JP2020068741A - Cell culture apparatus and cell culture method - Google Patents
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Abstract
Description
本発明は細胞培養装置および細胞培養方法に関する。 The present invention relates to a cell culture device and a cell culture method.
抗体医薬品などに用いられるタンパク質の大量生産や、医薬品、化粧品、食品などの評価に用いられる細胞の大量生産などのために、ガス透過性を有する有機高分子製の柔軟なシートからなる細胞培養バッグが開発されている（例えば、特許文献１、２など）。
前記のような細胞培養バッグは、従来、浮遊細胞の培養に用いられていたが、近年では、内面に親水処理や接着コートを施すことによって接着性細胞（足場依存性細胞とも呼ばれる）の培養にも用いられている。
Cell culture bag made of flexible sheet made of organic polymer with gas permeability for mass production of proteins used for antibody drugs and mass production of cells used for evaluation of drugs, cosmetics, foods, etc. Have been developed (for example, Patent Documents 1 and 2).
The cell culture bag as described above has been conventionally used for culturing floating cells, but in recent years, it has been cultivated for adhesive cells (also called anchorage-dependent cells) by subjecting the inner surface to hydrophilic treatment or an adhesive coat. Is also used.
このような細胞培養バッグを用いれば、出入口に接続されたチューブを介して他のバッグや容器、種々の処理装置（例えば、試薬、培地、細胞などを注入するための外部装置、培養後の細胞や廃液を回収するための外部装置）などと接続して閉鎖系の細胞培養システムを構成することができる。このような細胞培養システムは、細胞の播種、継代、培地交換、細胞の回収などを、外界に露出することなしに行うことができるので、汚染の危険性がより低減された好ましいシステムである。 If such a cell culture bag is used, other bags and containers, various processing devices (for example, external devices for injecting reagents, culture medium, cells, etc.) via tubes connected to the inlet and outlet, cells after culture Or an external device for collecting waste liquid) or the like to form a closed cell culture system. Since such a cell culture system can perform cell seeding, subculture, medium exchange, cell recovery, etc. without exposure to the outside world, it is a preferable system with a further reduced risk of contamination. ..
また、接着性細胞の培養にあたっては、剛性が高い足場があることが好ましく、一般にＴフラスコと呼ばれるような細胞培養用のフラスコが汎用されている。Ｔフラスコは、厚い容器で、底部に培地を収容し、上部は空気層となるように用いられている。 Further, in culturing the adhesive cells, it is preferable to have a scaffold having high rigidity, and a flask for cell culture generally called a T-flask is generally used. The T-flask is a thick container, which is used to contain a culture medium at the bottom and an air layer at the top.
細胞培養にともない、細胞が増殖することで培地中のガス濃度は大きく変動する。典型的には細胞が増殖することに伴い、酸素を消費して培地中の酸素濃度が低下する。特許文献１や２では、バッグを構成するフィルムにガス透過性を有するものを採用することで、ある程度のガス供給は可能である。また、Ｔフラスコでは上部の空気層と培地とでガス交換しながら培養される。 The gas concentration in the medium greatly changes as the cells grow due to the cell culture. Typically, as cells grow, they consume oxygen and reduce the oxygen concentration in the medium. In Patent Documents 1 and 2, it is possible to supply gas to some extent by adopting a film having gas permeability as a film forming the bag. Further, in the T-flask, the culture is performed while exchanging gas between the upper air layer and the medium.
また、このような細胞培養に必要なガス濃度を調整するために、特許文献３には、主壁部の一部におよび／または側壁部の一部または全部にガス透過部を有する細胞培養容器が開示されている。また、特許文献４には、２つの外側板状部および各側壁部から選ばれる１以上の部分にはガス透過部が設けられ、また、２つの外側板状部および各側壁部から選ばれる１以上の部分には培養液のための出入口が設けられる細胞培養容器が開示されている。 Further, in order to adjust the gas concentration necessary for such cell culture, Patent Document 3 discloses that a cell culture container having a gas permeable portion in a part of the main wall and / or a part or all of the side wall. Is disclosed. Further, in Patent Document 4, a gas permeable portion is provided at one or more portions selected from the two outer plate-shaped portions and the respective side wall portions, and one selected from the two outer plate-shaped portions and the respective side wall portions is provided. The above part discloses a cell culture container provided with an inlet / outlet for a culture solution.
細胞培養にあたっては、培地中のガス濃度の調整等が重要となる。前述の特許文献１〜４においても、ガス透過部などを有することで培養容器内の培地のガス濃度を調整することはできる。また、Ｔフラスコを用いる場合、容器内の上部の空気層を適宜調整することで培地中のガス濃度も調整できるが、このために十分な容量の空気層を設けることも必要となる。なお、従来の使用法におけるＴフラスコは、空気層には細胞が接着できないため、細胞が培養できる場は、培地が収容されている底部側のみであった。
しかしながら、細胞培養の需要が高度化し、より高濃度の細胞濃度での培養や、大容量の容器による大量培養、長期期間の培養、培養中の培地条件等の制御など、より高度な制御が必要になる場合がある。
In cell culture, adjustment of gas concentration in the medium is important. Also in the above-mentioned Patent Documents 1 to 4, it is possible to adjust the gas concentration of the medium in the culture container by having the gas permeable portion and the like. When using a T-flask, the gas concentration in the medium can be adjusted by appropriately adjusting the air layer in the upper part of the container, but it is also necessary to provide an air layer having a sufficient capacity for this purpose. In the conventional T-flask, cells cannot adhere to the air layer, so cells can be cultured only on the bottom side where the medium is contained.
However, the demand for cell culture is increasing, and more advanced control is required, such as culturing at higher cell concentration, large-scale culturing in large-capacity vessels, long-term culturing, control of medium conditions during culturing, etc. May be.
従来の培養容器などでは、ガス透過部が接する雰囲気とのガス交換が生じる。すなわち、培養容器を配置する環境すべての雰囲気となる庫内全体のガス濃度等を制御する必要があった。例えば、培地中の水分の蒸発を避けるために高湿度の雰囲気ガスを広範な室内で維持したり、酸素濃度等もその室内で十分に維持されるように管理する必要があった。 In a conventional culture container or the like, gas exchange with the atmosphere in contact with the gas permeable portion occurs. That is, it was necessary to control the gas concentration and the like in the entire chamber that became the atmosphere of all the environments in which the culture vessels were placed. For example, in order to avoid evaporation of water in the medium, it is necessary to maintain a high-humidity atmosphere gas in a wide range of rooms, and to manage oxygen concentration and the like so that it is sufficiently maintained in the room.
