recovery vessel 40 with the bottom area of the culture vessel 10 reduced. In this manner, by transferring the cell suspension in a state where the bottom area of the culture vessel 10 is reduced, blockage of the culture vessel 10 due to a decrease in the liquid amount (liquid thickness) of the cell suspension is suppressed. be able to. For this reason, the cell suspension remaining in the culture vessel 10 can be reduced, and the cell recovery efficiency can be improved.

Furthermore, after the supernatant discharge step, it is also preferable to perform a cell recovery step of transferring the concentrated cell suspension from the culture vessel 10 to the cell recovery vessel 40, and then perform a reduction step and a reduction recovery step. Thereby, it is also possible to collect the cells while suppressing the blockage of the culture vessel 10.
As described above, according to the cell recovery method of the present embodiment, it is possible to reduce the cell suspension remaining in the culture vessel 10 by preventing the culture vessel from being blocked. It is possible to improve the cell recovery efficiency without using it.

[Second Embodiment]
Next, a second embodiment of the cell recovery method of the present invention will be described with reference to FIG. The cell collection method of this embodiment is different from the first embodiment in that washing and collection are performed instead of reduced collection as follows. About another point, it is the same as that of 1st embodiment. In the present specification, the cell recovery method including the following supernatant return step and re-recovery step may be referred to as washing recovery.

(1) Supernatant discharge step First, as in the first embodiment, after culturing the culture vessel 10 and allowing the cells to settle downward, the supernatant of the cell suspension is transferred from the culture vessel 10 to the waste liquid container. .

(2) Cell recovery step Next, the cell suspension is transferred from the culture vessel 10 to the cell recovery vessel. In the state where the transfer is completed, a part of the cell suspension containing the cultured cells remains in the culture vessel 10.

(3) Supernatant return step Next, a part of the culture supernatant is returned from the waste liquid container to the culture container 10, and the culture container 10 is stirred by the stirring member 13. Thereby, the cells remaining in the culture container 10 such as the cells attached to the wall surface in the culture container 10 can be suspended in the cell suspension. In addition, what is necessary is just to perform stirring as needed.

(4) Recollection step Next, the cell suspension in which the remaining cells are suspended is transferred again from the culture vessel 10 to the cell collection vessel. Thereby, according to the cell collection method of the present embodiment, cells remaining in the culture vessel 10 in the cell collection step can be collected without using a new culture solution or washing solution, and the cell collection efficiency is improved. It is possible.

  Moreover, in the said cell collection method, it is also preferable to repeat a supernatant return process and a re-recovery process twice or more. If the supernatant return step and the re-recovery step are repeated twice or more in this way, the cells remaining in the culture vessel 10 can be further recovered each time, and thus the recovery efficiency can be further improved.

  Furthermore, in the cell recovery method, it is also preferable to perform the supernatant return step and the re-recovery step at the same time. If the supernatant return step and the re-recovery step are performed simultaneously in this way, the cells can be recovered again while flowing the culture supernatant, and the cells remaining in the culture vessel 10 can be easily transferred to the cell recovery vessel 40. It becomes possible.

[Third embodiment]
Next, a third embodiment of the cell recovery method of the present invention will be described with reference to FIG.
The cell collection method of this embodiment is different from the first embodiment and the second embodiment in that it has a stationary step before performing the supernatant discharging step as follows. About another point, it can be set as the same thing as 1st embodiment or 2nd embodiment, respectively. Hereinafter, the case of the second embodiment will be described.

(1) Standing step Before performing the supernatant discharging step in the cell recovery method of the second embodiment, the partition member 14 is used to reduce the bottom area of the culture vessel 10. Specifically, the bottom area of the culture vessel 10 can be reduced by moving the partition member 14 on the culture vessel 10 while bringing the front and back surfaces into close contact from the end of the culture vessel 10 with the partition member 14. . Then, the culture vessel 10 is allowed to stand to sediment the cells downward.
Thereafter, the supernatant discharge step, cell recovery step, supernatant return step, and re-recovery step described in the second embodiment are performed. Moreover, it is also preferable to repeat a supernatant return process and a re-recovery process.

  Thus, by carrying out washing | cleaning collection | recovery, after reducing the bottom area of the culture container 10, the quantity of the supernatant with respect to a bottom area can be increased, and the discharge | emission amount of a culture supernatant can be increased. For this reason, the cell concentration of the cell suspension recovered in the cell recovery step can be increased, and the cell recovery efficiency can be improved.

[Fourth embodiment]
Next, a fourth embodiment of the cell recovery method of the present invention will be described. The cell collection method of this embodiment is different from the first embodiment in that the first embodiment and the second embodiment are combined as described below, and the washing and collection are performed after the reduced collection. About another point, it is the same as that of 1st embodiment.

