WO2016121295A1 - 細胞培養における複数容器間の送液方法 - Google Patents
細胞培養における複数容器間の送液方法 Download PDFInfo
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- WO2016121295A1 WO2016121295A1 PCT/JP2016/000006 JP2016000006W WO2016121295A1 WO 2016121295 A1 WO2016121295 A1 WO 2016121295A1 JP 2016000006 W JP2016000006 W JP 2016000006W WO 2016121295 A1 WO2016121295 A1 WO 2016121295A1
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- C—CHEMISTRY; METALLURGY
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- C—CHEMISTRY; METALLURGY
- 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
- C12M23/40—Manifolds; Distribution pieces
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- C—CHEMISTRY; METALLURGY
- 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
- C12M23/58—Reaction vessels connected in series or in parallel
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- C—CHEMISTRY; METALLURGY
- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
- F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
- F16K11/00—Multiple-way valves, e.g. mixing valves; Pipe fittings incorporating such valves
- F16K11/02—Multiple-way valves, e.g. mixing valves; Pipe fittings incorporating such valves with all movable sealing faces moving as one unit
- F16K11/08—Multiple-way valves, e.g. mixing valves; Pipe fittings incorporating such valves with all movable sealing faces moving as one unit comprising only taps or cocks
Definitions
- the present invention relates to a cell culture technique, and more particularly, to a liquid feeding method between containers when a plurality of culture containers and a medium supply container are used.
- a method may be employed in which a plurality of containers are connected by a tube to form a closed environment, and liquid is fed between the plurality of containers.
- a plurality of culture bags and a medium supply bag are connected by a tube, the medium is transferred from the medium supply bag to the culture bag, the cells are cultured in the culture bag, and the cultured cells and the culture solution containing the medium (Hereinafter, what includes cells and a medium is referred to as a culture solution) is transferred to a culture bag having a larger culture area and further proliferated.
- Patent Document 1 discloses a cell culture container kit for use in an automatic cell culture apparatus. According to this cell culture kit, it is possible to carry out the cell injection, medium addition, sampling, and collection in the cell culture while maintaining the closed system.
- this cell culture kit has not been devised to simplify the flow path control mechanism in cell culture. For this reason, in order to control the flow path between the plurality of containers, it is necessary to use one or a plurality of pumps and a large number of clips, and there is a problem that the control mechanism of the flow path becomes somewhat complicated. Therefore, the present inventors diligently researched, connected a plurality of containers using a three-way stopcock, and placed a culture container with a drop in the order of liquid feeding, using only one pump, The present invention was completed by successfully controlling the flow path.
- the medium can be transferred from the medium supply container to the culture container by the pump, and the culture liquid can be transferred between the plurality of culture containers by natural liquid supply. Moreover, a pinch valve, a clip, etc. can be made unnecessary, and the control mechanism can be greatly simplified.
- the present invention has been made in view of the above circumstances, and provides a liquid feeding method between a plurality of containers in cell culture capable of simplifying the flow path control between the containers when cell culture is performed using the plurality of containers. With the goal.
- the liquid feeding method between a plurality of containers in the cell culture according to the present invention includes a medium supply container having one port, and a plurality of contents transfer ports each having a content transfer port to which a medium is supplied from the medium supply container.
- the tube between the culture vessel connected immediately before and the three-way stopcock is newly provided with a three-way stopcock, Repeatedly connecting the three-way stopcock and the next culture container with a tube, connecting all the culture containers to the three-way stopcock, and each culture in each
- the liquid feeding method between a plurality of containers in the cell culture according to the embodiment of the present invention includes a medium supply container having one port and a content transfer port to which the medium is supplied from the medium supply container.
- a method of feeding liquid between a plurality of containers in cell culture using a plurality of culture containers wherein the first culture container, the three-way stopcock and the medium supply container in the plurality of culture containers are connected by a tube, and the three-way stopcock and the medium supply
- the three-way stopcock and the second culture container in the plurality of culture containers are connected by a tube, and a plurality of cultures
- the number of culture vessels in the container is 3 or more
- a tube between the culture vessel connected immediately before and the three-way stopcock is newly provided with a three-way stopcock, and the new three-way stopcock and the next culture vessel are provided. It is characterized in that all the culture containers are connected to a three-way stopcock by repeatedly connecting with a tube, and the medium is transferred from the culture medium supply container to each culture container in a plurality of culture containers via the three-way stopcock.
