WO2013114859A1 - 細胞回収方法 - Google Patents
細胞回収方法 Download PDFInfo
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- WO2013114859A1 WO2013114859A1 PCT/JP2013/000462 JP2013000462W WO2013114859A1 WO 2013114859 A1 WO2013114859 A1 WO 2013114859A1 JP 2013000462 W JP2013000462 W JP 2013000462W WO 2013114859 A1 WO2013114859 A1 WO 2013114859A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/14—Bags
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/18—Rollers
Definitions
- 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.
- a recovery method used in the cell culture method described in Patent Document 1 can be mentioned.
- 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.
- 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.
- washing solution physiological saline or phosphate buffer
- a method of suspending and collecting again is also conceivable.
- 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. .
- An object of the present invention is to provide a cell recovery method capable of improving the cell recovery efficiency.
- 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 cell recovery step for transferring the suspension from the culture container to the cell recovery container, returning a part of the culture supernatant discharged to the waste liquid container to the culture container, and suspending the cells remaining in the culture container in the cell suspension
- the method includes a refining step and a re-recovery step of transferring the cell suspension in which the remaining cells are suspended from the culture vessel to the cell recovery vessel again.
- a method for recovering cells cultured using a cell culture kit comprising: a supernatant discharge step of transferring a cultured culture supernatant from a culture container to a waste liquid container, reducing the bottom area of the culture container; The method includes a reduction step of collecting the concentrated cell suspension to the cell collection port side of the culture vessel, and a reduction collection step of transferring the collected cell suspension from the culture vessel to the cell collection vessel.
- 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.
- 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.
- the cell culture kit used in the cell collection method of the present embodiment is not limited to such a configuration.
- 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 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.
- 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 required 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.
- the configuration of the cell culture apparatus in which the cell recovery method of this embodiment is performed will be described with reference to FIG.
- a cell culture apparatus is schematically shown.
- the culture vessel 10 in the above-described cell culture kit is arranged, and cells are cultured.
- 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.
- 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.
- 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.
- 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.
- 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.
- the shrinking step described later it is possible to prevent the culture vessel 10 from being clogged 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.
- the culture supernatant of the suspension can be transferred to a waste container.
- 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 constituted 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.
- 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.
- the tube 60 connects the culture container 10, the cell collection container, the waste liquid container, and the like.
- 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.
- B can be used as what connects with a sampling container, for example.
- 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, 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.
- 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.
- the cell suspension in which the remaining cells are suspended is transferred again from the culture vessel 10 to the cell collection vessel.
- the cell collection method it is also preferable to repeat the supernatant return step and the recollection step two or more times. 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.
- the culture vessel 10 having two or more ports can be suitably used. One port is connected to the cell collection vessel 40 through the tube 60, and the other port is connected to the waste liquid vessel. Is done.
- the cell collection method of the present embodiment is different from the first embodiment in that it has a stationary step before performing the supernatant discharging step as follows. About another point, it is the same as that of 1st 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, the cell recovery step, the supernatant return step, and the recollection step described in the first embodiment are performed. Moreover, it is also preferable to repeat a supernatant return process and a re-recovery process.
- the amount of the supernatant with respect to the bottom area can be increased, and the discharge amount of the 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.
- 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.
- 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.
- 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.
- 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.
- the cell suspension remaining in the culture vessel 10 can be reduced, and the cell recovery efficiency can be improved.
- the reduction process and the reduction recovery process simultaneously. That is, it is also preferable to transfer the cell suspension from the culture container 10 to the cell collection container 40 while reducing the bottom area of the culture container 10 using the partition member 14.
- the blockage of the culture vessel 10 can also be suppressed by simultaneously performing the reduction process and the reduction recovery process.
- 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.
- the supernatant discharge step, the reduction step, and the reduction recovery step of the third embodiment are first performed, and then the supernatant return step and the re-recovery step of the first embodiment are performed.
- the 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.
- the second embodiment is further combined with the cell collection method of the present embodiment, a reduced collection is performed after the stationary step, and then washing and collection are performed, and then the cell collection efficiency is further improved. Is possible.
- Example 1 By performing the supernatant discharge step, cell recovery step, supernatant return step, and re-recovery step in the cell recovery method of the first 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%.
