JPH06141850A - Culture system for animal cell and method for culture of animal cell - Google Patents
Culture system for animal cell and method for culture of animal cellInfo
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- JPH06141850A JPH06141850A JP4296825A JP29682592A JPH06141850A JP H06141850 A JPH06141850 A JP H06141850A JP 4296825 A JP4296825 A JP 4296825A JP 29682592 A JP29682592 A JP 29682592A JP H06141850 A JPH06141850 A JP H06141850A
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- culture
- tank
- cells
- cell
- culture tank
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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
- C12M29/10—Perfusion
-
- 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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/22—Settling tanks; Sedimentation by gravity
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- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は動物細胞の培養方法及び
装置に係り、特に、細胞を長期間連続的に良好な状態で
培養する方法及び装置に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for culturing animal cells, and more particularly to a method and apparatus for culturing cells in a good condition continuously for a long period of time.
【０００２】[0002]
【従来の技術】動物細胞の培養は、ワクチン、インター
フェロン、モノクローナル抗体等の有用物質の製造にと
って重要な技術であり、これらの有用物質は、ハイブリ
ドーマや遺伝子組替ＣＨＯ（チャイニーズハムスター卵
巣）細胞等の動物細胞を大量培養することにより生産さ
れる。2. Description of the Related Art Culturing of animal cells is an important technique for producing useful substances such as vaccines, interferons, and monoclonal antibodies. These useful substances include hybridoma and genetically modified CHO (Chinese hamster ovary) cells. It is produced by mass-culturing animal cells.
【０００３】有用物質の生産効率を高めるためには、高
い細胞密度を維持しつつ動物細胞を培養する技術が必須
である。それには、細胞を培養する際、酸素を十分供給
し、かつ細胞に必要な栄養物質を十分に供給すると同時
に培養液中に蓄積してくる代謝老廃物質を速やかに除去
して培地環境を良好にすることが要求され、そのための
具体的な手法として、連続的に培養液を細胞と分離して
培養系外に排出し、新鮮培地を培養槽に供給しながら連
続培養する、いわゆる灌流培養法が有効であると知られ
ている。特に浮遊性細胞の培養での培養液の交換手段と
しては、培養槽内に設けられた細胞沈殿管、培養槽内若
しくは培養槽に付設される培養液循環路に設けられた濾
過手段によるものが知られている。In order to increase the production efficiency of useful substances, a technique for culturing animal cells while maintaining a high cell density is essential. To this end, when culturing cells, oxygen is sufficiently supplied, and nutrients necessary for cells are sufficiently supplied, and at the same time, metabolic waste substances accumulated in the culture medium are rapidly removed to improve the medium environment. As a specific method for that, there is a so-called perfusion culture method, in which the culture solution is continuously separated from the cells and discharged to the outside of the culture system and continuously cultured while supplying a fresh medium to the culture tank. Known to be effective. In particular, as a means for exchanging the culture solution in the culture of floating cells, there is a cell settling tube provided in the culture tank, or a filtration means provided in the culture tank or a culture solution circulation path attached to the culture tank. Are known.
【０００４】一般にハイブリドーマ等の細胞では、細胞
の増殖が定常状態での長期培養において細胞当りの物質
生産性が向上すると知られており、高密度での定常状態
を安定に保つ培養方法がとられるが、細胞株の種類によ
っては、細胞が増殖期にある方が生産性が高い場合もあ
り、公開特許公報平３−１７２１７２号に述べられてい
るように、細胞を含む培養液を一部抜き出しながら、細
胞を増殖状態に保って生産に良好な条件での連続培養を
行なう方法も知られている。It is generally known that in cells such as hybridomas, the substance productivity per cell is improved in long-term culture in which the cell growth is in a steady state, and a culture method for keeping the steady state in a high density stable is adopted. However, depending on the type of cell line, the productivity may be higher when the cells are in the growth phase, and as described in Japanese Patent Laid-Open No. 3-172172, a part of the culture solution containing the cells is extracted. However, a method is also known in which cells are maintained in a proliferating state and continuously cultured under conditions favorable for production.
【０００５】一方で、動物細胞はハイブリドーマのよう
な浮遊性細胞とＣＨＯ細胞等の付着性細胞に大別される
が、浮遊性であっても株によっては付着性を持ち、細胞
同志あるいは器壁に対して付着し、細胞塊を形成しやす
い性状を持つものも多い。例えば、本来付着性であるよ
うな宿主細胞に対し、遺伝子組替を行って有用物質を生
産させる場合も、浮遊培養の方がスケールアップ性等工
業的物質生産において有効と考えられ、浮遊培養へ適用
することが多い。しかし、その場合上記のように培養中
その付着性によって培養槽内の器壁等へ付着する等して
細胞塊を形成しやすい。細胞塊は浮遊中に形成されるも
の、器壁等に付着して形成されるものがあり、生きた細
胞あるいは死んだ細胞により形成されているが、細胞塊
の周りの細胞をかき集めさらに大きな細胞塊へと成長
し、培養液中のばらばらで浮遊状態にある適性細胞の濃
度は減少する。細胞塊が生きた細胞である場合、物質生
産性が低下したり、酸素や栄養が行き届かず中の細胞か
ら死滅してしまう。また、培養液交換手段においても濾
過手段の目詰まりを起こす等、その妨げとなるという問
題がある。On the other hand, animal cells are roughly classified into floating cells such as hybridomas and adherent cells such as CHO cells. Many of them have the property that they are attached to and easily form a cell mass. For example, even when host cells that are originally adherent are genetically recombined to produce useful substances, suspension culture is considered to be more effective in industrial substance production such as scale-up. Often applied. However, in that case, as described above, due to the adhesiveness during the culture, it is likely to adhere to the vessel wall or the like in the culture tank to form a cell mass. Some cell clusters are formed in suspension, some are formed by attaching to the vessel wall, etc., and are formed by living or dead cells. Larger cells are collected by scraping cells around the cell cluster. It grows into clumps and the concentration of loose, floating aptitude cells in the culture is reduced. If the cell mass is a living cell, the productivity of the substance will be reduced, and oxygen and nutrients will not be fully delivered and the cell will die from the cell. In addition, there is a problem in that the culture medium exchange means also interferes with the clogging of the filtration means.
【０００６】従来の培養技術ではそういった細胞に対し
て、より良好な状態での連続培養を行うため、培養中形
成された細胞塊を除去する手段を設けた培養装置も開発
されている。例えば、公開特許公報平１−２０６９８８
号には培養槽に付設される培養液循環路に設けられた濾
過手段により、培養液中に浮遊する細胞塊を除去し適性
細胞は培養槽へ戻すことで培養を継続するものが開示さ
れている。この装置によれば浮遊する細胞塊は有効に除
去することはできるが、培養槽内の器壁等へ付着して形
成された細胞塊に対しての配慮はなされておらず、付着
して存在する細胞塊の除去は依然として課題として残さ
れている。In the conventional culturing technology, in order to carry out continuous culturing of such cells in a better condition, a culturing apparatus provided with a means for removing cell aggregates formed during culturing has been developed. For example, Japanese Unexamined Patent Publication No. 1-206988
No. 1 discloses that culture is continued by removing the cell mass floating in the culture solution and returning the suitable cells to the culture tank by a filtering means provided in the culture solution circulation path attached to the culture tank. There is. With this device, floating cell clusters can be effectively removed, but no consideration is given to cell clusters that have formed by adhering to the vessel walls in the culture tank. The removal of the resulting cell mass remains an issue.
【０００７】[0007]
【発明が解決しようとする課題】上記のように、付着性
を持ち細胞塊を形成しやすい性状を持つ細胞によって、
従来の浮遊培養で連続培養を行なう場合、濾過手段によ
って培養液中の細胞塊はある程度除去しつつ培養を行う
ことができるが、培養槽の器壁等に付着した細胞塊はそ
のまま成長を続けることから、前記のように連続培養に
対する支障は完全には解決していない。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention As described above, by the cells having the property of being adherent and easily forming a cell mass,
When continuous culture is performed by conventional suspension culture, it is possible to perform the culture while removing the cell clumps in the culture solution to some extent by the filtration means, but the cell clumps attached to the vessel wall of the culture tank should continue to grow. Therefore, as described above, the obstacle to continuous culture has not been completely solved.