- the method for feeding between a plurality of containers in the cell culture according to the embodiment of the present invention each culture container in the plurality of culture containers, the horizontal position of the culture container connected earlier, the horizontal position of the culture container connected later It is preferable to use a method in which the liquid is arranged so as to be higher than the position, and the liquid feeding from the culture vessel connected first to the culture vessel connected later is performed by natural liquid feeding based on the drop in the horizontal position. Furthermore, in the cell culture method according to the embodiment of the present invention, the culture solution supernatant or the culture solution is transferred from the culture vessel connected last to the medium supply container by a pump through the three-way stopcock from the connected culture vessel. It is also preferable to adopt a method of transferring.
- the liquid feeding method between a plurality of containers is provided with a three-way stopcock in a tube between a medium supply container and a pump, and the medium supply container is provided with one or two ports.
- the above-mentioned other medium supply containers are connected by tubes via three-way stopcocks, and the medium is transferred from these medium supply containers to each culture container in a plurality of culture containers via a three-way stopcock. Is also preferable.
- the liquid feeding method between a plurality of containers in the cell culture it is possible to appropriately control the flow path by using only one pump in the closed cell culture system. It becomes. That is, in this method, a plurality of containers are connected to a three-way stopcock using a single pump, and the culture containers are arranged with a drop in the order of liquid feeding. For this reason, the culture medium can be transferred from the culture medium supply container to the culture container by a pump, and the culture liquid can be transferred between a plurality of culture containers by natural liquid feeding based on the drop in the horizontal position of the culture container. Is possible. Further, according to this method, since it is not necessary to use a pinch valve, a clip, or the like, it is possible to greatly simplify the flow path control mechanism that has been complicated conventionally.
- FIG. 1 is a front view showing the connection relationship of containers used in the liquid feeding method between a plurality of containers in the cell culture of this embodiment
- FIG. 2 is a plan view of the same.
- the cell culture system used in this embodiment includes two culture containers.
- the cell culture system used in this embodiment includes a culture vessel 1, a culture vessel 1a, a medium supply vessel 2, a three-way cock 3, a pump 4, and a tube connecting them. 5 is provided.
- the culture container 1, the culture container 1a, and the culture medium supply container 2 are connected by a tube 5 through a three-way cock 3.
- Each container is provided with one port for transferring contents with other containers, and a tube 5 is connected to this port.
- a pump 4 is attached to the tube 5 between the three-way stopcock 3 and the medium supply container 2. Further, the horizontal position of the culture container 1 for first culture (connected earlier) is arranged higher than the horizontal position of the culture container 1a for subsequent culture (connected later). Is done.
- a culture solution containing cells and a medium is injected into the culture container 1 in a sterile environment, and the culture container 1, the culture container 1a, the medium supply container 2, and the three-way stopcock 3 are connected to the tube 5
- a closed system is configured by connecting using A pump 4 is attached between the culture medium supply container 2 and the three-way stopcock 3. Furthermore, it arrange
- the culture solution is then transferred naturally from the culture vessel 1 to the culture vessel 1a, and the medium is transferred from the culture medium supply vessel 2 to the culture vessel 1a by the pump 4 via the three-way cock 3. Then, the cells are cultured in the culture vessel 1a. At this time, it is preferable to stir the culture solution in the culture vessel prior to the natural feeding. Moreover, when performing natural liquid feeding, it is preferable to incline the mount on which the culture vessel is placed so that the port in the culture vessel is downward. If it does in this way, it will become possible to perform natural liquid feeding between culture vessels efficiently. Such a method related to natural liquid feeding can be similarly performed in the following embodiments.