さらには、このような管理を行っても、細胞培養容器内の培地は流動しにくいなどの理由から、そのガス濃度の勾配が生じやすかった。ガス透過部に近い細胞培養器の外周付近の培地は、雰囲気ガスとガス交換しガス濃度が制御されるが、ガス透過部から離れた細胞培養容器の内部の培地は培地中のガスが細胞培養に伴い消費・産生されたガスの影響が大きい状態となっていた。具体的には、ガス透過部近傍の外周付近では溶存酸素濃度が高く細胞培養を安定して継続できるものの、ガス透過部から離れた内奥では溶存酸素濃度が低く細胞増殖が停止したり遅くなる場合があった。 Furthermore, even if such a control is performed, a gradient of the gas concentration is likely to occur because the medium in the cell culture container is difficult to flow. The medium near the outer periphery of the cell incubator near the gas permeation part exchanges gas with the atmospheric gas to control the gas concentration. As a result, the effect of gas consumed and produced was large. Specifically, the dissolved oxygen concentration is high near the outer periphery near the gas permeation part and cell culture can be stably continued, but the dissolved oxygen concentration is low in the inner part far from the gas permeation part and cell growth is stopped or slowed down. There were cases.
係る状況下、本発明は、細胞培養容器内の培地のガス濃度の制御に適した細胞培養装置および細胞培養方法を提供することを目的とする。 Under such circumstances, an object of the present invention is to provide a cell culture device and a cell culture method suitable for controlling the gas concentration of a medium in a cell culture container.
本発明者は、上記課題を解決すべく鋭意研究を重ねた結果、下記の発明が上記目的に合致することを見出し、本発明に至った。すなわち、本発明は、以下の発明に係るものである。 As a result of earnest studies to solve the above problems, the present inventor has found that the following invention meets the above object, and has reached the present invention. That is, the present invention relates to the following inventions.
＜１＞ 培地および細胞を充填する中空状の内層を有する細胞培養容器と、
前記細胞培養容器に取り付けられ、前記培地を循環するための循環ラインと、
前記循環ライン内に設けられ前記細胞培養容器に前記培地を送液する送液ポンプと、
前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段と、を有する細胞培養装置。
＜２＞ 前記ガス交換手段の前記調整用ガスを調整するためのガス調整手段を有する前記＜１＞記載の細胞培養装置。
＜３＞ 前記調整用ガスが、酸素濃度および、窒素濃度、二酸化炭素濃度、湿度からなる群から選択される少なくとも１以上が調整されたガスである前記＜１＞または＜２＞に記載の細胞培養装置。
＜４＞ 前記中空糸膜が、シリコーン製の膜である前記＜１＞〜＜３＞のいずれかに記載の細胞培養装置。
＜５＞ 前記細胞培養容器は、所定の間隔をおいて互いに対面するよう配置された２枚の板状部を有する前記＜１＞〜＜４＞のいずれかに記載の細胞培養装置。
＜６＞ 前記細胞培養容器が、積層された板状部を有する前記＜１＞〜＜５＞のいずれかに記載の細胞培養装置。
＜７＞ 中空状の内層を有する細胞培養容器に培地および細胞液を充填する充填工程と、
前記細胞培養容器に充填された前記細胞を培養する培養工程と、
前記細胞培養容器に取り付けられた前記培地を循環するための循環ライン内に設けられた送液ポンプにより前記循環ライン内の前記培地を送液することで前記循環ライン内の前記培地を循環する循環工程と、
前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段によりガス交換するガス交換工程と、を有する細胞培養方法。
<1> A cell culture container having a hollow inner layer for filling a medium and cells,
A circulation line attached to the cell culture vessel for circulating the medium,
A liquid feed pump that is provided in the circulation line and feeds the medium to the cell culture container,
A cell culture device comprising: a gas exchange unit having a hollow fiber membrane, which is provided in the circulation line and exchanges a gas contained in the culture medium circulated in the circulation line with a regulating gas.
<2> The cell culture device according to <1>, further including a gas adjusting unit for adjusting the adjusting gas of the gas exchanging unit.
<3> The cell according to <1> or <2>, wherein the adjusting gas is a gas in which at least one selected from the group consisting of oxygen concentration, nitrogen concentration, carbon dioxide concentration, and humidity is adjusted. Incubator.
<4> The cell culture device according to any one of <1> to <3>, wherein the hollow fiber membrane is a silicone membrane.
<5> The cell culture device according to any one of <1> to <4>, wherein the cell culture container has two plate-shaped portions that are arranged so as to face each other at a predetermined interval.
<6> The cell culture device according to any one of <1> to <5>, in which the cell culture container has stacked plate-shaped portions.
<7> A filling step of filling a cell culture container having a hollow inner layer with a medium and a cell liquid,
A culturing step of culturing the cells filled in the cell culture container,
A circulation in which the medium in the circulation line is circulated by feeding the medium in the circulation line by a liquid feeding pump provided in a circulation line attached to the cell culture container for circulating the medium. Process,
A gas exchange step of performing gas exchange by a gas exchange means having a hollow fiber membrane for performing gas exchange between the gas contained in the medium and the adjusting gas provided in the circulation line and circulated in the circulation line, A method for culturing cells.
本発明によれば、細胞培養容器内の培地のガス濃度を制御しながらの培養を行うことができる。さらに、本発明によれば、細胞培養容器内に培地が充填された状態で、充填されている培地はガス交換しながら培養できるため、細胞培養容器内に空気層を必要とせずに培養できる。このため、細胞培養容器内の上下面など広範に接着性細胞を培養できる場が得られる。さらに、ガス交換する場が限られた閉鎖系の培養ができる。このため、比較的小型の培養装置や培養設備でも大量の細胞培養ができる。 According to the present invention, it is possible to perform culture while controlling the gas concentration of the medium in the cell culture container. Furthermore, according to the present invention, in the state where the medium is filled in the cell culture container, the filled medium can be cultured while exchanging the gas, so that the cell culture container can be cultured without requiring an air layer. Therefore, a place where the adhesive cells can be cultured in a wide range such as the upper and lower surfaces in the cell culture container can be obtained. Furthermore, closed-system culture with limited gas exchange is possible. Therefore, a large amount of cells can be cultured with a relatively small culture device or culture equipment.
以下に本発明の実施の形態を詳細に説明するが、以下に記載する構成要件の説明は、本発明の実施態様の一例（代表例）であり、本発明はその要旨を変更しない限り、以下の内容に限定されない。なお、本明細書において「〜」という表現を用いる場合、その前後の数値を含む表現として用いる。 BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail, but the description of the constituent elements described below is an example (representative example) of the embodiments of the present invention, and the present invention is as follows unless the gist thereof is changed. The content is not limited to. In addition, when using the expression "to" in this specification, it is used as an expression including numerical values before and after the expression.