【０００８】本発明の目的は、細胞塊を形成しやすい性
状を持つ細胞であっても、適正な細胞を保持した状態
で、細胞塊特に培養槽内の器壁等へ付着する等して形成
した細胞塊をも選択的に除去しつつ、良好な状態で連続
的に培養することができる培養方法と培養装置を提供す
ることにある。The object of the present invention is to form even a cell having a property of easily forming a cell mass by attaching it to a cell mass, in particular, a vessel wall in a culture tank, etc., while holding an appropriate cell. It is an object of the present invention to provide a culturing method and a culturing device capable of continuously culturing in a good state while selectively removing the formed cell mass.
【０００９】[0009]
【課題を解決するための手段】上記目的を達成するため
に、本発明による動物細胞を培養する方法は、基本的に
培養の際細胞の一部を排出しながら連続的に培養を行う
培養方法であり、その培養の途中で間歇的に、必要量の
適正細胞を含む培養液を培養槽と連結した別槽に一時保
留する工程と、培養槽あるいは培養槽内培養液に振動を
生じさせ培養槽内の器壁等に付着した細胞塊を剥離させ
る工程と、剥離した細胞塊を培養槽に残留した培養液と
共に培養槽外に排出する工程と、及び前記保留した培養
液を培養槽に戻し培養を継続する工程、とを有する。In order to achieve the above object, the method of culturing animal cells according to the present invention is basically a culturing method of continuously culturing while discharging a part of cells during culturing. The process of temporarily suspending the culture solution containing the required amount of appropriate cells in a separate tank connected to the culture tank during the culture, and culturing by shaking the culture tank or the culture solution in the culture tank The step of peeling off the cell clumps adhering to the vessel walls in the tank, the step of discharging the separated cell clumps together with the culture solution remaining in the culture tank to the outside of the culture tank, and returning the suspended culture solution to the culture tank And a step of continuing the culture.
【００１０】また、本発明による動物細胞培養装置は基
本的に、少なくとも、１）培養液の一部を取り出す手段
と残りの培養液を排出する手段とを有する培養槽、２）
前記培養槽及び／又は培養槽内の培養液に振動を生じさ
せる手段、及び３）前記培養液の一部を取り出す手段と
取り出された培養液を培養槽に戻す手段とに無菌的に接
続した細胞一時保留槽、とを有している。The animal cell culture device according to the present invention basically has at least 1) a culture tank having a means for taking out a part of the culture solution and a means for discharging the remaining culture solution, 2)
Aseptically connected to the culture tank and / or means for generating vibration in the culture solution in the culture tank, and 3) means for extracting a part of the culture solution and means for returning the removed culture solution to the culture tank. It has a cell temporary storage tank.
【００１１】本発明によれば、浮遊してあるいは器壁等
に付着して成長した大きな細胞塊を培養系外に除去し、
適正細胞すなわち細胞塊を形成しておらずばらばらで浮
遊状態にある細胞及び細胞塊のうち極小なものを必要な
量だけ、培養槽に残して培養することにより培養が継続
される。そのためには、適正細胞を含む培養液を必要な
量だけ、一時的に培養槽から無菌的に接続された別の槽
に移し、その後培養槽に残留した細胞塊を培養液と共に
更に別に設けた槽に排出し、先の一時的に保留している
適正細胞を含む培養液を培養槽に戻し、培養を継続する
ものである。According to the present invention, a large cell mass that has grown floating or attached to a vessel wall or the like is removed from the culture system,
Cultivation is continued by culturing the appropriate cells, i.e., cells and cell aggregates that do not form cell aggregates and are in a floating state and are extremely small, in the required amount in the culture tank. For that purpose, the required amount of culture solution containing appropriate cells was temporarily transferred from the culture tank to another tank that was aseptically connected, and then the cell mass remaining in the culture tank was provided separately together with the culture solution. The culture solution containing the appropriate cells temporarily discharged is returned to the culture tank, and the culture is continued.
【００１２】また、培養槽に残留した細胞塊を完全に排
出するために、培地あるいは生体の細胞に適した浸透圧
及びｐＨに調整した緩衝液等を注入して槽内を洗浄した
後に保留している培養液を培養槽に戻し、培養を継続す
ることは好ましい態様である。また、器壁等に付着して
成長してしまった大きな細胞塊を除去するために、まず
培養槽自体あるいは培養液に振動を与えるか、又は強度
の機械攪拌、あるいは強攪拌と停止とを数回繰返すこと
により器壁等より細胞塊を剥離させ、付着した細胞塊を
剥離させた状態で除去するようにしてもよい。若しくは
必要な細胞を回収後、洗浄用の培地あるいは緩衝液を培
養槽に満たし、培養槽に振動又は超音波を与えるか、又
は強度の機械攪拌、あるいは強攪拌と停止とを数回繰返
すことにより、細胞塊を破壊し完全に剥離させ、除去す
るようにしてもよい。Further, in order to completely discharge the cell mass remaining in the culture tank, a buffer solution or the like adjusted to an osmotic pressure and pH suitable for the medium or living cells is injected and the tank is washed and then suspended. It is a preferred embodiment that the culture solution is returned to the culture tank and the culture is continued. Further, in order to remove a large cell mass that has adhered and grown on a vessel wall or the like, first, vibrate the culture tank itself or the culture solution, or perform strong mechanical stirring or strong stirring and stopping. The cell mass may be peeled off from the vessel wall or the like by repeating the cycle, and the adhered cell mass may be removed in a peeled state. Alternatively, after collecting the required cells, the culture medium or buffer solution for washing is filled in the culture tank, and the culture tank is subjected to vibration or ultrasonic waves, or by vigorous mechanical stirring, or by repeating strong stirring and stopping several times. Alternatively, the cell mass may be destroyed and completely removed to be removed.
【００１３】また、細胞塊を形成していないばらばらの
適正細胞と、成長した巨大な細胞塊とを分離するために
は、培養槽の攪拌を一時停止して、巨大に成長した細胞
塊のみを沈降させうる時間、細胞及び細胞塊を含む培養
液を静置し、それにより適正細胞を含む上澄み培養液
を、沈降した巨大細胞塊を抜き出してしまわない高さに
開口部を設定した配管より抜き出し、別槽に一時保留す
るようにしてもよい。In order to separate the proper cells that have not formed cell clusters from the grown giant cell clusters, stirring of the culture tank is temporarily stopped and only the giant cell clusters are grown. Leave the culture medium containing cells and cell aggregates for a period of time to allow them to settle, and then extract the supernatant culture medium containing appropriate cells from the pipe that has an opening at a height that does not extract the sedimented giant cell clusters. Alternatively, it may be temporarily held in another tank.
【００１４】また、適正細胞を含む培養液を必要な量だ
け保留槽に移すためには、培養槽の底面から、特定量の
培養液を抜き出すに十分な高さであり、かつ沈降させた
巨大細胞塊を抜き出してしまうに至らない高さだけ離れ
た位置に開口部を持つ配管を設けることにより、特定量
抜き出すようにすることが好ましい。更に、培養系外に
排出する残留した培養液及び巨大細胞塊及び洗浄液をほ
ぼ完全に抜き出す為には、培養槽の底面から、残留した
培養液をほぼ完全に抜き出すに十分であり、かつ巨大細
胞塊を抜き出すに十分な高さだけ離れた位置に開口部を
持つ配管より、残留した培養液及び細胞塊を抜き出すよ
うにすることが好ましい。Further, in order to transfer a required amount of the culture solution containing the appropriate cells to the holding tank, the culture solution is of a height sufficient to withdraw a specific amount of the culture solution from the bottom of the culture tank, and the sedimented giant solution is used. It is preferable to extract a specific amount by providing a pipe having an opening at a position separated by a height that does not cause the cell mass to be extracted. Furthermore, in order to almost completely remove the residual culture solution and the giant cell mass and the washing solution discharged to the outside of the culture system, it is sufficient to almost completely remove the remaining culture solution from the bottom of the culture tank, and It is preferable to extract the remaining culture solution and cell clumps from a pipe having an opening at a position separated by a height sufficient to extract the clumps.