That is, in the cell recovery method of the present embodiment, the supernatant discharge step, the reduction step, and the reduction recovery step of the first embodiment are first performed, and then the supernatant return step and the re-recovery step of the second embodiment are performed. Do.
As a result, blockage of the culture vessel is suppressed, and not only the cell suspension remaining in the culture vessel 10 can be reduced to improve cell recovery efficiency, but also the cells adhered to the wall surface by washing the culture vessel 10 are removed. Cell recovery efficiency can be improved by suspending in a suspension.
Therefore, according to the cell collection method of the present embodiment, the cell collection efficiency can be further improved without using a new washing culture solution or washing solution.

  In addition, combining the third embodiment with the cell collection method of the present embodiment, performing a stationary collection step, performing reduced collection, and then washing and collecting can further improve cell collection efficiency. It becomes possible.

  Compare the case of recovering cells from the culture vessel by performing reduced recovery and / or washing and recovery by the cell recovery method of the above embodiment, and the case of recovering cells from the culture vessel without performing reduced recovery and washing and recovery, These effects were verified. The result is shown in FIG. This experiment was performed by using a culture container of 22 × 66 cm and enclosing 2000 mL of the culture solution and 8.00E + 09 cells.

(Comparative Example 1)
Only the supernatant discharge step and the cell recovery step in the cell recovery method of the first embodiment were performed, and the cells were recovered from the culture vessel without performing reduced recovery and washing recovery.
As a result, the culture supernatant discharged to the waste liquid container was 1600 mL, which contained 2.00E + 08 cells, and the percentage of the number of cells was 2.5%. The cell suspension recovered in the cell recovery container was 350 mL, which contained 6.83E + 09 cells, and the percentage of the number of cells was 85.31%. The cell suspension remaining in the culture vessel was 50 mL, which contained 9.75E + 08 cells, and the percentage of the number of cells was 12.19%.

(Reference Example 1)
By performing each of the supernatant discharge step, cell recovery step, supernatant return step, and re-recovery step in the cell recovery method of the second embodiment once, washing and recovery were performed once to recover the cells from the culture vessel. .
The culture supernatant discharged to the waste liquid container was 1550 mL, which contained 1.94E + 08 cells, and the percentage of the number of cells was 2.42%. The cell suspension recovered in the cell recovery container was 400 mL, which contained 7.32E + 09 cells, and the percentage of the number of cells was 91.45%. The cell suspension remaining in the culture vessel was 50 mL, which contained 4.91E + 08 cells, and the percentage of the number of cells was 6.13%.

(Reference Example 2)
By performing the supernatant discharge step, the cell recovery step, the supernatant return step, and the recollection step once each in the cell recovery method of the second embodiment, and then repeating the supernatant return step and the recollection step once more The cells were collected from the culture vessel by washing and collecting twice.
The culture supernatant discharged into the waste liquid container was 1500 mL, which contained 1.88E + 08 cells, and the percentage of the number of cells was 2.34%. The cell suspension recovered in the cell recovery container was 450 mL, which contained 7.56E + 09 cells, and the percentage of the number of cells was 94.55%. In addition, the cell suspension remaining in the culture vessel was 50 mL, which contained 2.48E + 08 cells, and the percentage of the number of cells was 3.11%.

Example 1
The supernatant discharge step, the reduction step, and the reduction recovery step in the cell recovery method of the first embodiment were each performed once to perform reduction recovery and recover the cells from the culture vessel.
The culture supernatant discharged to the waste liquid container was 1600 mL, which contained 2.00E + 08 cells, and the percentage of the number of cells was 2.5%. The cell suspension recovered in the cell recovery container was 390 mL, which contained 7.61E + 09 cells, and the percentage of the number of cells was 95.06%. The cell suspension remaining in the culture vessel was 10 mL, which contained 1.95E + 08 cells, and the percentage of the number of cells was 2.44%.

(Example 2)
By combining the cell recovery method of the first and second embodiments, performing the supernatant discharge step, the reduction step, and the reduction recovery step once, respectively, then performing the supernatant return step and the re-recovery step once, Washing and collection were performed once to collect cells from the culture vessel.
The culture supernatant discharged to the waste liquid container was 1590 mL, which contained 1.99E + 08 cells, and the percentage of the number of cells was 2.48%. The cell suspension recovered in the cell recovery container was 400 mL, which contained 7.70E + 09 cells, and the percentage of the number of cells was 96.29%. In addition, the cell suspension remaining in the culture vessel was 10 mL, which contained 9.81E + 07 cells, and the percentage of the number of cells was 1.23%.

In Comparative Example 1, 50 ml of the cell suspension remained in the culture container. This is because the amount of liquid was reduced, the culture vessel was blocked, and the cell suspension could no longer be transferred to the cell collection vessel.
In Reference Example 1, after 50 ml of the cell suspension remained in the culture container, 50 ml of the culture supernatant was returned from the waste container to the culture container, the culture container was stirred and washed, and another 50 ml was collected in the cell collection container. As a result, 50 ml of the cell suspension remained in the culture container. In Reference Example 2, the supernatant return and re-recovery of Reference Example 1 was repeated once more, and washing and recovery were performed twice.