- the medium in order to warm the medium, the medium is first transferred from the medium supply container 2 to the culture container 1 by the pump 4 through the three-way cock 3, and after the medium is sufficiently heated in the culture container 1.
- the medium can also be transferred to the culture container 1a by natural liquid feeding. Such heating of the culture medium can be similarly performed for each culture vessel in the following embodiments.
- the supernatant of the culture solution is transferred from the culture vessel 1a through the tube 5 and the three-way stopcock 3 to the empty medium supply vessel 2 by the pump 4, and the culture vessel 1a
- the culture solution in can be concentrated.
- the culture solution can be transferred from the culture container 1a to the culture medium supply container 2.
- the capacity of the culture vessel and the culture area be increased as the cells are connected later so that the culture vessels connected later can culture more cells.
- the culture vessel 1 and the culture vessel 1a are preferably formed into a bag shape (bag shape) using a soft packaging material as a material, and part or all of them are preferably transparent so that the contents can be confirmed.
- these culture vessels must have gas permeability (oxygen and carbon dioxide permeability) necessary for cell culture, and are used in a culture environment of 37 ° C. and 5% carbon dioxide concentration. It is preferable to do.
- these culture vessels preferably have low cytotoxicity, low elution properties, and suitability for radiation sterilization in order to achieve high cell growth efficiency.
- a polyethylene resin As a material for the culture vessel satisfying such conditions, a polyethylene resin is preferable.
- the polyethylene resin include polyethylene, a copolymer of ethylene and ⁇ -olefin, a copolymer of ethylene and vinyl acetate, an ionomer using an ethylene and acrylic acid or methacrylic acid copolymer and a metal ion.
- polyolefin, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone resin and the like can also be used.
- the four sides of the culture vessel can be sealed by heat sealing.
- the shape of the accommodating part of the culture vessel 2 may be a rectangular shape, and as shown in FIG. 2, it is also preferable that the shape is gradually narrowed toward the port.
- the culture container may be an integrally formed bag by blow molding.
- the medium supply container 2 preferably has a gas barrier property against oxygen and carbon dioxide so that the pH of the stored medium does not change greatly during the culture period. This is because the high-concentration carbon dioxide contained in the culture medium escapes into the air, and the carbon dioxide concentration in the culture medium decreases, resulting in an increase in pH. This is because it is desirable to minimize the leakage of carbon dioxide from the outside. It is also desirable to prevent the culture medium from being oxidized.
- the type of the three-way stopcock 3 is not particularly limited, but a closed three-way stopcock can be suitably used because it is easy to realize a closed system in the cell culture system.
- the kind of pump 4 is not specifically limited, From the same viewpoint, tube roller pumps, such as a peristaltic pump (R), can be used conveniently.
- the material of the tube 5 may be appropriately selected in accordance with the use environment, but when used in a CO 2 incubator, a material having excellent gas permeability to oxygen and carbon dioxide is particularly desirable. When used outside the CO 2 incubator, it is desirable to have excellent gas barrier properties.
- silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, etc. are used. be able to.
- styrene elastomer examples include SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene), and the like.
- SBS styrene / butadiene / styrene
- SIS styrene / isoprene / styrene
- SEBS styrene / ethylene / butylene / styrene
- SEPS styrene / ethylene / propylene / styrene
- the type of cultured cells in the present embodiment is not particularly limited, and for example, suspension cells such as lymphocytes, and adhesive cells such as skin cells, corneal cells, vascular endothelial cells, and mesenchymal stem cells can be preferably cultured. Is possible.
- FIG. 3 is a front view showing the connection relation of containers used in the liquid feeding method between a plurality of containers in the cell culture of this embodiment.
- the cell culture system used in this embodiment includes three culture containers.
- the cell culture system used in the present embodiment includes a culture vessel 1, a culture vessel 1a, a culture vessel 1b, a medium supply vessel 2, a three-way stopcock 3, a three-way stopcock 3a, a pump 4, and The tube 5 which connects these is provided.
- the culture container 1, the culture container 1a, and the culture medium supply container 2 are connected by a tube 5 through a three-way cock 3 as in the first embodiment.