- 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 first 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%.
- 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 3 By performing each of the supernatant discharge step, the reduction step, and the reduction collection step in the cell recovery method of the third embodiment once, reduction recovery was performed, and the cells were recovered 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 4 By combining the cell recovery methods of the first and third embodiments, and performing the supernatant discharge step, the reduction step, and the reduction recovery step once, respectively, the supernatant return step and the re-recovery step are performed once, respectively. 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%.
- 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%.
- Example 1 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.
- 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 50 ml of the cell suspension was further added to the cell collection container. Was recovered. As a result, 50 ml of the cell suspension remained in the culture container. In Example 2, the supernatant return and re-recovery of Example 1 were further repeated once, and washing and recovery were performed twice.
- Example 3 the bottom area of the culture vessel 10 was reduced and the cell suspension was recovered, so that the blockage of the culture vessel was suppressed, and the remaining amount of the cell suspension in the culture vessel was reduced to 10 ml.
- Example 4 after recovering the cell suspension by reducing the bottom area of the culture vessel 10, 10 ml of the culture supernatant is returned to the culture vessel from the waste vessel, and the culture vessel is stirred and washed, Furthermore, 10 ml of cell suspension was collected. 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, Example 1, Example 2, Example 3 and Example 4. , 95.06%, 96.29%.
- the percentage of the number of cells discharged to the waste liquid container was 2.5%, 2.42%, and 2.34 in the order of Comparative Example 1, Example 1, Example 2, Example 3, and Example 4, respectively. %, 2.5%, and 2.48%.
- 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, Example 1, Example 2, Example 3, Example 4. , 2.44% and 1.23%.