In Example 1, by collecting the culture container 10 while reducing the bottom area, the culture container was prevented from being blocked, and the remaining amount of the cell suspension in the culture container was reduced to 10 ml.
In Example 2, after collecting while reducing the bottom area of the culture vessel 10, 10 ml of the culture supernatant was returned to the culture vessel from the waste solution vessel, the culture vessel was stirred and washed, and another 10 ml was collected in the cell collection vessel. . As a result, 10 ml of the cell suspension remained in the culture container.

The percentage of the number of cells collected in the cell collection container was 85.31%, 91.45%, 94.55% in the order of Comparative Example 1, Reference Example 1, Reference Example 2, Example 1, and Example 2. , 95.06%, 96.29%.
In addition, the percentage of the number of cells discharged to the waste liquid container is 2.5%, 2.42%, 2.34 in the order of Comparative Example 1, Reference Example 1, Reference Example 2, Example 1, and Example 2, respectively. %, 2.5%, and 2.48%.
Furthermore, the percentage of the number of cells remaining in the culture vessel was 12.19%, 6.13%, 3.11% in the order of Comparative Example 1, Reference Example 1, Reference Example 2, Example 1, and Example 2. , 2.44% and 1.23%.

As described above, the cell recovery efficiency of Reference Example 1 in which washing and recovery was performed only once is increased by 6% or more compared to Comparative Example 1. In addition, the cell recovery efficiency of Reference Example 2 in which the washing and recovery were performed twice increased by 9% or more compared to Comparative Example 1.
Furthermore, the cell recovery efficiency of Example 1 in which the reduced recovery was performed is increased by nearly 10% compared to Comparative Example 1. Further, the cell recovery efficiency of Example 2 in which the reduced recovery and the cleaning recovery were performed once increased by nearly 11% as compared with Comparative Example 1.

  As described above, the cell recovery efficiency of the above embodiment was able to be improved without using a new washing culture solution or washing solution. In particular, the cell collection efficiency is remarkably increased by performing reduced collection in the cell collection method of the above embodiment. Therefore, it can be seen that by performing the reduced collection, the cell suspension remaining in the culture container can be reduced by blocking the culture container, and the cell recovery efficiency can be greatly improved.

It goes without saying that the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
For example, in FIGS. 2 and 3, the supernatant returning process is performed using a roller-type stirring member, but the culture vessel may be stirred by other methods such as shaking the loading table, and washed three times or more. It is possible to make appropriate changes such as collecting.

  The present invention can be suitably used in the fields of biopharmaceuticals, regenerative medicine, immunotherapy and the like that require culturing a large amount of cells.

DESCRIPTION OF SYMBOLS 10 Culture container 11 Loading stand 12 Fixed member 13 Stirring member 14 Partition member 20 Medium storage container (waste liquid container)
30 Cell injection container 40 Cell collection container 50 Sampling container 60 Tube

Claims (9)

Culture using a cell culture kit in which at least a culture container for culturing cells, a cell collection container for collecting cultured cells, and a waste liquid container for collecting the cultured medium are connected by a conduit. A method for recovering the collected cells
Transferring the cultured culture supernatant from the culture container to the waste liquid container, and concentrating the cell suspension in the culture container ;
Reduction of the bottom area of the culture container to increase the thickness of the concentrated cell suspension in the culture container , and the concentrated cell suspension is gathered to the cell collection port side of the culture container And a reduced recovery step of transferring the collected cell suspension from the culture vessel to the cell recovery vessel.   The cell according to claim 1, wherein after the supernatant discharging step, a cell recovery step of transferring the concentrated cell suspension from the culture vessel to the cell recovery vessel is performed, and then the reduction step is performed. Collection method. After the reduced recovery process,
A part of the culture supernatant discharged into the waste container is returned to the culture container, a supernatant returning step for suspending cells remaining in the culture container in a cell suspension, and a cell suspension in which the remaining cells are suspended. re recovery step of transferring again said cell collection container from the culture vessel Nigoeki, cell recovery method according to claim 1 or 2, wherein it contains. 4. The cell recovery method according to claim 3, wherein the supernatant return step and the re-recovery step are repeated twice or more. The cell collection method according to any one of claims 1 to 4 , wherein a medium storage container for storing a medium or the like is used as the waste liquid container. Prior to the supernatant discharging step, the culture vessel is allowed to stand until the cells settle in a state where the bottom area of the culture vessel is reduced and the thickness of the cell suspension in the culture vessel is increased. The cell collection method according to any one of claims 1 to 5 , wherein a stationary step is performed. In the stationary step, the partition member is moved on the culture vessel while pressing the culture vessel so that the front surface and the back surface are in close contact with each other using a predetermined partition member, and the bottom of the culture vessel The cell collection method according to claim 6, wherein the area is reduced. In the reduction step, the partition member is moved on the culture vessel while pressing the culture vessel from the outside of the culture space so that the front surface and the back surface are in close contact with each other using a predetermined partition member, cell recovery method according to any one of claims 1 to 7, characterized in that to reduce the bottom area. The cell recovery method according to claim 7 or 8 , wherein the predetermined partition member is a roller.

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