［本発明の細胞培養装置］
本発明の細胞培養装置は、培地および細胞を充填する中空状の内層を有する細胞培養容器と、前記細胞培養容器に取り付けられ、前記培地を循環するための循環ラインと、前記循環ライン内に設けられ前記細胞培養容器に前記培地を送液する送液ポンプと、前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段とを有する。本発明の細胞培養装置により細胞を培養することで、細胞培養容器内の培地のガス濃度を制御しながらの培養を行うことができる。また、本発明の細胞培養装置は、細胞培養容器内に空気層を必要とせずに培養でき、閉鎖系で、大量の細胞培養に適した装置である。
[Cell culture device of the present invention]
The cell culture device of the present invention is provided with a cell culture container having a hollow inner layer for filling a medium and cells, a circulation line attached to the cell culture container for circulating the medium, and provided in the circulation line. A liquid feed pump for feeding the medium to the cell culture container, and a gas provided in the circulation line for performing gas exchange between the gas contained in the medium circulated in the circulation line and the adjusting gas. And a gas exchange means having a hollow fiber membrane. By culturing cells with the cell culture device of the present invention, it is possible to perform culture while controlling the gas concentration of the medium in the cell culture container. Further, the cell culture device of the present invention is a device that can perform culture without requiring an air layer in the cell culture container, is a closed system, and is suitable for large-scale cell culture.
［本発明の細胞培養方法］
本発明の細胞培養方法は、中空状の内層を有する細胞培養容器に培地および細胞を充填する充填工程と、前記細胞培養容器に充填された前記細胞を培養する培養工程と、前記細胞培養容器に取り付けられた前記培地を循環するための循環ライン内に設けられた送液ポンプにより前記循環ライン内の前記培地を送液することで前記循環ライン内の前記培地を循環する循環工程と、前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段によりガス交換するガス交換工程とを有する。
本発明の細胞培養方法により細胞を培養することで、細胞培養容器内の培地のガス濃度を制御しながらの培養を行うことができる。また、本発明の細胞培養方法は、細胞培養容器内に空気層が生じない培養に適しており、閉鎖系で、大量の細胞培養に適した方法である。
なお、本願において本発明の細胞培養装置により本発明の細胞培養方法を行うことができ、本願においてそれぞれに対応する構成は相互に利用することができる。
[Cell culture method of the present invention]
The cell culture method of the present invention comprises a filling step of filling a medium and cells into a cell culture container having a hollow inner layer, a culture step of culturing the cells filled in the cell culture container, and a cell culture container A circulating step of circulating the medium in the circulation line by feeding the medium in the circulation line by a liquid feed pump provided in a circulation line for circulating the attached medium; A gas exchange step of performing gas exchange by a gas exchange means provided in a line and having a hollow fiber membrane for performing a gas exchange between the gas contained in the culture medium and the adjusting gas circulated in the circulation line.
By culturing cells by the cell culturing method of the present invention, it is possible to perform culturing while controlling the gas concentration of the medium in the cell culture container. Further, the cell culture method of the present invention is suitable for culture in which an air layer does not occur in the cell culture container, is a closed system, and is suitable for large-scale cell culture.
In the present application, the cell culture method of the present invention can be performed by the cell culture device of the present invention, and configurations corresponding to each other in the present application can be mutually utilized.
［第一の実施形態］
図１は、本発明の細胞培養装置の第一の実施形態を示す概要図である。図１は、細胞培養装置１０を示すものである。細胞培養装置１０は、細胞培養容器２１と、循環ライン３１１、３１２、３１３と、送液ポンプ４１と、ガス交換手段５１とを有する。
細胞培養容器２１は、開口部２１１、２１２を有し、開口部２１１は循環ライン３１１と接続され、開口部２１２は循環ライン３１３に接続されている。
循環ライン３１１はガス交換手段５１の開口部５１１に接続されている。ガス交換手段の開口部５１２は、循環ライン３１２に接続されている。循環ライン３１２は、送液ポンプ４１に接続され、送液ポンプ４１は循環ライン３１３にも接続されている。循環ライン３１１、３１２、３１３は細胞培養容器２１内の培地を移送することができるチューブである。
送液ポンプ４１は、培地を送液することができ、循環ライン３１２側から循環ライン３１３側へのＦ１１方向へ送液するものでもよいし、逆方向のＦ１２方向へ送液するものでもよい。送液ポンプ４１の送液により、循環ライン３１１、３１２、３１３や、これらと適宜接続されている細胞培養容器２１内や、ガス交換手段５１内の培地が順に移送され、培地はこれらの系内で循環する。
ガス交換手段５１は、開口部５１１、５１２のように培地が循環する部分とは別に、培地が漏出しない開口部５１３、５１４を有している。この開口部５１３、５１４は、ガスが通過することができる。開口部５１１、５１２から導入・導出された培地は、開口部５１３、５１４から導入・導出されたガスと、ガス交換し、培地中のガス濃度が調整される。
この培地は、前述のように系内を循環するため、ガス交換手段５１内でガス濃度が調整された状態で、細胞培養容器２１にも移送される。これにより、細胞培養容器２１内で培養されている細胞は、ガス濃度が調整された培地により培養される。また、細胞培養装置１０は、細胞培養容器２１等の系内の培地がガス交換できる部分を実質ガス交換手段５１内のみの閉鎖系として運転でき、開口部５１３と開口部５１４とから導入・導出するガスの湿度やガス濃度を管理するだけで、培地のガス交換を制御することができる。
[First embodiment]
FIG. 1 is a schematic diagram showing a first embodiment of the cell culture device of the present invention. FIG. 1 shows a cell culture device 10. The cell culture device 10 includes a cell culture container 21, circulation lines 311, 312, 313, a liquid feed pump 41, and a gas exchange means 51.
The cell culture container 21 has openings 211 and 212, the opening 211 is connected to the circulation line 311, and the opening 212 is connected to the circulation line 313.
The circulation line 311 is connected to the opening 511 of the gas exchange means 51. The opening 512 of the gas exchange means is connected to the circulation line 312. The circulation line 312 is connected to the liquid feed pump 41, and the liquid feed pump 41 is also connected to the circulation line 313. The circulation lines 311, 312, 313 are tubes that can transfer the medium in the cell culture vessel 21.
The liquid feed pump 41 can feed the medium, and may feed the medium in the F11 direction from the circulation line 312 side to the circulation line 313 side, or may feed the medium in the opposite F12 direction. By the liquid feeding of the liquid feeding pump 41, the culture medium in the circulation lines 311, 312, 313, the cell culture vessel 21 appropriately connected to these, or the gas exchange means 51 is sequentially transferred, and the medium is in these systems. Circulate in.
The gas exchange means 51 has openings 513 and 514 through which the culture medium does not leak, in addition to the portions where the culture medium circulates, such as the openings 511 and 512. Gas can pass through the openings 513 and 514. The medium introduced / derived from the openings 511, 512 exchanges gas with the gas introduced / derived from the openings 513, 514 to adjust the gas concentration in the medium.
Since this medium circulates in the system as described above, it is also transferred to the cell culture vessel 21 in a state where the gas concentration is adjusted in the gas exchange means 51. As a result, the cells cultured in the cell culture vessel 21 are cultured in the medium whose gas concentration is adjusted. Further, the cell culture device 10 can operate a portion of the cell culture container 21 or the like in which the medium can exchange gas as a closed system only inside the substantial gas exchange means 51, and is introduced / extracted from the opening 513 and the opening 514. The gas exchange of the culture medium can be controlled only by controlling the humidity and the gas concentration of the gas to be used.