【００１５】さらに、培養系外に排出する、残留した培
養液及び巨大細胞塊及び洗浄液をほぼ完全に抜き出すた
めには、培養槽の底面から、残留した培養液をほぼ完全
に抜き出すに十分であり、かつ巨大細胞塊を抜き出すに
十分な高さだけ離れた位置に開口部を持つ配管より、残
留した培養液及び細胞塊を抜き出すようにすることが好
ましい。Further, in order to almost completely withdraw the remaining culture solution and the giant cell mass and the washing solution discharged to the outside of the culture system, it is sufficient to almost completely withdraw the remaining culture solution from the bottom of the culture tank. Moreover, it is preferable to extract the remaining culture solution and cell mass from a pipe having an opening at a position separated by a height sufficient to extract the giant cell mass.
【００１６】また、細胞又は細胞塊を含む培養液を、培
養槽から保留槽又は廃棄細胞の貯留槽へと無菌的に移動
させるためには、一時保留槽又は廃棄細胞の貯留槽を培
養槽とチューブで連結し、一時保留槽あるいは廃棄槽側
を陰圧にあるいは加圧することにより、細胞を培養槽か
ら一時保留槽へあるいは一時保留槽から培養槽へと移動
させることもできる。あるいは、連結するチューブの間
にポンプを組み込み、ポンプにより細胞を培養槽から一
時保留槽へあるいは一時保留槽から培養槽へと移動する
ようにしてもよい。In order to aseptically move the culture solution containing cells or cell clusters from the culture tank to the holding tank or the waste cell storage tank, the temporary holding tank or the waste cell storage tank is used as the culture tank. It is also possible to transfer cells from the culture tank to the temporary storage tank or from the temporary storage tank to the culture tank by connecting with a tube and applying negative pressure or pressurizing the temporary storage tank or the waste tank side. Alternatively, a pump may be incorporated between the tubes to be connected, and the cells may be moved from the culture tank to the temporary storage tank or from the temporary storage tank to the culture tank by the pump.
【００１７】[0017]
【作用】本発明においては、培養中、浮遊してあるいは
器壁等に付着して成長した大きな細胞塊が培養系外に除
去されることで、周囲の細胞を付けながら成長する細胞
塊がそれ以上大きな細胞塊に成長することが防がれ、細
胞塊の成長による培養液中の浮遊細胞濃度の減少が抑え
られる。さらに細胞塊の形成によって起こっていた物質
生産性の低下が改善される。In the present invention, a large cell mass that grows while floating or adheres to a vessel wall or the like is removed out of the culture system during culture, so that a cell mass that grows with surrounding cells It is possible to prevent the growth of a large cell mass as described above, and to suppress a decrease in the concentration of suspended cells in the culture medium due to the growth of the cell mass. Further, the decrease in substance productivity caused by the formation of cell clusters is improved.
【００１８】培養槽から無菌的に接続された別の槽は、
細胞塊を形成していないばらばらの細胞や細胞塊のうち
極小さなものを必要な量だけ、培養槽から移して一時的
に保留し、更に別に設けた培養槽から無菌的に接続され
た槽は、培養槽に残留した不要な細胞及び巨大な細胞塊
を廃棄し貯留する。器壁等に付着して成長してしまった
大きな細胞塊に対して、培養槽自体に振動を与えるか、
又は強度の機械攪拌、あるいは強攪拌と停止とを数回繰
返すことにより、付着した大きな細胞塊は器壁等より剥
離する。更には必要な細胞を回収後、洗浄用の培地ある
いは生体の細胞に適した浸透圧及びｐＨに調整した緩衝
液を培養槽に満たし、培養槽に振動及び超音波を与える
か、又は強度の機械攪拌、あるいは強攪拌と停止とを数
回繰返すことにより、細胞塊は破壊され完全に剥離させ
る。特に生体の細胞に適した浸透圧及びｐＨに調整した
リン酸緩衝液（ＰＢＳ）を満たすことにより細胞塊はよ
り剥がれやすくなる。以上により効果的に細胞塊を除去
することができる。Another tank aseptically connected from the culture tank is
A very small amount of loose cells or cell aggregates that do not form cell aggregates are temporarily transferred from the culture tank and temporarily retained, and a tank that is aseptically connected from a separate culture tank is used. , Discard and store unnecessary cells and huge cell clusters remaining in the culture tank. Whether the culture tank itself is vibrated for a large cell mass that has grown by attaching to the vessel wall,
Alternatively, the adhered large cell mass is peeled off from the vessel wall or the like by repeating mechanical stirring with high intensity, or repeating strong stirring and stopping several times. Furthermore, after collecting the necessary cells, the culture medium is filled with a washing medium or a buffer solution adjusted to an osmotic pressure and pH suitable for living cells, and the culture vessel is subjected to vibration and ultrasonic waves, or a mechanical strength By agitating or repeating agitation and vigorous agitation several times, the cell mass is destroyed and completely detached. Particularly, by filling a phosphate buffer solution (PBS) adjusted to an osmotic pressure and a pH suitable for cells of a living body, the cell mass becomes more likely to come off. As described above, the cell mass can be effectively removed.
【００１９】また、培養槽の攪拌を一時停止して細胞及
び細胞塊を含む培養液を静置すると、巨大に成長した細
胞塊は、浮遊状態のばらばらの細胞及び極小さな細胞塊
に比べ、沈降速度が早いので、細胞塊のみが沈降した段
階で、細胞及び極小さな細胞塊が浮遊状態の上澄み液を
別槽に抜き出すことで、浮遊状態の細胞を選択的に回収
することができる。When the agitation of the culture tank is temporarily stopped and the culture solution containing cells and cell aggregates is left to stand still, the hugely grown cell aggregates are sedimented as compared to the floating discrete cells and extremely small cell aggregates. Since the speed is high, the floating cells can be selectively collected by extracting the supernatant of the floating cells and the extremely small cell clusters into a separate tank when only the cell clusters have settled.
【００２０】また、培養液のグルコース濃度より算出さ
れるグルコース消費速度が細胞濃度に対して所定の比例
関係にある場合には、培養液中に浮遊して計数される細
胞数が正確であり、細胞の付着及び細胞塊の形成が進ん
でいないが、これより外れた場合、細胞の付着及び細胞
塊の形成が進んでいるため、付着細胞の剥離及び細胞塊
の除去作業が必要となる。すなわち、グルコース消費速
度と細胞濃度との関係を検知することで、より効果的な
タイミングで付着細胞の剥離及び細胞塊の除去操作を行
うことができる。When the glucose consumption rate calculated from the glucose concentration of the culture medium is in a predetermined proportional relationship with the cell concentration, the number of cells suspended and counted in the culture medium is accurate, Although the attachment of cells and the formation of cell aggregates have not progressed, but when the amount of the cells is out of this range, the attachment of cells and the formation of cell aggregates have progressed, and therefore the work of detaching adherent cells and removing the cell aggregates becomes necessary. That is, by detecting the relationship between the glucose consumption rate and the cell concentration, the detachment of adherent cells and the removal of cell aggregates can be performed at more effective timing.
【００２１】以上のように、細胞塊を形成しやすい性状
を持つ浮遊性細胞でも、形成された細胞塊が除去され、
選択的に浮遊化した細胞を残し培養及び生産に良好な状
態で培養を継続することができる。As described above, even in the floating cells having the property of easily forming cell clusters, the formed cell clusters are removed,
It is possible to continue the culture in a favorable state for the culture and the production while leaving the selectively suspended cells.