- Example 1 the cell recovery efficiency of Example 1 in which washing and recovery was performed only once is increased by 6% or more compared to Comparative Example 1.
- the cell recovery efficiency of Example 2 in which the washing and recovery were performed twice increased by 9% or more compared to Comparative Example 1.
- the cell recovery efficiency of Example 3 in which the reduced recovery was performed is increased by nearly 10% compared to Comparative Example 1.
- the cell recovery efficiency of Example 4 in which the reduced recovery and the cleaning recovery were performed once increased by nearly 11% as compared with Comparative Example 1.
- the cell collection efficiency is improved without using a new washing culture solution or washing solution by carrying out washing and collecting in the cell collection method of the present embodiment.
- cell collection efficiency is significantly increased by performing reduced collection in the cell collection method of the present embodiment. Therefore, according to the reduced recovery in the cell recovery method of the present embodiment, it can be understood that 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. .
- the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
- the supernatant returning step 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 washing and recovery may be performed three times or more. It can be changed as appropriate.
- 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.
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Abstract
Description
このような細胞の大量培養では、一般に培養容器を用いて細胞を培養することが行われている。この培養容器を用いた細胞培養においては、培養後に培養容器から細胞を回収する必要がある。
培養細胞(特に浮遊系細胞)を回収する場合、従来は培養容器から細胞懸濁液を全量回収し、遠心分離により細胞を分離する方法が行われていた。しかし、この方法では、細胞を大量に培養して細胞懸濁液が多量にある場合、何回かに分けて遠心分離する必要があり、時間と手間がかかるという問題点があった。
すなわち、この回収方法によれば、濃縮された細胞懸濁液を回収することができるが、細胞懸濁液を回収しても、培養容器内の壁面などに付着した状態で残ってしまう細胞が存在する。また、培養容器が可撓性材料からなる場合には、細胞懸濁液が少量になると培養容器内が閉塞し易く、比較的多量の細胞懸濁液が培養容器に残る結果、細胞が培養容器内に残存してしまうという問題もあった。
本実施形態の細胞回収方法は、このような細胞培養用キットを使用して細胞培養を行った後に、細胞培養用キットにおける培養容器10から細胞回収容器40に細胞を回収する場合に用いられる方法である。
この培養容器10は、培養液や細胞の注入、回収などを行うためのポートを備えている。ポートの数は特に限定されないが、1~3ポートのものが好適に用いられる。図1には1ポートの細胞培養用キットが示されており、このポートを通じてその他の容器と閉鎖系を維持しつつ、培養液や細胞懸濁液等の移送を行うことができるようになっている。
なお、培養対象の細胞としては、浮遊系細胞を好適に用いることができる。培養液としては、一般的な各種培地を用いることができる。
また、本実施形態の細胞培養用キットにおいて、培地貯蔵容器20を2以上備えることもできる。これにより、同一の培養液を2倍量以上貯蔵できるほか、それぞれに異なる培養液を貯蔵することで、培養容器10に様々なバリエーションで培養液を供給することもできる。