［細胞培養容器２１］
細胞培養容器２１は、培地および細胞を充填する中空状の内層を有する。図２〜図４は細胞培養容器２１の詳しい構造を説明するための図である。
[Cell culture vessel 21]
The cell culture vessel 21 has a hollow inner layer that is filled with a medium and cells. 2 to 4 are views for explaining the detailed structure of the cell culture container 21.
図２は、細胞培養容器２１の平面図である。また、図３（ａ）は細胞培養容器２１を右側面図であり、図３（ｂ）は正面図である。細胞培養容器２１は、略直方体状とすることができる。平面視したとき、その形状は正方形状でもよいし、長方形状でもよく、細胞培養ができる任意の大きさとすることができる。例えば、縦辺２０ｍｍ〜５００ｍｍ、横辺２０ｍｍ〜５００ｍｍ程度の大きさとすることができる。面積として４００ｍｍ2〜２．５×１０5ｍｍ2程度としてもよい。細胞培養容器は、略直方体状に限定されるものではなく、円柱状や多角柱状などその内部で細胞培養することができる任意の他の形状でもよい。 FIG. 2 is a plan view of the cell culture container 21. Further, FIG. 3A is a right side view of the cell culture vessel 21, and FIG. 3B is a front view. The cell culture container 21 can be made into a substantially rectangular parallelepiped shape. When viewed in a plan view, the shape may be a square shape or a rectangular shape, and may be any size capable of culturing cells. For example, the size may be about 20 mm to 500 mm on the vertical side and about 20 mm to 500 mm on the horizontal side. The area may be about 400 mm 2 to 2.5 × 10 5 mm 2 . The cell culture container is not limited to a substantially rectangular parallelepiped shape, and may have any other shape such as a columnar shape or a polygonal pillar shape that allows cell culture therein.
細胞培養容器２１は、開口部２１１、２１２を有する。開口部２１１、２１２は、対角線上に設けて、細胞培養容器２１内の培地を循環させるとき、より効率よく入れ替えが生じるように配置されている。なお、ここでは開口部は２つの場合を示したが、開口部は３つ以上設けてもよく、培地の循環ラインを繋ぎ変えながら利用したり、他の成分等を入れ替えるために用いたりしてもよい。また、培養中に使用しない開口部は蓋部を設けて密閉することが好ましい。細胞培養容器２１は、開口部以外から液が漏出しない密閉性が高いものであることが好ましい。 The cell culture container 21 has openings 211 and 212. The openings 211 and 212 are provided on a diagonal line and are arranged so that replacement can be performed more efficiently when the medium in the cell culture container 21 is circulated. Although two openings are shown here, three or more openings may be provided, and it may be used by connecting the circulation lines of the medium or by replacing other components. Good. Further, it is preferable that the opening not used during the culture is closed by providing a lid. It is preferable that the cell culture container 21 has a high hermeticity so that the liquid does not leak from other than the opening.
細胞培養容器２１の材質は、細胞培養の容器に用いることができる各種材料を用いることができる。例えば、樹脂や、ガラス、金属などを用いることができる。好ましくは、培養状況を外部から観察しやすく、材料の評価試験を行う必要性が低い、ポリスチレンや、ポリカーボネート、ポリエチレンテレフタレートなど樹脂を用いることが好ましい。 As the material of the cell culture vessel 21, various materials that can be used for the cell culture vessel can be used. For example, resin, glass, metal or the like can be used. It is preferable to use a resin such as polystyrene, polycarbonate, or polyethylene terephthalate, which makes it easy to observe the culture condition from the outside and has a low necessity for conducting a material evaluation test.
細胞培養容器２１は、形状が変形しにくいものであることが好ましい。前述したような材料を用いて、例えば１ｍｍ以上や１．５ｍｍ以上などの十分な厚みとすることで、細胞培養容器として使用したり、取り扱うときなどの外力でも変形しないものとすることができる。このような変形しにくいもののとき、細胞培養容器２１の内部の壁に細胞が接着した状態で安定して培養され増殖や成長しやすいものとすることができる。 It is preferable that the shape of the cell culture container 21 does not easily change. By using a material as described above and having a sufficient thickness of, for example, 1 mm or more or 1.5 mm or more, it can be used as a cell culture container or can not be deformed by an external force when it is handled. When such a material is hard to be deformed, the cells can be stably cultured and easily proliferate and grow in a state where the cells are adhered to the inner wall of the cell culture container 21.
図４は細胞培養容器２１のＡ−Ａ断面図（図３（ａ）参照）である。細胞培養容器２１は、上下の板２１３と側壁２１４間に中空状の内層があり、細胞培養の場となる。中空状の内層は、開口部２１１においては、中空部２１１１により外部に開口されている。同様に開口部２１２側でも開口している。細胞培養容器２１の中空状の内層は、平板上の上下の板２１３の内面２１３１に細胞２１６が接着し、その内側で培地２１５が循環される。この培地２１５は流動性がある培地であり、液体培地やゼリー状の培地である。なお、細胞が接着する場となる内面２１３１は、培養する細胞の種類に応じて、適宜火炎処理（フレーム処理）や、コロナ処理、プラズマ処理、ＶＵＶ（真空紫外光）等の表面処理を行い接着しやすい状態としておくことが好ましい。 FIG. 4 is a cross-sectional view of the cell culture container 21 taken along the line AA (see FIG. 3A). The cell culture container 21 has a hollow inner layer between the upper and lower plates 213 and the side walls 214, and serves as a place for cell culture. In the opening 211, the hollow inner layer is opened to the outside by the hollow portion 2111. Similarly, the opening 212 is opened. In the hollow inner layer of the cell culture container 21, the cells 216 adhere to the inner surfaces 2131 of the upper and lower plates 213 on the flat plate, and the medium 215 is circulated inside thereof. The medium 215 is a fluid medium, and is a liquid medium or a jelly-like medium. It should be noted that the inner surface 2131, which is a place where cells adhere, is appropriately subjected to surface treatment such as flame treatment (frame treatment), corona treatment, plasma treatment, VUV (vacuum ultraviolet light), etc., depending on the type of cells to be cultured. It is preferable to make it easy to do.
細胞培養容器２１は、所定の間隔をおいて互いに対面するよう配置された２枚の板状部（上下の板２１３）を有する。この２枚の板状部は、開口部を設ける部分を除き、その周囲を密閉するように閉じたものとする。開口部２１１、２１２は、循環ラインに接続されるため、細胞培養容器２１の内層は、密閉された空間となる。
この内層には、容器内には培地や細胞が充填され、さらに培養中は培地が循環ラインにより循環されるため、液体や細胞による充填率が高い状態で用いることができる。細胞培養を行うときの細胞培養容器２１内の培地および細胞の充填率（培地および細胞の総量／内層の容量）は８０容積％以上や９０容積％以上、９５容積％以上、９９容積％以上とすることができる。すなわち、実質的に、細胞培養容器２１内に空気層を含まない状態で培養を行うことができる。これにより、２枚の板状部の内層側のそれぞれの面（上下の面）が、細胞が接着する場となり、大量の細胞を効率よく培養することができる。
The cell culture container 21 has two plate-shaped portions (upper and lower plates 213) arranged so as to face each other at a predetermined interval. The two plate-shaped portions are closed so as to hermetically seal the periphery except for the portion where the opening is provided. Since the openings 211 and 212 are connected to the circulation line, the inner layer of the cell culture container 21 becomes a sealed space.