【００２２】[0022]
〔実施例１〕図１は、本発明によるによる灌流培養装置
の実施例１を概略的に示した説明図であり、小スケール
のスピンナフラスコである培養槽１は恒温槽２中に設置
される。培養槽１はこれを密閉し得る着脱可能な蓋体４
を有し、該蓋体４には攪拌子３、２本の液面レベルセン
サ７、７、通気管１３、排気管１４、サンプリング管１
６、及び後述する廃液用管路６ａ及び回収細胞用管路６
ｂとが、それぞれ所定の位置に設定されている。通気管
１３及び排気管１４には除菌フィルタ１５、１５が設け
られ、また、液面レベルセンサ７、７は最高位液面レベ
ル及び最低位液面レベルを制御するものでありレベルコ
ントローラ８に接続している。[Example 1] Fig. 1 is an explanatory view schematically showing Example 1 of a perfusion culture apparatus according to the present invention, in which a culture tank 1 which is a small-scale spinner flask is installed in a constant temperature tank 2. . The culture tank 1 is a removable lid 4 capable of sealing the culture tank 1.
The lid 4 has a stirrer 3, two liquid level sensors 7, 7, a ventilation pipe 13, an exhaust pipe 14, and a sampling pipe 1.
6, and a pipeline 6a for waste liquid and a pipeline 6 for recovered cells, which will be described later.
b and b are set at predetermined positions. The ventilation pipe 13 and the exhaust pipe 14 are provided with sterilization filters 15 and 15, and the liquid level sensors 7 and 7 are for controlling the highest liquid level and the lowest liquid level, and the level controller 8 Connected.
【００２３】廃液用管路６ａは、培養槽１内においてそ
の底部近傍まですなわち培養槽１の底面から残留した培
養液をほぼ完全に抜き出すに十分でありかつ巨大細胞塊
を抜き出すに十分な高さだけ離れた位置にその一方端を
延出し、他方端は培養槽１とは別個に配置された廃液槽
１０に開口している。回収細胞用管路６ｂはその一方端
を培養槽１内に培養液が注入されたときの液面より幾分
下方位置となる位置まで、すなわち培養槽の底面から特
定量の培養液を抜き出すに十分な高さでありかつ沈降さ
せた巨大細胞塊を抜き出してしまうに至らない高さだけ
離れた位置まで延出しており、他方端は培養槽１とは別
個に配置された回収細胞のための一時保留槽９に開口し
ている。The waste liquid conduit 6a has a height sufficient to almost completely extract the remaining culture solution from the bottom of the culture tank 1 to the vicinity of the bottom of the culture tank 1, that is, to extract a giant cell mass. One end thereof is extended to a position separated by a distance, and the other end is opened to a waste liquid tank 10 arranged separately from the culture tank 1. The collected cell conduit 6b has one end thereof at a position slightly lower than the liquid level when the culture solution is injected into the culture tank 1, that is, for extracting a specific amount of the culture solution from the bottom surface of the culture tank. It has a sufficient height and extends to a position separated by a height that does not allow the sedimented giant cell mass to be extracted, and the other end is for collecting cells arranged separately from the culture tank 1. It is open to the temporary holding tank 9.
【００２４】培養槽１外には新鮮培地槽５及び洗浄液槽
（緩衝液槽）１２が位置しており、それぞれポンプ１
１、１１及び配管６ｃを介して培養槽１内に連通してい
る。新鮮培地槽５に接続したポンプ１１には液面レベル
センサ７、７からの情報がレベルコントローラ８を介し
て伝達される。また、上記した廃液槽１０、回収細胞一
時保留槽９、新鮮培地槽５、及び洗浄液槽１２にはそれ
ぞれ除菌フィルタ１５、１５を介して大気側に連通して
いる。Outside the culture tank 1, a fresh medium tank 5 and a washing solution tank (buffer solution tank) 12 are located.
It communicates with the inside of the culture tank 1 through 1, 11 and a pipe 6c. Information from the liquid level sensors 7, 7 is transmitted to the pump 11 connected to the fresh medium tank 5 via the level controller 8. In addition, the waste liquid tank 10, the recovered cell temporary holding tank 9, the fresh medium tank 5, and the washing liquid tank 12 are connected to the atmosphere side through sterilizing filters 15 and 15, respectively.
【００２５】この実施例において、培養槽１は、恒温槽
２内に位置する振動発生器１７により外側から包持され
ており、振動発生器１７の作動により培養槽１には振動
が与えられる。この灌流培養装置の作動に当たって、新
鮮培地槽５からポンプ１１及び管路６ｃを介して新鮮培
地を培養槽１内に導入しかつ細胞を導入し、恒温槽２等
の培養環境を整えたのち、培養を開始する。その間に必
要に応じて攪拌子３により培養液の攪拌を行う。In this embodiment, the culture tank 1 is wrapped from the outside by a vibration generator 17 located in the constant temperature tank 2, and the operation of the vibration generator 17 gives vibration to the culture tank 1. In the operation of this perfusion culture device, after introducing the fresh medium from the fresh medium tank 5 into the culture tank 1 via the pump 11 and the conduit 6c and introducing the cells, the culture environment such as the constant temperature tank 2 is prepared, Start culturing. In the meantime, the culture solution is stirred by the stirrer 3 as needed.
【００２６】培養の経過と共に培養液中に浮遊したある
いは培養槽１の器壁に付着した細胞塊が発生する。後記
するように例えば図示しないグルコースセンサからの情
報により細胞塊の除去が必要となったと判断された時点
において、攪拌子３の作動を一時停止し、細胞塊を培養
槽１の底部に沈降させる。その後、回収細胞一時保留槽
９をポンプ１１により陰圧状態にし、適正細胞を含む上
澄み培養液を回収細胞用管路６ｂを介して吸引して一時
保留槽９に一時保留する。As the culture progresses, cell aggregates floating in the culture medium or adhering to the vessel wall of the culture tank 1 are generated. As will be described later, when it is determined that the cell mass needs to be removed based on information from a glucose sensor (not shown), the operation of the stirring bar 3 is temporarily stopped and the cell mass is allowed to settle on the bottom of the culture tank 1. After that, the collected cell temporary holding tank 9 is brought into a negative pressure state by the pump 11, and the supernatant culture solution containing appropriate cells is sucked through the collected cell pipe 6b and temporarily held in the temporary holding tank 9.
【００２７】次に、必要に応じて洗浄槽１２からの洗浄
液（緩衝液）を培養槽１内に満たした後、振動発生装置
１７を作動させて培養槽１に振動を与え、器壁等に付着
して成長した大きな細胞塊を機壁から剥離させる。剥離
した細胞塊と浮遊状態にある細胞塊とをポンプ１１を作
動させて残りの培養液と共に管路６ａを介して廃液槽１
０に排出し培養槽１を空の状態とする。Next, if necessary, after the cleaning solution (buffer solution) from the cleaning tank 12 is filled in the culture tank 1, the vibration generator 17 is activated to vibrate the culture tank 1 to cause the culture chamber 1 to vibrate. The large cell mass that has adhered and grown is detached from the machine wall. The pump 11 is operated to operate the separated cell mass and the floating cell mass together with the rest of the culture solution via the conduit 6a to the waste liquid tank 1
It is discharged to 0 and the culture tank 1 is emptied.
【００２８】なお、機壁等に付着した細胞塊の付着力が
小さいものである場合には、前記振動発生装置１７を作
動させずに、攪拌子３の強攪拌と一時停止の操作を繰り
返えすことにより付着した細胞塊を剥離するようにして
もよい。また、付着細胞の種類によっては洗浄液を導入
しなくても初期の目的を達成することができる場合もあ
り得る。If the cell clusters adhering to the machine wall have a small adhesive force, the vibrating device 17 is not operated and the agitating operation of the agitator 3 is repeated. The cell mass that has adhered may be removed by removing it. Further, depending on the type of adherent cells, it may be possible to achieve the initial purpose without introducing a washing solution.
【００２９】その後、配管６ｂに配置したポンプ１１を
作動して、先に保留しておいた上澄み液中の適正細胞を
一時保留槽９から培養槽１に戻す。排出した分の培養液
の補充は新鮮培地槽５より配管６ｃを介して新鮮培地を
培養槽１に注入することで行う。液量はレベルセンサ７
及びレベルコントローラ８により制御される。なお、通
気及び排気は、通気管１３及び排気管１４より除菌フィ
ルタ１５を通して行われる。Thereafter, the pump 11 arranged in the pipe 6b is operated to return the appropriate cells in the supernatant liquid previously reserved from the temporary storage tank 9 to the culture tank 1. The discharged culture medium is replenished by injecting the fresh medium into the culture tank 1 from the fresh medium tank 5 through the pipe 6c. Level sensor 7
And level controller 8. The ventilation and the exhaust are performed through the sterilization filter 15 from the ventilation pipe 13 and the exhaust pipe 14.