- the three-way stopcock 3 and the three-way stopcock 3 a are connected by a tube 5, and the culture container 1 a, the three-way stopcock 3 a, and the culture container 1 b are connected by a tube 5.
- Each container is provided with one port for transferring contents with other containers, and a tube 5 is connected to this port.
- a pump 4 is attached to the tube 5 between the three-way stopcock 3 and the medium supply container 2. Further, the horizontal position of the culture container 1 for first culture (connected earlier) is arranged higher than the horizontal position of the culture container 1a for subsequent culture (connected later). Then, the horizontal position of the culture vessel 1a is arranged so as to be higher than the horizontal position of the culture vessel 1b for further culturing (and connected later).
- four or more culture containers may be provided and connected to the culture container 1 and the medium supply container 2 via the three-way stopcock via the tube 5 in the same manner as the culture containers 1a and 1b.
- the culture vessel connected first is arranged so that the horizontal position of the culture vessel connected earlier is higher than the horizontal position of the culture vessel connected later. It is preferable that the cell suspension can be naturally fed from the culture vessel to the culture vessel connected later. It is also preferable to provide a sampling port in the culture vessel in which culture is performed last, and connect a sampling tube to this port.
- a culture solution containing cells and a medium is injected into the culture container 1 in a sterile environment, and the culture container 1, the culture container 1a, the culture container 1b, the culture medium supply container 2, and the three-way stopcock 3 are used. And the three-way stopcock 3a are connected using a tube 5 to constitute a closed system.
- a pump 4 is attached between the culture medium supply container 2 and the three-way stopcock 3.
- the culture container 1 is arranged so that the horizontal position of the culture container 1a is higher than the horizontal position of the culture container 1a, and the culture container 1a is arranged so that the horizontal position is higher than the horizontal position of the culture container 1b.
- the medium is transferred from the medium supply container 2 to the culture container 1 by the pump 4 through the three-way cock 3, and the cells are cultured in the culture container 1.
- the culture solution is then transferred from the culture vessel 1 to the culture vessel 1a by natural feeding, and from the culture medium supply vessel 2 to the culture vessel 1a via the three-way stopcocks 3 and 3a.
- the medium is transferred, and the cells are cultured in the culture vessel 1a.
- the culture solution is then transferred from the culture vessel 1a to the culture vessel 1b by natural feeding, and the culture vessel is supplied from the medium supply vessel 2 through the three-way stopcocks 3 and 3a by the pump 4.
- the medium is transferred to 1b, and the cells are cultured in the culture vessel 1b.
- the culture medium supernatant is transferred from the culture container 1b to the emptied medium supply container 2 by the pump 4 through the three-way stopcock 3a and the three-way stopcock 3, and the culture container
- the culture solution in 1b can be concentrated.
- the culture solution can be transferred from the culture container 1b to the culture medium supply container 2.
- the culture solution is transferred by natural feeding from the previously connected culture vessel to the later connected culture vessel in the same manner as described above. Then, the cells are cultured in a culture vessel connected later. When cell culture in the last connected culture vessel is completed, the supernatant of the culture solution is transferred from the culture vessel to the emptied medium supply vessel 2 by the pump 4 through a plurality of three-way stopcocks. The culture solution in the culture vessel can be concentrated. Similarly, the culture solution can be transferred from the culture vessel to the culture medium supply container 2.
- the type of cultured cells in the present embodiment is not particularly limited, and for example, suspension cells such as lymphocytes can be suitably cultured.
- the culture vessel 1 can be suitably used as a vessel for performing curing culture and expansion culture for recovering the function of damaged cells.
- cells that have been damaged due to damage such as cells collected from patients and cells thawed after cryopreservation, cannot be performed sufficiently even if they are activated as they are.
- the culture vessel 1 has a culture area that can be expanded, and the method is not particularly limited.
- the culture vessel 1a is preferably used as an activated culture vessel used in a culture step (activation culture step) for activating cells.
- a substance that activates cells such as anti-CD3 antibody, is immobilized on the bottom surface of the culture container 1a, and the cells contained in the culture container 1a are activated by binding to the substance.