The inner layer is filled with a medium and cells in a container, and further, the medium is circulated by a circulation line during culturing, so that the medium and the cells can be used in a high filling rate. The filling rate of the medium and cells in the cell culture vessel 21 (total amount of medium and cells / volume of inner layer) when performing cell culture is 80% by volume or more, 90% by volume or more, 95% by volume or more, 99% by volume or more. can do. That is, it is possible to perform the culture substantially without containing the air layer in the cell culture container 21. As a result, the respective surfaces (upper and lower surfaces) on the inner layer side of the two plate-shaped portions serve as a place for cells to adhere, and a large amount of cells can be efficiently cultured.
細胞培養容器２１の内層の厚みｄ１は、細胞が培養できる厚さで任意の厚みとすることができる。この厚みｄ１は、互いに対面するように配置された２枚の板状部の所定の間隔に相当する。厚みｄ１は、好ましくは、例えば２ｍｍ〜２０ｍｍ程度である。厚みの下限は、３ｍｍ以上や４ｍｍ以上としてもよい。特に培養が進んだ接着性細胞は２ｍｍ程度の厚みとなる場合があり、細胞培養容器の上下面で培養しやすいように厚くしてもよい。一方、厚みの上限は厚くても細胞が接着する場は上下の面となるため、循環させるための培地量が過剰となる場合もあるため、１５ｍｍ以下や、１２ｍｍ以下、１０ｍｍ以下としてもよい。このような厚みのとき、細胞培養容器２１内で循環する培地２１５が比較的少量でも内層の上下に、細胞が接着して培養されやすい。 The thickness d1 of the inner layer of the cell culture vessel 21 may be any thickness that allows cells to be cultured. The thickness d1 corresponds to a predetermined distance between the two plate-shaped portions arranged so as to face each other. The thickness d1 is preferably, for example, about 2 mm to 20 mm. The lower limit of the thickness may be 3 mm or more or 4 mm or more. In particular, adherent cells that have undergone culturing may have a thickness of about 2 mm, and may be thickened so that they can be easily cultured on the upper and lower surfaces of the cell culture container. On the other hand, the upper limit of the thickness is 15 mm or less, 12 mm or less, and 10 mm or less, because the cells adhere to the upper and lower surfaces even if they are thick, and the amount of the medium for circulation may be excessive. With such a thickness, even if the medium 215 circulated in the cell culture vessel 21 is relatively small, cells are easily adhered to and cultivated above and below the inner layer.
細胞培養容器２１は、積層したものとすることもできる。細胞培養容器２１は薄い直方体状のため、その上下に積み重ねるように配置して積層にすることができる。そして、各開口部を並列的につなぐ配管を設けて循環ラインとつなぎ、一つの送液ポンプやガス交換手段により循環、ガス交換を行うものとすることができる。細胞培養容器は、薄い容器とすることができるため積層したものとしても全体として小容量で高密度な培養を行うことができる。このような細胞培養容器２１は、特開２０１８−３３３８７号公報や、特開２０１８−１４３２１０号公報に開示される細胞培養容器を参照して、これらに準じるものを用いることもできる。 The cell culture vessel 21 may be laminated. Since the cell culture container 21 has a thin rectangular parallelepiped shape, the cell culture container 21 can be arranged so as to be stacked above and below to be laminated. A pipe for connecting the openings in parallel may be provided to connect with the circulation line, and circulation and gas exchange may be performed by one liquid feed pump or gas exchange means. Since the cell culture container can be a thin container, it is possible to perform high-density culture with a small volume as a whole even when they are stacked. As such a cell culture container 21, with reference to the cell culture containers disclosed in JP-A-2018-33387 and JP-A-2018-143210, those similar thereto can be used.
［循環ライン３１１〜３１３］
細胞培養装置１０は、細胞培養容器２１に取り付けられ、細胞培養容器内の培地を循環するための循環ライン３１１、３１２、３１３を有する。循環ライン３１１〜３１３には、樹脂製のチューブ等を用いることができる。
[Circulation lines 311 to 313]
The cell culture device 10 is attached to the cell culture container 21 and has circulation lines 311, 312, and 313 for circulating the medium in the cell culture container. A resin tube or the like can be used for the circulation lines 311 to 313.
［送液ポンプ４１］
細胞培養装置１０は、循環ライン３１２、３１３間に設けられ細胞培養容器２１に培地を送液するための送液ポンプ４１を有する。送液ポンプ４１は、培地を送液できる任意のポンプを用いることができる。細胞培養装置１０、特に細胞培養容器２１の内容量や、培養する細胞の種類、培養する量等にもよるが、本発明の細胞培養装置で培養するような通常の細胞培養容器２１の大きさに対して、０．０１ｍＬ／ｍｉｎ〜１００ｍＬ／ｍｉｎ程度の送液ができるものなどを用いることができる。この送液は、培養中、連続的に送液し続けるものでもよいし、断続的に送液するものでもよい。また、培養初期は、送液量を少なくしてガス交換手段５１でガス交換する培地の量を抑制し、培養後期は送液量を多くしてガス交換手段５１でガス交換する培地の量を増加させることができるように、送液量を適宜変更できるものや、プログラムして運転することができるものが好ましい。
送液ポンプ４１の送液方向は、ガス交換手段５１から吸い込み方向となるＦ１１方向でもよいし、ガス交換手段５１に対して送り出し方向となるＦ１２方向でもよい。ガス交換手段５１から培地が送出されにくい条件のとき、ガス交換手段５１側に送り出すＦ１２方向として、積極的に送り出されるものとしてもよいし、ガス交換手段５１で、培地中に開口部５１３、５１４から導入されている調整用ガスを吸い込む負圧となりやすいようにガス交換手段５１から吸い込み方向となるＦ１１方向としてもよい。なお、送液ポンプは、１台でもよいが送液量やガス交換の効率等を考慮して、適宜複数台設けてもよい。
[Liquid feeding pump 41]
The cell culture device 10 has a liquid feed pump 41 provided between the circulation lines 312 and 313 for feeding the medium to the cell culture vessel 21. As the liquid feed pump 41, any pump that can feed the medium can be used. The size of a normal cell culture container 21 for culturing with the cell culture device of the present invention depends on the inner volume of the cell culture device 10, particularly the cell culture container 21, the type of cells to be cultured, the amount of culture, and the like. On the other hand, it is possible to use a material capable of sending a liquid of about 0.01 mL / min to 100 mL / min. The liquid may be continuously fed during the culture or may be intermittently fed. Further, in the initial stage of the culture, the liquid feeding amount is reduced to suppress the amount of the medium to be gas exchanged by the gas exchange means 51, and in the latter stage of the culture, the liquid feeding amount is increased to increase the amount of the medium to be gas exchanged by the gas exchange means 51. It is preferable that the amount of liquid to be sent can be appropriately changed or can be programmed and operated so that the amount can be increased.