【００３０】以上の操作を培養の途中で間歇的に行うこ
とによって、細胞塊を形成しやすい性状を持つ浮遊性細
胞でも、形成された細胞塊が除去され、選択的に浮遊化
した細胞を残し培養及び生産に良好な状態で培養を継続
することが可能となる。なお、既に記したように、付着
した細胞塊を器壁より剥離するための洗浄液には、培地
あるいは生体の細胞に適した浸透圧及びｐＨに調整した
緩衝液を用いることが好ましく、特に迅速さが要求され
る培養中の洗浄操作においてはリン酸緩衝液（ＰＢＳ）
が好ましい。By performing the above operation intermittently during the culturing, even in the case of floating cells having the property of easily forming cell clusters, the formed cell clusters are removed and the selectively suspended cells remain. It becomes possible to continue culturing in a good condition for culturing and production. As described above, it is preferable to use a buffer solution adjusted to an osmotic pressure and pH suitable for the medium or cells of the living body as the washing solution for peeling the adhered cell mass from the vessel wall. Phosphate buffer solution (PBS) in washing operation during culture
Is preferred.
【００３１】表１は浮遊化ＣＨＯ細胞を用いて行った培
地及びＰＢＳによる付着細胞の剥離実験の結果を示して
いる。本細胞は付着性であるＣＨＯ細胞を無血清でスク
リーニングし浮遊化させたものであるが、器壁に対し
て、また細胞同志で非常に付着しやすい。実験でφ35デ
ィッシュを用い、37.5時間培養後培養液を除去し、ディ
ッシュ底面に付着した細胞に対し洗浄液（培地、Ｐ
ＢＳ）を注入し、一定時間（10分、40分）、(a) 振盪
（振幅２cm、100 ストローク/min) 、あるいは(b)静置
して、洗浄液中に剥離した細胞とディッシュ底面に残っ
た細胞数を計測した。表中、カッコ内に全付着細胞数に
対する割合を示した。Table 1 shows the results of the detachment experiment of adherent cells by the medium and PBS performed using the suspended CHO cells. The present cells are adherent CHO cells screened and suspended in serum-free, but they are very likely to adhere to the organ wall and between cells. In the experiment, a φ35 dish was used, and after the culture for 37.5 hours, the culture solution was removed, and a washing solution (medium, P was added to the cells attached to the bottom of the dish.
(BS) is injected for a certain period of time (10 minutes, 40 minutes), (a) Shaking (amplitude 2 cm, 100 strokes / min), or (b) Allowing to stand, leaving the cells detached in the washing solution and the bottom of the dish. The number of cells was measured. In the table, the ratio to the total number of adherent cells is shown in parentheses.
【００３２】その結果、特に短期間振盪を行う場合で
は、培地では36％剥離したのに対してＰＢＳでは67％が
剥離し、ＰＢＳの方がより効果的であるが、長時間経過
すると培地、ＰＢＳで効果はほぼ同程度となった。従っ
て、迅速さが要求される培養中の洗浄操作においては、
ＰＢＳを用いることが好ましいことがわかる。As a result, particularly when shaking was performed for a short period of time, 36% of the medium was peeled off, whereas 67% of PBS was peeled off, and PBS is more effective. The effect was almost the same with PBS. Therefore, in the washing operation during culturing, which requires quickness,
It turns out that it is preferable to use PBS.
【００３３】[0033]
【表１】 [Table 1]
【００３４】〔実施例２〕図２は本発明による培養装置
の実施例２を概略的に示した説明図であり、培養槽１に
振動を与える手段として培養槽１を従来知られた振盪機
１８に固定したものである。他の構成は図１の実施例の
ものと同様であり、この実施例においても必要に応じて
洗浄用の培地等を満たした後、適正細胞を一時保留槽９
に一時保留した後、前記振盪機１８を作用させて細胞塊
を剥離する。 〔実施例３〕図３は本発明による培養装置の実施例３を
概略的に示した説明図であり、サンプリング管１６の途
中にさらにグルコースセンサ１９を取り付け、培養液の
グルコース濃度をグルコースアナライザ２０により分析
できるようにしたものである。前記のように、培養液の
グルコース濃度より算出されるグルコース消費速度が細
胞濃度に対して所定の比例関係にある場合には、培養液
中に浮遊して計数される細胞数は正確であり、細胞の付
着及び細胞塊の形成が進んでいないが、これより外れた
場合、細胞の付着及び細胞塊の形成が進んでいることと
なるため、この実施例のものにおいてはグルコース消費
速度と細胞濃度との関係を検知することで、より効果的
なタイミングで付着細胞の剥離及び細胞塊の除去操作を
行うことが可能となる。 〔実施例４〕図４は本発明による培養装置の実施例４を
概略的に示した説明図である。全体としてはエアリフト
型の灌流培養装置であり、培養槽４１に超音波を与える
手段を具備したことを特徴とする。図において、４１は
エアリフト型培養槽であり、上槽４１ａ、上槽４１ａに
細管部を介して連通する下槽４１ｂ、及び上槽４１ａと
下槽４１ｂを連通するバイパス管路４１ｃとから構成さ
れる。上槽４１ａは恒温槽ジャケット４２を具備してお
り、さらに、上槽４１ａにはこれを密閉しうる着脱可能
な蓋体４４が設けられ、該蓋体４４には２本の液面レベ
ルセンサ７、７、排気管１４、及び温度センサ２４とが
それぞれ所定の位置に設定されている。排気管１４には
除菌フィルタ１５が設けられ、また、液面レベルセンサ
７、７はレベルコントローラ８に接続している。[Embodiment 2] FIG. 2 is an explanatory view schematically showing Embodiment 2 of the culturing apparatus according to the present invention. As a means for vibrating the culturing tank 1, the culturing tank 1 is a conventionally known shaker. It is fixed to 18. The other structure is the same as that of the embodiment of FIG. 1, and in this embodiment as well, after filling a washing medium and the like as needed, appropriate cells are temporarily retained in the tank 9.
Then, the shaker 18 is actuated to separate the cell mass. [Embodiment 3] FIG. 3 is an explanatory view schematically showing Embodiment 3 of the culture apparatus according to the present invention. A glucose sensor 19 is further attached in the middle of the sampling tube 16, and the glucose concentration of the culture solution is measured by a glucose analyzer 20. It can be analyzed by. As described above, when the glucose consumption rate calculated from the glucose concentration of the culture medium has a predetermined proportional relationship to the cell concentration, the number of cells suspended and counted in the culture medium is accurate, The cell attachment and the formation of the cell mass have not progressed, but if the cell detachment is out of this range, the cell attachment and the formation of the cell mass have progressed. Therefore, in this example, the glucose consumption rate and the cell concentration were By detecting the relationship with, it becomes possible to perform the detachment of adherent cells and the removal of cell mass at more effective timing. [Embodiment 4] FIG. 4 is an explanatory view schematically showing Embodiment 4 of the culture apparatus according to the present invention. The whole is an air-lift type perfusion culture device, and is characterized in that it is provided with means for applying ultrasonic waves to the culture tank 41. In the figure, reference numeral 41 is an air-lift type culture tank, which is composed of an upper tank 41a, a lower tank 41b communicating with the upper tank 41a via a narrow tube portion, and a bypass conduit 41c connecting the upper tank 41a and the lower tank 41b. It The upper tank 41a is provided with a constant temperature tank jacket 42, and further, the upper tank 41a is provided with a detachable lid member 44 capable of sealing the same, and the lid member 44 has two liquid level sensors 7 , 7, the exhaust pipe 14, and the temperature sensor 24 are set at predetermined positions. The exhaust pipe 14 is provided with a sterilizing filter 15, and the liquid level sensors 7, 7 are connected to a level controller 8.