チューブ60は、細胞培養用キットにおける各容器を連結する導管である。
固定部材12は、培養容器10を積載台11に固定するための部材である。図2において、固定部材12は、クランプとして示されているが、培養容器10の四隅に孔を備え、これに通して固定するためのピンなどの止め部材として構成することもできる。
なお、図示していないが、攪拌部材13は、支持台などによって培養容器10上に配置させることができる。そして、この支持台を、駆動装置によって上下方向と水平方向に移動させ、攪拌部材13による攪拌を行うことが可能である。
培養容器10が可撓性素材からなるため、仕切部材14によって培養容器10の底面積を縮小させると、培養容器10内では細胞懸濁液が上方に押し上げられて、培養容器10の厚みがやや増加した状態となる。
このようにして培養上清の廃液容器への移送を行うと、培養容器10の底面積を縮小させることなく培養上清を廃液容器へ移送する場合に比較して、上清排出後の培養容器10に残る細胞懸濁液の細胞濃度を高めることができる。
これは、培養容器10における底面積に対する上清の量を増やすことができ、培養上清の排出量を増やすことができるためである。その結果、細胞回収容器40へ回収される細胞懸濁液の細胞濃度が高まり、回収する細胞懸濁液量を少量にすることが可能となる。
次に、本発明の細胞回収方法の第一実施形態について、図2を参照して説明する。本明細書において、以下の上清戻し工程、及び再回収工程を含む細胞回収方法を、洗浄回収と称する場合がある。
まず、培養容器10を静止させて細胞を下方に沈降させた後に、細胞懸濁液の上清を培養容器10から廃液容器へ移送させる。
これによって、培養容器10における細胞懸濁液を濃縮することができ、細胞の濃度を高めることが可能となる。この培養上清の排出は、チューブ60におけるチューブポンプを作動させて行うことができる。なお、以下に説明する細胞懸濁液の移送や、培養上清の戻しについても、チューブ60におけるチューブポンプを作動させることにより行うことができる。
次に、培養容器10から細胞懸濁液を細胞回収容器へ移送する。この移送を終了した状態において、培養容器10内には、培養細胞が含まれる細胞懸濁液が一部残存している。
次に、廃液容器から培養容器10へ培養上清を一部戻して、培養容器10を攪拌部材13により攪拌する。これにより、培養容器10内の壁面に付着した細胞など、培養容器10内に残存した細胞を細胞懸濁液内に懸濁させることができる。なお、攪拌は必要に応じて行えばよい。
次に、残存細胞が懸濁した細胞懸濁液を培養容器10から細胞回収容器へ再度移送する。これにより、本実施形態の細胞回収方法によれば、新たな培養液や洗浄液を用いることなく、細胞回収工程において培養容器10に残存した細胞を回収することができ、細胞の回収効率を向上させることが可能となっている。
次に、本発明の細胞回収方法の第二実施形態について、図3を参照して説明する。
本実施形態の細胞回収方法は、以下のように、上清排出工程を行う前に静置工程を有している点で第一実施形態と異なる。その他の点については、第一実施形態と同様である。
第一実施形態の細胞回収方法における上清排出工程を行う前に、仕切部材14を用いて、培養容器10の底面積を縮小させる。具体的には、仕切部材14により培養容器10の端部から表面と裏面を密着させながら、仕切部材14を培養容器10上で移動させることで、培養容器10の底面積を縮小させることができる。そして、培養容器10を静置させて細胞を下方に沈降させる。
その後、第一実施形態で説明した上清排出工程、細胞回収工程、上清戻し工程、及び再回収工程を行う。また、上清戻し工程、及び再回収工程を繰り返し行うことも好ましい。
次に、本発明の細胞回収方法の第三実施形態について、図4を参照して説明する。本明細書において、以下の縮小工程、及び縮小回収工程を含む細胞回収方法を、縮小回収と称する場合がある。
まず、第一実施形態と同様に、培養容器10を静止させて細胞を下方に沈降させた後に、細胞懸濁液の上清を培養容器10から廃液容器へ移送させる。
次に、仕切部材14を用いて、培養容器10の底面積を縮小させる。具体的には、仕切部材14により培養容器10の端部から表面と裏面を密着させながら、仕切部材14を培養容器10上で移動させることで、培養容器10の底面積を縮小させることができる。これによって、細胞懸濁液を培養容器10における細胞回収ポート付近又は細胞回収ポート側へ寄せ集めることができる。
さらに、培養容器10の底面積を縮小させた状態で、培養容器10から細胞懸濁液を細胞回収容器40に移送する。このように、培養容器10の底面積を縮小させた状態で、細胞懸濁液の移送を行うことで、細胞懸濁液の液量(液厚み)の低下による培養容器10の閉塞を抑制することができる。このため、培養容器10内に残存する細胞懸濁液を低減でき、細胞の回収効率を向上させることが可能となっている。
さらに、上清排出工程の後に、濃縮された細胞懸濁液を培養容器10から細胞回収容器40に移送する細胞回収工程を行い、次いで縮小工程、及び縮小回収工程を行うことも好ましい。これにより、培養容器10の閉塞を抑制して細胞を回収することも可能である。