- the anti-CD3 antibody is preferably used for activating lymphocytes.
- the culture vessel 1b is preferably used as an amplification culture vessel used in a culture step (amplification culture step) for growing cells in large quantities.
- the volume of the culture vessel 1a is preferably larger than the volume of the culture vessel 1, and for example, a volume more than twice the volume of the culture vessel 1 can be suitably used.
- the culture container 1b is used for growing a large amount of cells, it is preferable to use a culture container having a particularly large capacity.
- a culture container having a capacity about 10 times the capacity of the culture container 1a can be suitably used.
- a tube 5 between the medium supply container 2 and the pump 4 is provided with a three-way stopcock, and through this three-way stopcock, an enzyme solution supply container in which an enzyme solution for separating cells from the inner surface of the culture container is enclosed. It is preferable to connect an inhibitor solution supply container in which an inhibitor solution for inhibiting the action of the enzyme is enclosed. Furthermore, it is possible to connect other containers such as a cleaning liquid supply container and a waste liquid recovery container in the same manner. In this case as well, it is preferable that the capacity and the culture area of the culture vessel be increased as those connected later so that subculture can be performed efficiently.
- the material, properties, form, and the like of the culture vessel can be the same as those in the first embodiment. Further, the properties of the medium supply container 2, the types of the three-way cock 3 and the pump 4, and the material of the tube 5 can be the same as those in the first embodiment.
- FIG. 4 is a front view showing the connection relationship of containers used in the liquid feeding method between a plurality of containers in the cell culture of this embodiment.
- the cell culture system used in the present embodiment includes three culture containers and two medium supply containers. About another point, it can be set as the thing similar to 2nd embodiment.
- the cell culture system used in this embodiment is further provided with a three-way stopcock 6 and a medium supply container 2a in addition to the configuration in the second embodiment, and the three-way stopcock 3 and the three-way stopcock
- the stopcock 6 is connected by a tube 5, and the culture medium supply container 2, the three-way stopcock 6 and the culture medium supply container 2 a are connected by a tube 5.
- the properties of these medium supply containers can be the same as those in the first embodiment.
- the medium supply container 2 and the medium supply container 2a may enclose the same medium or different mediums.
- three or more medium supply containers may be provided and connected to the medium supply container 2 and each culture container via the tube 5 via a three-way stopcock, similarly to the medium supply container 2a.
- Such this embodiment can be used especially suitably when supplying different culture media to a plurality of culture vessels.
- a culture medium is supplied from the culture medium supply container 2a to the culture container 1 and the culture container 1a, the curing culture process and the expansion culture process described above are performed in the culture container 1, and the activation culture process described above is performed in the culture container 1a.
- Can do It is also possible to supply the culture medium from the culture medium supply container 2 to the culture container 1b and perform the above-described amplification culture process in the culture container 1b.
- the present invention can be suitably used in gene therapy, immunotherapy, regenerative medicine, antibody drug production, etc., in which cells are cultured in large quantities in a closed system using a plurality of culture vessels.