【００３５】さらに、上槽４１ａの側壁部から上槽４１
ａ内部に向けてＤＯ・ｐＨセンサ２３、及びサンプリン
グ管１６が延出しており、サンプリング管の途中に設け
られたグルコースセンサ１９はグルコールアナライザ２
０に、ＤＯ・ｐＨセンサ２３は後述するコントローラ２
５に接続している。下槽４１ｂには細胞分離フィルタ２
８が配置されていると共に、最下方には通気ノズル１８
が設けられ該通気ノズル１８は図示しない適宜の送気源
に接続している。通気ノズル１８に近接してその上方に
は廃液用管路６ａの一端が開口しておりその他方端は廃
液槽１０に開口している。さらに、下槽４１ｂの上方部
には回収細胞用管路６ｂの一方端が特定量の培養液を抜
き出すに十分な高さでありかつ沈降させた巨大細胞塊を
抜き出してしまうに至らない位置まで延出して設けられ
ており、他方端は回収細胞のための一時保留槽９に開口
している。各管路にはポンプ１１が配置されている。Further, from the side wall of the upper tank 41a to the upper tank 41a
The DO / pH sensor 23 and the sampling tube 16 extend toward the inside of the a, and the glucose sensor 19 provided in the middle of the sampling tube is a glucose analyzer 2
The DO / pH sensor 23 is a controller 2 which will be described later.
Connected to 5. The cell separation filter 2 is provided in the lower tank 41b.
8 is arranged, and the ventilation nozzle 18 is provided at the bottom.
Is provided and the ventilation nozzle 18 is connected to an appropriate air supply source (not shown). One end of the waste liquid pipe 6a is opened in the vicinity of and close to the ventilation nozzle 18, and the other end is opened to the waste liquid tank 10. Further, in the upper part of the lower tank 41b, one end of the recovered cell conduit 6b is at a position that is high enough to extract a specific amount of the culture solution and does not lead to the extraction of the sedimented giant cell mass. It is provided so as to extend, and the other end is opened to a temporary storage tank 9 for collected cells. A pump 11 is arranged in each pipeline.
【００３６】また、下槽４１ｂの下方部には管路６ｃの
一端が開口しており、その他端は培養槽４１外に設けら
れた新鮮培地槽５、培養ろ液槽６、及び洗浄液槽（緩衝
液槽）１２にそれぞれポンプ１１、１１を介して連通し
ている。新鮮培地槽５及び培養ろ液槽６に接続したポン
プ１１には液面レベルセンサ７、７からの情報がレベル
コントローラ８を介して伝達される。Further, one end of the conduit 6c is opened at the lower portion of the lower tank 41b, and the other end is provided with a fresh medium tank 5, a culture filtrate tank 6, and a washing liquid tank (outside the culture tank 41). The buffer solution tank 12 is in communication with each other via pumps 11 and 11. Information from the liquid level sensors 7, 7 is transmitted to the pump 11 connected to the fresh medium tank 5 and the culture filtrate tank 6 via the level controller 8.
【００３７】また、上記した廃液槽１０、一時保留槽
９、新鮮培地槽５、培養ろ液槽６、及び洗浄液槽１２に
はそれぞれ除菌フィルタ１５、１５を介して大気側に連
通している。この実施例において、器壁等に付着して成
長してしまった大きな細胞塊を除去する手段として、培
養槽４１に超音波発振機２６とその端子２７組み込んで
いる。端子２７は上槽４１ａ及び下槽４１ｂに各１個設
置されているが、２つ以上であってもよい。Further, the waste liquid tank 10, the temporary holding tank 9, the fresh medium tank 5, the culture filtrate tank 6, and the washing liquid tank 12 are connected to the atmosphere side through sterilizing filters 15 and 15, respectively. . In this embodiment, the ultrasonic oscillator 26 and its terminal 27 are incorporated in the culture tank 41 as a means for removing large cell clusters that have adhered and grown on the vessel wall or the like. Although one terminal 27 is installed in each of the upper tank 41a and the lower tank 41b, the number of terminals 27 may be two or more.
【００３８】この培養槽４１においては、通気ノズル１
８より通気することにより、培養液の攪拌が行われる。
通気により生じる泡沫は上槽４１ａに上部に配置した消
泡ネット２２により除去される。また、ＤＯ・ｐＨセン
サ２３、温度センサ２４はコントーラ２５に接続してお
り、コントーラ２５は灌流培養時に培養状態をモニタリ
ングして温度、通気量を制御する。In this culture tank 41, the ventilation nozzle 1
By aeration from No. 8, the culture solution is stirred.
The foam generated by aeration is removed by the defoaming net 22 arranged on the upper part of the upper tank 41a. Further, the DO / pH sensor 23 and the temperature sensor 24 are connected to a controller 25, and the controller 25 controls the temperature and aeration rate by monitoring the culture state during perfusion culture.
【００３９】本実施例においては、培養の途中で、通気
攪拌を一時停止し、細胞塊を沈降させた後、適正細胞を
含む上澄み培養液を一時保留槽９に吸引して一時保留
し、培養槽４１に残った細胞塊を廃液槽１０に排出した
後、前述の適正細胞を一時保留槽９から培養槽４１に戻
し、培養を継続することができる。若しくは、細胞塊を
廃液槽１０に出した後に、洗浄用の緩衝液を注入し、通
気速度を強化し、強攪拌した後一時停止する操作を繰返
して、器壁等に付着して成長してしまった大きな細胞塊
を剥離し、その後、その細胞塊を含む洗浄液を排出し、
一時保留していた細胞を培養槽４１に戻し培養を継続す
ることもできる。In this example, aeration and agitation were temporarily stopped during the culture to settle the cell mass, and then the supernatant culture solution containing the appropriate cells was sucked into the temporary storage tank 9 and temporarily stored therein to perform the culture. After discharging the cell mass remaining in the tank 41 to the waste liquid tank 10, the appropriate cells described above can be returned from the temporary holding tank 9 to the culture tank 41 to continue the culture. Alternatively, after the cell clumps are taken out to the waste liquid tank 10, the washing buffer solution is injected, the aeration rate is enhanced, and the operation of temporarily stirring after vigorous stirring is repeated to grow by adhering to the vessel wall or the like. Peel off the large cell mass that has accumulated, then drain the washing solution containing the cell mass,
It is also possible to return the temporarily held cells to the culture tank 41 and continue the culture.
【００４０】若しくは、細胞塊を廃液槽１０に出した後
に、洗浄用の緩衝液を注入し、超音波発振機２６及びそ
の端子２７により緩衝液に超音波を与え、器壁等に付着
して成長してしまった大きな細胞塊を破壊、剥離し、そ
の後、その細胞塊を含む緩衝液を排出し、一時保留して
いた細胞を培養槽４１に戻し、培養を継続することがで
きる。Alternatively, after the cell clumps are taken out to the waste liquid tank 10, a washing buffer solution is injected, ultrasonic waves are applied to the buffer solution by the ultrasonic oscillator 26 and its terminal 27, and adhered to the vessel wall or the like. The large cell mass that has grown can be destroyed and peeled off, then the buffer solution containing the cell mass can be discharged, and the temporarily suspended cells can be returned to the culture tank 41 to continue the culture.
【００４１】以上の操作を培養の途中で間歇的に行うこ
とによって、細胞塊を形成しやすい性状を持つ浮遊性細
胞でも、形成された細胞塊が除去され、選択的に浮遊化
した細胞を残し培養及び生産に良好な状態で培養を継続
することが可能となる。 〔実施例５〕図５は本発明による培養装置を用いて医薬
品製造を製造するシステムを概略的に示すフロー図であ
る。図において、本発明による培養システム５１より回
収された培養液は分離、精製カラム５２を経て有用物質
が精製され、製剤システム５３において、製剤された医
薬品が製造される。By performing the above operation intermittently during the culturing, even in the case of floating cells having the property of easily forming cell clusters, the formed cell clusters are removed and the selectively suspended cells remain. It becomes possible to continue culturing in a good condition for culturing and production. [Embodiment 5] FIG. 5 is a flow chart schematically showing a system for manufacturing pharmaceutical products using the culture apparatus according to the present invention. In the figure, the culture solution recovered from the culture system 51 according to the present invention is separated and purified through a purification column 52 to purify useful substances, and in a formulation system 53, a formulated drug is manufactured.