The liquid feed direction of the liquid feed pump 41 may be the F11 direction which is the suction direction from the gas exchange means 51 or the F12 direction which is the feed direction from the gas exchange means 51. When the medium is difficult to be delivered from the gas exchange means 51, it may be positively delivered as the F12 direction to be delivered to the gas exchange means 51 side. In the gas exchange means 51, openings 513, 514 are formed in the medium. The F11 direction, which is the suction direction from the gas exchanging means 51, may be set so that a negative pressure for sucking the adjustment gas introduced from is easily generated. It should be noted that the liquid supply pump may be one, but a plurality of liquid supply pumps may be appropriately provided in consideration of the liquid supply amount, gas exchange efficiency, and the like.
［ガス交換手段５１］
細胞培養装置１０は、循環ライン３１１、３１２内に設けられ循環される培地に含まれるガスを調整用ガスとガス交換するためのガス交換手段５１を有する。
このガス交換手段５１は中空糸膜を有し、中空糸膜の内外に、それぞれ、培地または調整用ガスを流通させることで、中空糸膜を介して、培地と調整用ガスとでガス交換させる。
このガス交換手段は、例えば、複数の中空糸膜を有する中空糸束の両端がその中空部分を開口状態に保持されたまま中空部材内に固定された中空糸膜カートリッジ（いわゆる、ホローファイバーカートリッジ）を用いることができる。中空糸膜の内層又は外層の一方に循環ラインにより循環される培地が流通され、その他方に調整用ガスが流通され、中空糸膜を介して培地と調整用ガスとのガス交換が行われる。
細胞培養装置１０では、ガス交換手段５１の開口部５１１、５１２は、中空糸膜の外層に流通する流体の流路に連通させ、開口部５１３、５１４は中空糸膜の内層に流通する流体の流路に連通するものとして設計されている。このため、培地は、中空糸膜の外層を流通し、調整用ガスは内層を流通する。
[Gas exchange means 51]
The cell culture device 10 has a gas exchange means 51 provided in the circulation lines 311, 312 for exchanging the gas contained in the circulated culture medium with the adjustment gas.
The gas exchanging means 51 has a hollow fiber membrane, and the medium or the adjusting gas is passed through the inside and the outside of the hollow fiber membrane to exchange gas between the medium and the adjusting gas through the hollow fiber membrane. ..
This gas exchange means is, for example, a hollow fiber membrane cartridge (so-called hollow fiber cartridge) in which both ends of a hollow fiber bundle having a plurality of hollow fiber membranes are fixed in a hollow member while holding the hollow portions in the open state. Can be used. The medium circulated by the circulation line is circulated in one of the inner layer and the outer layer of the hollow fiber membrane, and the adjusting gas is circulated in the other side, and gas exchange between the medium and the adjusting gas is performed through the hollow fiber membrane.
In the cell culture device 10, the openings 511, 512 of the gas exchange means 51 are communicated with the flow path of the fluid flowing in the outer layer of the hollow fiber membrane, and the openings 513, 514 of the fluid flowing in the inner layer of the hollow fiber membrane. It is designed to communicate with the flow path. Therefore, the medium flows through the outer layer of the hollow fiber membrane, and the adjusting gas flows through the inner layer.
図５は、細胞培養装置の第一の実施形態の変形例を示すものである。細胞培養装置１０１は、ガス交換手段５１への循環ラインの接続状態を変更したものであり、循環ライン３１１は開口部５１３に接続し、循環ライン３１２は開口部５１４に接続した状態である。このように中空糸膜の内外のいずれに培地または調製用ガスを流通させるかは適宜選択して培養することができる。 FIG. 5 shows a modification of the first embodiment of the cell culture device. The cell culture device 101 is a device in which the connection state of the circulation line to the gas exchange means 51 is changed, the circulation line 311 is connected to the opening 513, and the circulation line 312 is connected to the opening 514. In this way, the inside or outside of the hollow fiber membrane through which the medium or the preparation gas is passed can be appropriately selected and cultured.
［中空糸膜の材質］
ガス交換手段５１内では、ガス交換のみ行われることが好ましい。このため中空糸膜は、液体の透過率が極めて低い、または透過しないものであり、ガスのみ選択的に透過するものであることが好ましい。このようなガス交換性を有するものとしては、例えば、シリコーン製の中空糸膜などを用いることができる。
[Material of hollow fiber membrane]
It is preferable that only gas exchange is performed in the gas exchange means 51. Therefore, the hollow fiber membrane has extremely low liquid permeability or does not permeate liquid, and it is preferable that only the gas selectively permeates. As a material having such a gas exchange property, for example, a hollow fiber membrane made of silicone can be used.
［第二の実施形態］
図６は、本発明の細胞培養装置の第二の実施形態を示す図である。細胞培養装置１１は、第一の細胞培養装置１０に加えて、さらに、培地の入れ替え等を行うことができる培地の移送ラインを有する。循環ライン３１１内に三方継手などを設けて配管６１１をつないでいる。また、循環ライン３１３にも三方継手などを設けて配管６１２をつないでいる。循環ライン３１１には開閉弁３２１が、配管６１１には開閉弁６２１が設けられており、循環ライン３１３には開閉弁３２２が、配管６１２には開閉弁６２２が設けられている。これらの開閉弁を調整することで、ガス交換手段５１を通る循環を停止して、配管６１１、配管６１２を介して細胞培養容器２１を系外とつなぐことができる。さらに、外部の送液ポンプ等を利用してＦ２１１方向およびＦ２２２方向で培地等を流通させたり、Ｆ２１２方向およびＦ２２１方向で培地等を流通させることができる。
この配管６１１、配管６１２を用いる構成は、運転開始時に、培養容器２１に培地等を充填するときや、細胞の種類や実験目的に応じて培養時期等に応じて、培養中に培地を入れ替えるとき、培養を終了し細胞を回収するときなどに用いることができる。
[Second embodiment]
FIG. 6 is a diagram showing a second embodiment of the cell culture device of the present invention. The cell culture device 11 has, in addition to the first cell culture device 10, a medium transfer line capable of changing the medium and the like. A three-way joint or the like is provided in the circulation line 311 to connect the pipe 611. The circulation line 313 is also provided with a three-way joint or the like to connect the pipe 612. The circulation line 311 is provided with an opening / closing valve 321, the pipe 611 is provided with an opening / closing valve 621, the circulation line 313 is provided with an opening / closing valve 322, and the pipe 612 is provided with an opening / closing valve 622. By adjusting these on-off valves, the circulation through the gas exchange means 51 can be stopped and the cell culture vessel 21 can be connected to the outside of the system via the pipe 611 and the pipe 612. Furthermore, the medium or the like can be circulated in the F211 direction and the F222 direction, or the medium or the like can be circulated in the F212 direction and the F221 direction by using an external liquid feed pump or the like.