Abstract
Description
具体的には、例えば複数の培養バッグと培地供給バッグをチューブで接続し、培地供給バッグから培養バッグに培地を移送して、培養バッグにおいて細胞を培養し、培養した細胞及び培地を含む培養液(以下、細胞と培地を含むものを培養液と言う)を培養面積のより大きい培養バッグに移送して、さらに細胞を増殖させることなどが行われている。
一方、チューブを分岐させることによって、ポンプの台数を減らすことは可能である。しかしながら、この場合には、分岐したチューブの流路を制御するためにピンチバルブなどが必要となり、やはり複雑な制御機構と多くの機器が必要になるという問題があった。
そこで、本発明者らは鋭意研究し、複数の容器を三方活栓を用いて接続すると共に、培養容器を送液の順番に落差をつけて配置することで、1台のポンプのみを用いて、流路を簡易に制御することに成功し、本発明を完成させた。
本発明は、上記事情に鑑みなされたものであり、複数の容器を用いて細胞培養を行う場合における容器間の流路制御を簡易化可能な、細胞培養における複数容器間の送液方法の提供を目的とする。
最初に、本発明の実施形態に係る細胞培養における複数容器間の送液方法の特徴について概説する。
本発明の実施形態に係る細胞培養における複数容器間の送液方法は、ポートを一つ備えた培地供給容器と、培地供給容器から培地が供給される内容物移送用のポートを各々一つ備えた複数の培養容器とを用いる細胞培養における複数容器間の送液方法であって、複数の培養容器における第一の培養容器と三方活栓と培地供給容器をチューブにより接続し、三方活栓と培地供給容器の間に1つのポンプを配置すると共に、複数の培養容器における培養容器の個数が2個である場合、三方活栓と複数の培養容器における第二の培養容器をチューブにより接続し、複数の培養容器における培養容器の個数が3個以上である場合、直前に接続された培養容器及び三方活栓の間のチューブに新たに三方活栓を備え、当該新たな三方活栓と次の培養容器をチューブにより接続することを繰り返して、全ての培養容器を三方活栓に接続し、培地供給容器から三方活栓を介して、ポンプにより複数の培養容器における各培養容器に培地を移送することを特徴とする。
さらに、本発明の実施形態に係る細胞培養における複数容器間の送液方法を、最後に接続された培養容器から三方活栓を介して、ポンプにより培地供給容器に培養液の上澄み液又は培養液を移送する方法とすることも好ましい。
すなわち、この方法では、複数の容器を三方活栓と1つのポンプを用いて接続すると共に、培養容器を送液の順番に落差をつけて配置している。
このため、培地供給容器から培養容器への培地の移送はポンプにより行うことができると共に、複数の培養容器間における培養液の移送は、培養容器の水平位置の落差にもとづく自然送液により行うことが可能となっている。また、この方法によれば、ピンチバルブやクリップなどを用いる必要がないため、従来は煩雑であった流路の制御機構を大きく簡易化することが可能となっている。
次に、本発明の第一実施形態に係る細胞培養における複数容器間の送液方法について、図1及び図2を参照して説明する。図1は、本実施形態の細胞培養における複数容器間の送液方法で用いられる容器の接続関係を示す正面図であり、図2は同平面図である。
本実施形態で用いられる細胞培養システムには、培養容器が2個備えられている。
培養容器1と培養容器1aと培地供給容器2は、三方活栓3を介してチューブ5により接続されている。各容器には、他の容器との内容物の移送を行うためのポートが一つ備えられており、このポートにチューブ5が接続されている。
さらに、最初に培養を行うための(先に接続された)培養容器1の水平位置が、次に培養を行うための(後に接続された)培養容器1aの水平位置よりも高くなるように配置される。
また、培地供給容器2と三方活栓3の間にポンプ4を取り付けて配置する。さらに、培養容器1の水平位置が、培養容器1aの水平位置よりも高くなるように配置する。そして、培地供給容器2から三方活栓3を介して、ポンプ4によって培養容器1に培地を移送し、培養容器1において細胞の培養を行う。
このとき、自然送液に先立って、培養容器内における培養液を攪拌することが好ましい。また、自然送液を行うにあたっては、培養容器を載置した架台を、培養容器におけるポートが下になるように傾斜させることが好ましい。このようにすれば、培養容器間の自然送液を効率的に行うことが可能となる。このような自然送液に関する手法は、以下の実施形態においても同様に行うことができる。