【００４２】以上の説明は本発明の培養装置及び培養方
法の幾つかの実施例についての説明であって、他に多く
の変形例が存在する。例えは、図１、２に示した実施例
の場合であっても、浮遊する細胞塊を含む残りの培養液
をすべて廃液槽に出した後に、洗浄用の緩衝液を注入し
かつ振動を与えることにより器壁等に付着して成長した
大きな細胞塊を剥離し、その後、その細胞塊を含む洗浄
液を排出し、一時保留していた細胞を培養槽１に戻し、
培養を継続するようにしてもよく、また、そこに超音波
発振器を付設してもよいものである。The above description is for some examples of the culture apparatus and the culture method of the present invention, and there are many other modified examples. For example, even in the case of the embodiments shown in FIGS. 1 and 2, after the remaining culture solution containing floating cell aggregates is all taken out to the waste liquid tank, a washing buffer is injected and vibration is applied. By doing so, the large cell clumps that have grown by adhering to the vessel wall or the like are peeled off, then the washing solution containing the cell clumps is discharged, and the temporarily suspended cells are returned to the culture tank 1,
The culture may be continued, or an ultrasonic oscillator may be attached thereto.
【００４３】[0043]
【発明の効果】本発明によれば、細胞同志あるいは器壁
に対して付着し、細胞塊を形成しやすい性状を持つ細胞
でも、浮遊してあるいは器壁等に付着して成長した大き
な細胞塊を培養系外に除去し、細胞塊を形成していない
ばらばらの適正な細胞を選択して必要な量だけ、培養槽
に残して培養を継続することができるので、細胞塊を形
成しやすい性状を持つ細胞の連続灌流培養による物質生
産が可能となる。INDUSTRIAL APPLICABILITY According to the present invention, even a cell having a property of easily adhering to cells or organ walls and forming a cell mass is a large cell mass grown in suspension or adhering to the organ wall. Can be removed from the culture system, and appropriate cells that do not form cell clusters can be selected and left in the culture tank for the required amount to continue culturing. It is possible to produce a substance by continuous perfusion culture of cells having.
【図１】 本発明による培養装置の実施例１を概略的に
示した説明図である。FIG. 1 is an explanatory view schematically showing Example 1 of a culture device according to the present invention.
【図２】 本発明による培養装置の実施例２を概略的に
示した説明図である。FIG. 2 is an explanatory view schematically showing Example 2 of the culture device according to the present invention.
【図３】 本発明による培養装置の実施例３を概略的に
示した説明図である。FIG. 3 is an explanatory view schematically showing Example 3 of the culture device according to the present invention.
【図４】 本発明による培養装置の実施例４を概略的に
示した説明図である。FIG. 4 is an explanatory view schematically showing Example 4 of the culture device according to the present invention.
【図５】 本発明による医薬品製造システムを概略的に
示すフロー図である。FIG. 5 is a flow diagram schematically showing a pharmaceutical manufacturing system according to the present invention.
１…培養槽、２…恒温槽、３…攪拌子、５…新鮮培地
槽、６…培養ろ液槽、７…レベルセンサ、８…レベルコ
ントローラ、９…一時保留槽、１０…廃液槽、１１、…
ポンプ、１３…通気管、１４…排気管、１５…除菌フィ
ルタ、１７…バイブレータ、１７…振盪機、１８…通気
ノズル、２３…ＤＯ・ｐＨセンサ、２４…温度センサ、
２５…コントーラ、２６…超音波発振機、２７…超音波
発振端子、５２…分離、精製カラム、５３…製剤システ
ム。1 ... Culture tank, 2 ... Constant temperature tank, 3 ... Stirrer, 5 ... Fresh culture medium tank, 6 ... Culture filtrate tank, 7 ... Level sensor, 8 ... Level controller, 9 ... Temporary holding tank, 10 ... Waste liquid tank, 11 , ...
Pump, 13 ... Ventilation pipe, 14 ... Exhaust pipe, 15 ... Sterilization filter, 17 ... Vibrator, 17 ... Shaker, 18 ... Ventilation nozzle, 23 ... DO / pH sensor, 24 ... Temperature sensor,
25 ... Controller, 26 ... Ultrasonic oscillator, 27 ... Ultrasonic oscillation terminal, 52 ... Separation and purification column, 53 ... Formulation system.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 村上 聖 山口県下松市東豊井794番地 株式会社日 立製作所笠戸工場内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor St. Murakami 794 Higashitoyoi, Kudamatsu City, Yamaguchi Prefecture Hitate Manufacturing Co., Ltd. Kasado Factory
Claims (13)
しながら連続的に培養を行う培養方法において、培養の
途中で間歇的に、必要量の適正細胞を含む培養液を培養
槽と連結した別槽に一時保留する工程、培養槽あるいは
培養槽内培養液に振動を生じさせ培養槽内の器壁等に付
着した細胞塊を剥離させる工程、剥離した細胞塊を培養
槽に残留した培養液と共に培養槽外に排出する工程、及
び前記保留した培養液を培養槽に戻し培養を継続する工
程、とを有することを特徴とする動物細胞の培養方法。1. In a culture method for continuously culturing animal cells while discharging a part of the cells, a culture solution containing a required amount of appropriate cells is intermittently used as a culture tank during the culture. A step of temporarily holding in a connected separate tank, a step of vibrating the culture tank or a culture solution in the culture tank to peel off cell clumps attached to the vessel wall in the culture tank, and the separated cell clumps remained in the culture tank A method for culturing animal cells, comprising the steps of discharging the culture solution together with the culture solution to the outside of the culture tank, and returning the retained culture solution to the culture tank and continuing the culture.
しながら連続的に培養を行う培養方法において、培養の
途中で間歇的に、培養液の攪拌を一時停止して培養中の
細胞塊のみを沈降させる工程、必要量の適正細胞を含む
上澄み培養液を培養槽と連結した別槽に一時保留する工
程、培養槽あるいは培養槽内培養液に振動を生じさせ培
養槽内の器壁等に付着した細胞塊を剥離させる工程、剥
離した細胞塊を培養槽に残留した培養液と共に培養槽外
に排出する工程、及び前記保留した培養液を培養槽に戻
し培養を継続する工程、とを有することを特徴とする動
物細胞の培養方法。2. A method for culturing animal cells, which comprises continuously culturing while discharging a part of the cells, the cells being cultivated by intermittently suspending the stirring of the culture solution during the culturing. Step of settling only lumps, step of temporarily holding supernatant culture solution containing necessary amount of appropriate cells in another tank connected to culture tank, vessel wall in culture tank by vibrating culture tank or culture solution in culture tank A step of exfoliating the cell mass attached to the etc., a step of discharging the exfoliated cell mass together with the culture solution remaining in the culture tank to the outside of the culture tank, and a step of returning the suspended culture solution to the culture tank and continuing the culture, A method for culturing animal cells, which comprises:
しながら連続的に培養を行う培養方法において、培養の
途中で間歇的に、培養槽あるいは培養槽内培養液に振動
を生じさせ培養槽内の器壁等に付着した細胞塊を剥離さ
せる工程、培養液の攪拌を一時停止して培養中の細胞塊
のみを沈降させる工程、必要量の適正細胞を含む上澄み
培養液を培養槽と連結した別槽に一時保留する工程、培
養槽に残留した細胞塊を含む培養液を排出する工程、及
び前記保留した培養液を培養槽に戻し培養を継続する工
程、とを有することを特徴とする動物細胞の培養方法。3. A method for culturing animal cells, which comprises continuously culturing while discharging a part of the cells, wherein the culture tank or the culture solution in the culture tank is vibrated intermittently during the culture. The step of peeling off the cell clumps adhering to the vessel walls in the culture tank, the step of suspending the stirring of the culture solution to settle only the cell clumps in the culture, and the supernatant culture solution containing the necessary amount of appropriate cells And a step of temporarily retaining the culture solution containing the cell mass remaining in the culture tank, and a step of returning the retained culture solution to the culture tank and continuing the culture. And a method for culturing animal cells.