このように、本実施形態の細胞回収方法によれば、培養容器の閉塞を抑止して培養容器10に残存する細胞懸濁液を減少させることができ、新たな洗浄用の培養液や洗浄液を用いることなく、細胞の回収効率を向上させることが可能となる。
次に、本発明の細胞回収方法の第四実施形態について説明する。本実施形態の細胞回収方法は、以下のように、第三実施形態に第一実施形態を組み合わせ、縮小回収を行った後に、洗浄回収を行う点で第三実施形態と異なる。その他の点については、第三実施形態と同様である。
これにより、培養容器の閉塞が抑止され、培養容器10に残存する細胞懸濁液を減少させて細胞回収効率を向上できるのみならず、培養容器10内を洗浄して壁面に付着した細胞を細胞懸濁液に懸濁させることで、細胞回収効率を向上させることができる。
よって、本実施形態の細胞回収方法によれば、新たな洗浄用の培養液や洗浄液を用いることなく、一層細胞回収効率を向上させることが可能となる。
第一実施形態の細胞回収方法における上清排出工程と細胞回収工程のみを行って、縮小回収及び洗浄回収を行うことなく培養容器から細胞を回収した。
その結果、廃液容器へ排出された培養上清は1600mLであり、これに2.00E+08の細胞が含まれており、その細胞数の百分率は2.5%であった。細胞回収容器に回収された細胞懸濁液は350mLであり、これに6.83E+09の細胞が含まれており、その細胞数の百分率は85.31%であった。また、培養容器に残存した細胞懸濁液は50mLであり、これに9.75E+08の細胞が含まれており、その細胞数の百分率は12.19%であった。
第一実施形態の細胞回収方法における上清排出工程、細胞回収工程、上清戻し工程、及び再回収工程をそれぞれ1回行うことにより、洗浄回収を1回行って、培養容器から細胞を回収した。
廃液容器へ排出された培養上清は1550mLであり、これに1.94E+08の細胞が含まれており、その細胞数の百分率は2.42%であった。細胞回収容器に回収された細胞懸濁液は400mLであり、これに7.32E+09の細胞が含まれており、その細胞数の百分率は91.45%であった。また、培養容器に残存した細胞懸濁液は50mLであり、これに4.91E+08の細胞が含まれており、その細胞数の百分率は6.13%であった。
第一実施形態の細胞回収方法における上清排出工程、細胞回収工程、上清戻し工程、及び再回収工程をそれぞれ1回行った後に、上清戻し工程と再回収工程をさらに1回繰り返すことにより、洗浄回収を2回行って、培養容器から細胞を回収した。
廃液容器へ排出された培養上清は1500mLであり、これに1.88E+08の細胞が含まれており、その細胞数の百分率は2.34%であった。細胞回収容器に回収された細胞懸濁液は450mLであり、これに7.56E+09の細胞が含まれており、その細胞数の百分率は94.55%であった。また、培養容器に残存した細胞懸濁液は50mLであり、これに2.48E+08の細胞が含まれており、その細胞数の百分率は3.11%であった。
第三実施形態の細胞回収方法における上清排出工程、縮小工程、及び縮小回収工程をそれぞれ1回行うことにより、縮小回収を行って、培養容器から細胞を回収した。
廃液容器へ排出された培養上清は1600mLであり、これに2.00E+08の細胞が含まれており、その細胞数の百分率は2.5%であった。細胞回収容器に回収された細胞懸濁液は390mLであり、これに7.61E+09の細胞が含まれており、その細胞数の百分率は95.06%であった。また、培養容器に残存した細胞懸濁液は10mLであり、これに1.95E+08の細胞が含まれており、その細胞数の百分率は2.44%であった。
第一及び三実施形態の細胞回収方法を組み合わせて、上清排出工程、縮小工程、及び縮小回収工程をそれぞれ1回行った後に、上清戻し工程と再回収工程をそれぞれ1回行うことにより、洗浄回収を1回行って、培養容器から細胞を回収した。
廃液容器へ排出された培養上清は1590mLであり、これに1.99E+08の細胞が含まれており、その細胞数の百分率は2.48%であった。細胞回収容器に回収された細胞懸濁液は400mLであり、これに7.70E+09の細胞が含まれており、その細胞数の百分率は96.29%であった。また、培養容器に残存した細胞懸濁液は10mLであり、これに9.81E+07の細胞が含まれており、その細胞数の百分率は1.23%であった。
実施例1では、培養容器に細胞懸濁液が50ml残存した後、廃液容器から培養上清50mlを培養容器に戻し、培養容器を攪拌して洗浄し、細胞回収容器にさらに細胞懸濁液50mlを回収した。その結果、培養容器には細胞懸濁液が50ml残存していた。実施例2では、実施例1の上清戻しと再回収をさらに1回繰り返して、洗浄回収を2回行った。
実施例4では、培養容器10の底面積を縮小して細胞懸濁液を回収した後に、廃液容器から培養上清10mlを培養容器に戻し、培養容器を攪拌して洗浄し、細胞回収容器にさらに細胞懸濁液10mlを回収した。その結果、培養容器には細胞懸濁液が10ml残存していた。
また、廃液容器へ排出された細胞の数の百分率は、比較例1,実施例1,実施例2,実施例3,実施例4の順に、2.5%,2.42%,2.34%,2.5%,2.48%であった。
さらに、培養容器に残存した細胞の数の百分率は、比較例1,実施例1,実施例2,実施例3,実施例4の順に、12.19%,6.13%,3.11%,2.44%,1.23%であった。