The configuration using the pipes 611 and 612 is used when the culture container 21 is filled with a medium or the like at the start of operation, or when the medium is replaced during the culture according to the type of cells or the culture time according to the purpose of the experiment. It can be used when the culture is completed and the cells are collected.
［第三の実施形態］
図７は、本発明の細胞培養装置の第三の実施形態を示す図である。細胞培養装置１２は、第二の細胞培養装置１１に加えて、さらに、ガスポンプ７１と、フィルター７２を有する。第一の実施形態の細胞培養装置１０等に示すように、ガス交換手段５１の開口部５１３、５１４は単に開放して、雰囲気の空気等が流通するものとしてもよく、空気中の酸素や窒素と、ガス交換手段５１に流通される培地とでガス交換し、空気中の酸素濃度等に対応したガス濃度に調整することもできる。
細胞培養装置１２は、ガスポンプ７１、フィルター７２により、さらに積極的にガス交換量の制御等を行うものである。ガスポンプ７１は、雰囲気の空気の流通量を上昇させてガス交換量を向上させるものでもよいし、ガスポンプ７１で送り出すガスを、ガス濃度を調整したものとしてもよい。
[Third embodiment]
FIG. 7 is a diagram showing a third embodiment of the cell culture device of the present invention. The cell culture device 12 has a gas pump 71 and a filter 72 in addition to the second cell culture device 11. As shown in the cell culture device 10 and the like of the first embodiment, the openings 513 and 514 of the gas exchange means 51 may be simply opened so that air or the like in the atmosphere can circulate, and oxygen or nitrogen in the air can be used. It is also possible to perform gas exchange between the medium and the medium that is circulated in the gas exchange means 51, and adjust the gas concentration to correspond to the oxygen concentration in the air.
The cell culture device 12 is configured to more positively control the amount of gas exchange and the like by using the gas pump 71 and the filter 72. The gas pump 71 may increase the flow rate of the air in the atmosphere to improve the gas exchange amount, or the gas delivered by the gas pump 71 may have the gas concentration adjusted.
［調整用ガス］
本発明の細胞培養装置は、ガス交換手段５１で、調整用ガスと培地とのガス交換を行う。この調製用ガスは、培地のガス濃度を調整するためのものである。調製用ガスは、培地で消費されている酸素を供給するために空気でもよい。また、培養対象に応じて、適宜、酸素濃度や、窒素濃度、二酸化炭素濃度、湿度などを調整したものを用いてもよい。このような各種ガス濃度等が調整されたガスを安定供給するために、前述したようなガスポンプ７１を用いて調整用ガスが収容されているタンク等のガスを供給する構成とすることができる。また、このとき、調整用ガスから不純物等が流入したり、ガス交換されたガス中の不純物や副生物を除去するために、適宜、フィルターを介してガス交換手段に導入・導出するガスを調整することもできる。
本発明の細胞培養装置は、細胞培養中に、実質的に細胞培養容器等に含まれる培地が外気と接する場が、ガス交換手段内でのみとする構成のため、このガス交換手段内のガスの制御や管理を行うだけで培養系の管理ができる点で優れている。
これは、例えば、細胞培養容器を常温低湿度の環境においたままの安定した培養もできることを意味し、細胞培養しながら顕微鏡観察を容易に行ったり、細胞培養環境の清浄度が低くともガス交換手段のみ清浄度を高くすることで細胞培養の系内は汚染されない培養ができるといった利点を有する。
[Adjusting gas]
In the cell culture device of the present invention, the gas exchange means 51 performs gas exchange between the adjusting gas and the medium. This adjusting gas is for adjusting the gas concentration of the medium. The conditioning gas may be air to supply the oxygen consumed in the medium. Moreover, you may use what adjusted oxygen concentration, nitrogen concentration, carbon dioxide concentration, humidity, etc. suitably according to the culture object. In order to stably supply the gas whose various gas concentrations are adjusted, the gas pump 71 as described above may be used to supply the gas in the tank or the like in which the adjusting gas is stored. Also, at this time, in order to remove impurities and by-products from the gas for adjustment and to remove impurities and by-products in the gas that has undergone gas exchange, the gas that is introduced into and discharged from the gas exchange means is appropriately adjusted through a filter. You can also do it.
In the cell culture device of the present invention, during the cell culture, the medium substantially contained in the cell culture container or the like is in contact with the outside air only in the gas exchange means, and therefore the gas in the gas exchange means is It is excellent in that the culture system can be managed simply by controlling and controlling the culture.
This means, for example, that stable culture can be performed while the cell culture vessel is kept in an environment of room temperature and low humidity, and microscopic observation can be easily performed while culturing cells, or gas exchange can be performed even if the cell culture environment is not clean. By increasing the cleanliness only by means, there is an advantage that the culture of the cell culture system can be performed without contamination.
［第四の実施形態］
図８は、本発明の細胞培養装置に用いられる細胞培養容器の他の実施形態を示すものである。本発明の細胞培養装置において、細胞培養容器は、積層された板状部を有するものであることが好ましい。細胞培養容器２１は、それぞれ板状部を有し、容器内の上下の各面が細胞培養時に細胞が接着する接着面となる。このような細胞培養容器２１は、複数層積層して用いてもよい。図８においては、６層積層したものである。このとき、各細胞培養容器２１の開口部２１１、２１２は、開口部２１１側を多岐管２１３で連結することができる。多岐管２１３は、細胞培養容器２１の開口部２１１側に分岐を有し、循環ライン３１１側は集約され接続口２１３１の１か所で連結される。同様に、開口部２１２側は、多岐管２１４を介して循環ライン３１３と連結することができる。このような構成とすれば、一の循環ラインや送液ポンプ、ガス交換手段でも、複数の細胞培養容器に、培地循環することができ、省スペースで効率的に多量の接着性の細胞培養を行うことができる。さらに、板状部を有する細胞培養容器を用いているため、培養後の細胞の回収も操作性に優れ、効率的に行うことができる。なお、細胞培養容器の中空状の内層が積層される数は、特に制限はなく、例えば、２以上や、３以上、５以上とすることができ、２０以下や、１５以下、１２以下とすることができる。このとき、各層を構成する部分ごとに分離できるものでもよいし、１枚の板が上下の仕切りとなるように共通するものとしてもよい。
[Fourth Embodiment]
FIG. 8 shows another embodiment of the cell culture container used in the cell culture device of the present invention. In the cell culture device of the present invention, it is preferable that the cell culture container has laminated plate-shaped portions. Each of the cell culture vessels 21 has a plate-shaped portion, and the upper and lower surfaces in the vessel serve as adhesion surfaces to which cells adhere during cell culture. Such a cell culture vessel 21 may be used by stacking a plurality of layers. In FIG. 8, 6 layers are laminated. At this time, the openings 211 and 212 of each cell culture vessel 21 can be connected to each other on the side of the opening 211 by the manifold 213. The manifold 213 has a branch on the side of the opening 211 of the cell culture vessel 21, and the side of the circulation line 311 is integrated and connected at one place of the connection port 2131. Similarly, the opening 212 side can be connected to the circulation line 313 via the manifold 214. With such a configuration, it is possible to circulate the medium in a plurality of cell culture vessels with a single circulation line, a liquid feed pump, or a gas exchange means, and a large amount of adhesive cell culture can be efficiently performed in a space-saving manner. It can be carried out. Further, since the cell culture container having the plate-shaped portion is used, the cells after the culture can be recovered with excellent operability and efficiently. The number of hollow inner layers of the cell culture container to be stacked is not particularly limited and may be, for example, 2 or more, 3 or more, 5 or more, and 20 or less, 15 or less, 12 or less. be able to. At this time, it may be one that can be separated for each part constituting each layer, or may be common so that one plate serves as an upper and lower partition.