液を培養槽と連結した別槽に一時保留する工程と、前記
別槽に一時保留した細胞を培養槽に戻す工程との間に、
培養槽の無菌状態を保ったままで培地あるいは生体の細
胞に適した浸透圧及びｐＨに調整した緩衝液を培養槽内
に導入する工程をさらに有することを特徴とする、請求
項１ないし３いずれか記載の動物細胞の培養方法。4. Between the step of temporarily holding the supernatant culture solution containing the required amount of appropriate cells in a separate tank connected to the culture tank and the step of returning the cells temporarily held in the separate tank to the culture tank,
4. The method according to claim 1, further comprising the step of introducing a culture medium or a buffer solution adjusted to an osmotic pressure and a pH suitable for living cells into the culture tank while keeping the culture tank sterile. The method for culturing animal cells described.
項４記載の動物細胞の培養方法。5. The method for culturing animal cells according to claim 4, wherein the buffer solution is a phosphate buffer solution.
せる振動は、培養液自体の強度の機械的あるいは通気に
よる攪拌、培養液への超音波の付与、あるいは培養槽に
付設された振動発生装置により生じる振動のいずれか又
はその組合せであることを特徴とする、請求項１ないし
４記載の動物細胞の培養方法。6. The vibration generated in the culture tank or the culture solution in the culture tank is agitated by mechanical or aeration of the strength of the culture solution itself, imparts ultrasonic waves to the culture solution, or generates vibration attached to the culture tank. The method for culturing animal cells according to any one of claims 1 to 4, characterized in that any one or a combination of vibrations generated by the device is used.
しグルコース濃度より産出されるグルコース消費速度が
細胞濃度に対して所定の比例関係より外れたことを検知
して、付着細胞塊の剥離及び細胞塊の除去操作を行うこ
とを特徴とする、請求項１ないし４記載の動物細胞の培
養方法。7. During culture, the glucose concentration of the culture solution is measured, and it is detected that the glucose consumption rate produced from the glucose concentration deviates from a predetermined proportional relationship with the cell concentration, and the detachment of adherent cell mass and The method for culturing animal cells according to any one of claims 1 to 4, characterized in that a cell mass removing operation is performed.
１ないし７記載の動物細胞の培養方法。8. The method for culturing animal cells according to claim 1, wherein the animal cells are floating cells.
する手段とを有する培養槽、 ２）前記培養槽及び／又は培養槽内の培養液に振動を生
じさせる手段、及び ３）前記培養液の一部を取り出す手段と取り出された培
養液を培養槽に戻す手段とに無菌的に接続した細胞一時
保留槽、とからなることを特徴とする動物細胞培養装
置。9. At least the following elements 1) to 3), 1) a culture tank having a means for extracting a part of the culture solution and a means for discharging the remaining culture solution, 2) the culture tank and / or the culture tank And a means for removing a part of the culture solution and a means for returning the removed culture solution to the culture tank aseptically connected to a temporary cell holding tank. An animal cell culture device characterized by:
液に振動を生じさせる手段が、培養槽内に設けた培養液
の機械的攪拌手段、培養液への通気手段、培養槽に付設
した振動発生手段、及び超音波発生手段のいずれか、又
はその組合せであることを特徴とする、請求項９記載の
動物細胞培養装置。10. The means for vibrating the culture tank and / or the culture solution in the culture tank is provided with a mechanical stirring means for the culture solution, an aeration means for the culture solution, and a culture tank provided in the culture tank. 10. The animal cell culture device according to claim 9, which is any one of the vibration generating means and the ultrasonic wave generating means, or a combination thereof.
り出された培養液を培養槽に戻す手段は、前記培養槽又
は細胞一時保留槽のいずれかを陰圧あるいは加圧する手
段であることを特徴とする、請求項８記載の動物細胞培
養装置。11. The means for extracting a part of the culture solution and the means for returning the removed culture solution to the culture tank are means for applying negative pressure or pressurization to either the culture tank or the temporary cell holding tank. 9. The animal cell culture device according to claim 8, which is characterized.
養方法を用いて動物細胞を培養し有用物質を生産させる
工程、前記工程より回収した有用物質を含む培養液を分
離し精製する工程、及び精製した有用物質を製剤化する
工程、とからなる医薬品製造システム。12. A step of culturing an animal cell to produce a useful substance by using the method for culturing an animal cell according to claim 1, a step of separating and purifying a culture solution containing the useful substance recovered from the step, And a step of formulating a purified useful substance into a pharmaceutical product.
養装置を用いて動物細胞を培養し有用物質を生産させる
手段、前記手段より回収した有用物質を含む培養液を分
離し精製する手段、及び精製した有用物質を製剤化する
手段、とからなる医薬品製造装置。13. A means for culturing animal cells using the animal cell culturing apparatus according to claim 9 to produce a useful substance, a means for separating and purifying a culture solution containing the useful substance recovered from said means, A pharmaceutical manufacturing device comprising: means for formulating a purified useful substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4296825A JPH06141850A (en) | 1992-11-06 | 1992-11-06 | Culture system for animal cell and method for culture of animal cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4296825A JPH06141850A (en) | 1992-11-06 | 1992-11-06 | Culture system for animal cell and method for culture of animal cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH06141850A true JPH06141850A (en) | 1994-05-24 |
Family
ID=17838648
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4296825A Pending JPH06141850A (en) | 1992-11-06 | 1992-11-06 | Culture system for animal cell and method for culture of animal cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06141850A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998044089A1 (en) * | 1997-04-02 | 1998-10-08 | Mitsubishi Chemical Corporation | Erythritol-producing microorganism and process for producing the same |
| WO2001025404A1 (en) * | 1999-10-05 | 2001-04-12 | Teijin Limited | Methods for inhibiting osteoclast formation or methods for inhibiting bone resorption |
| JP2006129765A (en) * | 2004-11-05 | 2006-05-25 | Tokyo Rika Kikai Kk | Culture apparatus and method for sterilizing the same |
| JP2007530037A (en) * | 2004-03-25 | 2007-11-01 | ヒューレット−パッカード デベロップメント カンパニー エル．ピー． | Cell transporter for biodevices |
| JP2008054674A (en) * | 2006-08-02 | 2008-03-13 | Becton Dickinson & Co | Bioreactor and method |
| US8114646B2 (en) | 2008-05-30 | 2012-02-14 | Corning Incorporated | Method for ultrasonic cell removal |
| EP3795671A3 (en) * | 2019-06-27 | 2021-06-16 | Schott Ag | Cultivation system and container attachment for a cultivation container |
-
1992
- 1992-11-06 JP JP4296825A patent/JPH06141850A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998044089A1 (en) * | 1997-04-02 | 1998-10-08 | Mitsubishi Chemical Corporation | Erythritol-producing microorganism and process for producing the same |
| WO2001025404A1 (en) * | 1999-10-05 | 2001-04-12 | Teijin Limited | Methods for inhibiting osteoclast formation or methods for inhibiting bone resorption |
| JP2007530037A (en) * | 2004-03-25 | 2007-11-01 | ヒューレット−パッカード デベロップメント カンパニー エル．ピー． | Cell transporter for biodevices |
| JP2006129765A (en) * | 2004-11-05 | 2006-05-25 | Tokyo Rika Kikai Kk | Culture apparatus and method for sterilizing the same |
| JP2008054674A (en) * | 2006-08-02 | 2008-03-13 | Becton Dickinson & Co | Bioreactor and method |
| US8114646B2 (en) | 2008-05-30 | 2012-02-14 | Corning Incorporated | Method for ultrasonic cell removal |
| EP3795671A3 (en) * | 2019-06-27 | 2021-06-16 | Schott Ag | Cultivation system and container attachment for a cultivation container |
| US11525710B2 (en) | 2019-06-27 | 2022-12-13 | Schott Ag | Multi-sensor component for bioprocess control |
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