さらに、この場合、培地を加温するため、培地供給容器2から三方活栓3を介して、ポンプ4によって培養容器1にまず培地を移送し、培養容器1にて培地が十分加温された後、培地を培養容器1aに自然送液により移送することもできる。このような培地の加温も以下の実施形態におけるそれぞれの培養容器について同様に行うことができる。
また、これらの培養容器は、細胞の培養に必要なガス透過性(酸素及び二酸化炭素透過性)を有していることが必要であり、37℃、5%二酸化炭素濃度の培養環境下で使用することが好ましい。さらに、これらの培養容器は、高い細胞増殖効率を実現するために、低細胞毒性、低溶出性、及び放射線滅菌適性を有することが好ましい。
また、ポンプ4の種類も特に限定されないが、同様の観点から、ペリスタポンプ(R)などのチューブローラポンプを好適に用いることができる。
特に、チューブ5におけるポンプを取り付ける部分にはシリコーンチューブを好適に用いることができ、その他の部分には軟質塩化ビニル樹脂チューブを好適に用いることができる。
次に、本発明の第二実施形態に係る細胞培養における複数容器間の送液方法について、図3を参照して説明する。図3は、本実施形態の細胞培養における複数容器間の送液方法で用いられる容器の接続関係を示す正面図である。
本実施形態で用いられる細胞培養システムには、培養容器が3個備えられている。
培養容器1と培養容器1aと培地供給容器2は、第一実施形態と同様に、三方活栓3を介してチューブ5により接続されている。また、三方活栓3と三方活栓3aが、チューブ5により接続され、培養容器1aと三方活栓3aと培養容器1bが、チューブ5により接続されている。各容器には、他の容器との内容物の移送を行うためのポートが一つ備えられており、このポートにチューブ5が接続されている。
さらに、最初に培養を行うための(先に接続された)培養容器1の水平位置が、次に培養を行うための(後に接続された)培養容器1aの水平位置よりも高くなるように配置され、培養容器1aの水平位置が、次に培養を行うための(さらに後に接続された)培養容器1bの水平位置よりも高くなるように配置される。
また、最後に培養が行われる培養容器に、サンプリング用の別個のポートを備え、このポートにサンプリングチューブを接続することも好ましい。
また、培地供給容器2と三方活栓3の間にポンプ4を取り付けて配置する。さらに、培養容器1の水平位置が、培養容器1aの水平位置よりも高くなるように配置し、培養容器1aの水平位置が、培養容器1bの水平位置よりも高くなるように配置する。そして、培地供給容器2から三方活栓3を介して、ポンプ4によって培養容器1に培地を移送し、培養容器1において細胞の培養を行う。
また、培養容器1aにおける細胞培養が終了すると、次いで培養容器1aから培養容器1bに培養液を自然送液により移送し、培地供給容器2から三方活栓3、3aを介して、ポンプ4によって培養容器1bに培地を移送し、培養容器1bにおいて細胞の培養を行う。
この場合、培養容器1を、ダメージを受けた細胞の機能を回復させるための養生培養及び拡大培養を行うための容器として好適に用いることができる。すなわち、患者から採取した細胞や、凍結保存後に解凍された細胞のような、ダメージを受けて機能が衰えた細胞は、そのままの状態では活性化させようとしても十分に行うことができず、細胞の大量培養に用いることができない。そこで、このような細胞を増殖させるため、細胞本来の機能が回復するように、細胞培養の初期の段階において当該細胞を養生しつつ培養する培養工程(養生培養工程)が行われる。そして、細胞本来の機能が回復し、細胞の増殖がある程度進んでから培養面積を拡大して、所定の細胞密度となるまで細胞の培養を継続する培養工程(拡大培養工程)が行われる。したがって、この場合、培養容器1は、培養面積を拡大可能に備えることが好ましく、その方法は特に限定されない。
さらに、この場合、培養容器1bは、細胞を大量に増殖させるための培養工程(増幅培養工程)に用いる増幅培養容器として用いることが好ましい。
この場合も、培養容器の容量及び培養面積は、継代培養などを効率的に行えるように、後に接続されるものほど大きくすることが好ましい。
次に、本発明の第三実施形態に係る細胞培養における複数容器間の送液方法について、図4を参照して説明する。図4は、本実施形態の細胞培養における複数容器間の送液方法で用いられる容器の接続関係を示す正面図である。