さらに、縮小回収を行った実施例3の細胞回収効率は、比較例1に比べて、10%近く増加している。また、縮小回収と洗浄回収を1回行った実施例4の細胞回収効率は、比較例1に比べて、11%近く増加している。
例えば、図2ではローラ型の攪拌部材を用いて上清戻し工程を行っているが、積載台を振とうするなどその他の方法で培養容器を攪拌しても良く、また3回以上洗浄回収を行うなど適宜変更することが可能である。
Claims (13)
- 少なくとも、細胞を培養するための培養容器と、培養後の細胞を回収する細胞回収容器と、培養後の培地を回収する廃液容器とが、導管によって連結されてなる細胞培養用キットを用いて培養された細胞の回収方法であって、
培養済みの培養上清を前記培養容器から前記廃液容器へ移送する上清排出工程、
濃縮された細胞懸濁液を前記培養容器から前記細胞回収容器へ移送する細胞回収工程、
前記廃液容器に排出された培養上清の一部を前記培養容器に戻し、前記培養容器に残存する細胞を細胞懸濁液に懸濁させる上清戻し工程、及び
残存細胞が懸濁した細胞懸濁液を前記培養容器から前記細胞回収容器に再度移送する再回収工程を、含む
ことを特徴とする細胞回収方法。 - 前記上清戻し工程において、所定の懸濁部材を用いて、前記培養容器の表面が所定の深さに下降する位置まで前記培養容器の表面を押圧しながら、前記懸濁部材を前記培養容器上で水平方向に往復移動させ、前記培養容器に残存する細胞を細胞懸濁液に分散させる
ことを特徴とする請求項1記載の細胞回収方法。 - 前記上清戻し工程における前記培養容器に残存する細胞の細胞懸濁液での分散と、前記再回収工程における培養容器から前記細胞回収容器への細胞懸濁液の移送を、同時に行うことを特徴とする請求項1又は2記載の細胞回収方法。
- 少なくとも、細胞を培養するための培養容器と、培養後の細胞を回収する細胞回収容器と、培養後の培地を回収する廃液容器とが、導管によって連結されてなる細胞培養用キットを用いて培養された細胞の回収方法であって、
培養済みの培養上清を前記培養容器から前記廃液容器に移送する上清排出工程、
前記培養容器の底面積を縮小させて、濃縮された細胞懸濁液を前記培養容器の細胞回収ポート側に寄せ集める縮小工程、及び
寄せ集められた細胞懸濁液を前記培養容器から前記細胞回収容器に移送する縮小回収工程を、含む
ことを特徴とする細胞回収方法。 - 前記縮小工程における濃縮された細胞懸濁液の寄せ集めと、前記縮小回収工程における寄せ集められた細胞懸濁液の前記細胞回収容器への移送とを、同時に行うことを特徴とする請求項4記載の細胞回収方法。
- 前記上清排出工程の後に、濃縮された細胞懸濁液を前記培養容器から前記細胞回収容器に移送する細胞回収工程を行い、次いで前記縮小工程を行うことを特徴とする請求項4又は5記載の細胞回収方法。
- 前記縮小回収工程の後に、
前記廃液容器に排出された培養上清の一部を前記培養容器に戻し、前記培養容器に残存する細胞を細胞懸濁液に懸濁させる上清戻し工程、及び
残存細胞が懸濁した細胞懸濁液を前記培養容器から前記細胞回収容器に再度移送する再回収工程を、含む
ことを特徴とする請求項4~6のいずれかに記載の細胞回収方法。 - 前記縮小工程において、所定の仕切部材を用いて、前記培養容器を培養空間の外部から表面と裏面が密着するように押圧しながら、前記仕切部材を前記培養容器上で移動させ、前記培養容器の底面積を縮小させる
ことを特徴とする請求項4~7のいずれかに記載の細胞回収方法。 - 前記上清戻し工程及び前記再回収工程を2回以上繰り返すことを特徴とする請求項1~3,7のいずれかに記載の細胞回収方法。
- 培地等を貯蔵しておくための培地貯蔵容器が、前記廃液容器として用いられることを特徴とする請求項1~9のいずれかに記載の細胞回収方法。
- 前記上清排出工程の前に、前記培養容器の底面積を縮小させ、細胞が沈降するまで前記培養容器を静置する静置工程を行う
ことを特徴とする請求項1~10のいずれかに記載の細胞回収方法。 - 前記静置工程において、所定の仕切部材を用いて、前記培養容器を端部から表面と裏面が密着するように押圧しながら、前記仕切部材を前記培養容器上で移動させ、前記培養容器の底面積を縮小させる
ことを特徴とする請求項11記載の細胞回収方法。 - 前記所定の仕切部材が、ローラであることを特徴とする請求項8又は12記載の細胞回収方法。
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CN104093828B (zh) | 2017-10-20 |
EP3056560A1 (en) | 2016-08-17 |
EP2811014A1 (en) | 2014-12-10 |
US20140335600A1 (en) | 2014-11-13 |
EP3056560B1 (en) | 2018-05-02 |
KR20140107672A (ko) | 2014-09-04 |
US9574168B2 (en) | 2017-02-21 |
EP2811014B1 (en) | 2017-05-03 |
EP2811014A4 (en) | 2015-09-16 |
CN104093828A (zh) | 2014-10-08 |
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