本実施形態で用いられる細胞培養システムには、培養容器が3個備えられ、培地供給容器が2個備えられている。その他の点については、第二実施形態と同様のものとすることができる。
また、本実施形態において、培地供給容器を3個以上設けて、培地供給容器2aと同様に、三方活栓を介して培地供給容器2及び各培養容器にチューブ5により接続する構成としても良い。
例えば、培地供給容器2aから培養容器1と培養容器1aに培地を供給して、培養容器1において上述した養生培養工程及び拡大培養工程を行い、培養容器1aにおいて上述した活性化培養工程を行うことができる。また、次いで培地供給容器2から培養容器1bに培地を供給して、培養容器1bにおいて上述した増幅培養工程を行うことなどが可能である。
例えば、第一実施形態において、第三実施形態のように複数の培地供給容器を備えたり、さらにその他の容器を備える構成とするなど適宜変更することが可能である。
2,2a 培地供給容器
3,3a,3b 三方活栓
4 ポンプ
5 チューブ
6 三方活栓
Claims (6)
- ポートを一つ備えた培地供給容器と、前記培地供給容器から培地が供給される内容物移送用のポートを各々一つ備えた複数の培養容器とを用いる細胞培養における複数容器間の送液方法であって、
前記複数の培養容器における第一の培養容器と三方活栓と前記培地供給容器をチューブにより接続し、前記三方活栓と前記培地供給容器の間に1つのポンプを配置すると共に、
前記複数の培養容器における培養容器の個数が2個である場合、
前記三方活栓と前記複数の培養容器における第二の培養容器をチューブにより接続し、
前記複数の培養容器における培養容器の個数が3個以上である場合、
直前に接続された培養容器及び三方活栓の間のチューブに新たに三方活栓を備え、当該新たな三方活栓と次の培養容器をチューブにより接続することを繰り返して、全ての培養容器を三方活栓に接続し、
前記培地供給容器から三方活栓を介して、ポンプにより前記複数の培養容器における各培養容器に培地を移送する
ことを特徴とする細胞培養における複数容器間の送液方法。 - 前記複数の培養容器が第一の培養容器と第二の培養容器からなり、
前記第一の培養容器と三方活栓と前記培地供給容器をチューブにより接続し、前記三方活栓と前記培地供給容器の間に1つのポンプを配置すると共に、
前記三方活栓と前記第二の培養容器をチューブにより接続し、
前記培地供給容器から三方活栓を介して、ポンプにより前記第一の培養容器と前記第二の培養容器に培地を移送する
ことを特徴とする請求項1記載の細胞培養における複数容器間の送液方法。 - 前記複数の培養容器が第一の培養容器と第二の培養容器と第三の培養容器からなり、
前記第一の培養容器と三方活栓と前記培地供給容器をチューブにより接続し、前記三方活栓と前記培地供給容器の間に1つのポンプを配置すると共に、
前記三方活栓と他の三方活栓をチューブにより接続し、かつ前記第二の培養容器と前記他の三方活栓と前記第三の培養容器をチューブにより接続し、
前記培地供給容器から三方活栓を介して、ポンプにより前記第一の培養容器と前記第二の培養容器と前記第三の培養容器に培地を移送する
ことを特徴とする請求項1記載の細胞培養における複数容器間の送液方法。 - 前記複数の培養容器における各培養容器を、先に接続された培養容器の水平位置が後に接続された培養容器の水平位置よりも高くなるように配置し、
先に接続された培養容器から後に接続された培養容器への送液を、水平位置の落差にもとづく自然送液により行う
ことを特徴とする請求項1~3のいずれかに記載の細胞培養における複数容器間の送液方法。 - 最後に接続された培養容器から三方活栓を介して、ポンプにより前記培地供給容器に培養液の上澄み液又は培養液を移送する
ことを特徴とする請求項1~4のいずれかに記載の細胞培養における複数容器間の送液方法。 - 前記培地供給容器とポンプの間のチューブに三方活栓を備え、
前記培地供給容器に、一つのポートを備えた一又は二以上の他の培地供給容器をそれぞれ三方活栓を介してチューブにより接続し、
これらの培地供給容器から三方活栓を介して、ポンプにより前記複数の培養容器における各培養容器に培地を移送する
ことを特徴とする請求項1~5のいずれかに記載の細胞培養における複数容器間の送液方法。
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