［培養方法］
本発明は、このような細胞培養装置を用いた培養方法に関する。本発明の培養方法は、一部前述したように、中空状の内層を有する細胞培養容器に培地および細胞を充填する充填工程と、前記細胞培養容器に充填された前記細胞を培養する培養工程と、前記細胞培養容器に取り付けられた前記培地を循環するための循環ライン内に設けられた送液ポンプにより前記循環ライン内の前記培地を送液することで前記循環ライン内の前記培地を循環する循環工程と、前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段によりガス交換するガス交換工程とを有する。
[Culturing method]
The present invention relates to a culture method using such a cell culture device. The culturing method of the present invention comprises, as described above in part, a filling step of filling a medium and cells into a cell culture container having a hollow inner layer, and a culturing step of culturing the cells filled in the cell culture container. , Circulating the medium in the circulation line by feeding the medium in the circulation line with a liquid feed pump provided in a circulation line attached to the cell culture container for circulating the medium. Gas for gas exchange by a circulation step and a gas exchange means having a hollow fiber membrane for gas exchange between the gas contained in the medium and the adjustment gas provided in the circulation line and circulated in the circulation line And a replacement process.
［充填工程］
本発明の培養方法は、中空状の内層を有する細胞培養容器に培地および細胞を充填する充填工程を有する。この充填工程は、細胞培養容器内や、循環ライン等の空気層を細胞懸濁液や培地で満たすように充填する。また、この充填にあたっては、培養対象となる細胞と培地とを細胞培養容器に充填する。細胞懸濁液は、培地を先に充填して細胞を播種することで細胞懸濁液としてもよいし、予め培地に細胞を播種して細胞懸濁液とした状態で、細胞培養容器等に充填してもよい。また、細胞培養容器等から空気層を取り除くために、適宜、脱気して充填したり、排気路を設けて除去してもよい。
[Filling process]
The culturing method of the present invention has a filling step of filling a cell culture container having a hollow inner layer with a medium and cells. In this filling step, the cell culture container and the air layer such as the circulation line are filled with the cell suspension and the medium. Further, in this filling, the cells to be cultured and the medium are filled in the cell culture container. The cell suspension may be made into a cell suspension by first filling the medium with cells and then seeding the cells, or in a state where the cells are previously seeded into the medium to give a cell suspension, and then placed in a cell culture container or the like. It may be filled. Further, in order to remove the air layer from the cell culture container or the like, it may be appropriately deaerated and filled, or an exhaust passage may be provided to remove the air layer.
［培養工程］
本発明の培養方法は、細胞培養容器に充填された細胞を培養する培養工程を有する。なお、培養工程で、細胞は、細胞懸濁液の状態で培養される必要はなく、細胞培養容器に接着した状態で培養される。
[Culture process]
The culturing method of the present invention has a culturing step of culturing cells filled in a cell culture container. In the culturing step, the cells need not be cultivated in the state of cell suspension, but are cultivated in the state of being adhered to the cell culture container.
［細胞］
培養の対象となる細胞は、特に限定しないでもよい。細胞培養容器内に接着して培養することが好ましいため、接着性を有する細胞などが好適な培養対象となる。例えば、ＥＳ細胞や、ｉＰＳ細胞、幹細胞、線維芽細胞、ＣＨＯ（Chinese Hamster Ovary）細胞、ＨＥＫ２９３（ヒト胎児腎細胞２９３：Human Embryonic Kidney cells 293）細胞、脂肪細胞などが挙げられる。
[cell]
The cells to be cultured may not be particularly limited. Since it is preferable that the cells are adhered and cultured in the cell culture container, cells having adhesiveness and the like are suitable culture targets. Examples thereof include ES cells, iPS cells, stem cells, fibroblasts, CHO (Chinese Hamster Ovary) cells, HEK 293 (Human Embryonic Kidney cells 293) cells, and adipocytes.
［培地］
本発明の細胞培養に当たって用いる培地は、循環させるため液状培地のような流動性が高い培地であることが好ましい。培地の具体的な組成は、培養対象や、培養目的に応じて適宜調製したものを用いることができる。例えば、ＤＭＥＭ培地（ダルベッコ改変イーグル培地）、ＭＥＭ培地（Minimum Essential. Medium）、ＳｔｅｍＦｉｔ（登録商標）培地などや、これらを基に適宜成分調整を行ったり、培養目的等に応じて任意の成分を混合したものなどを用いることができる。
[Culture medium]
The medium used for cell culture of the present invention is preferably a medium having high fluidity such as a liquid medium for circulation. The specific composition of the medium may be appropriately prepared according to the subject of culture and the purpose of culture. For example, DMEM medium (Dulbecco's modified Eagle medium), MEM medium (Minimum Essential. Medium), StemFit (registered trademark) medium, and the like, and appropriate components are adjusted based on these, or any component may be added depending on the purpose of culture and the like. A mixture or the like can be used.
［播種］
培養開始時は、細胞懸濁液などを調整して培地および細胞を細胞培養容器に充填させる。このとき、培養対象の細胞が細胞培養容器の内壁に接着するまでは、培地の循環を停止して培養することが好ましい。播種する細胞量は、細胞の種類や培地との相性、培養期間等を鑑みて適宜設定される。
[Seeding]
At the start of culturing, a cell suspension or the like is prepared and a medium and cells are filled in a cell culture container. At this time, it is preferable to stop the circulation of the medium and culture until the cells to be cultured adhere to the inner wall of the cell culture container. The amount of cells to be seeded is appropriately set in consideration of the type of cells, the compatibility with the medium, the culture period, and the like.
細胞培養するときの温度は、その細胞の至適温度や、実験条件とする温度などで制御することができる。なお、細胞培養中の培地は外気と接する場が、ガス交換手段に限られるため、湿度などのガスの条件を制御する必要性は低く、ガス交換手段雰囲気に限定した管理でもよい。 The temperature at which the cells are cultured can be controlled by the optimum temperature of the cells or the temperature used as an experimental condition. Since the medium in which the cell culture is in contact with the outside air is limited to the gas exchange means, there is little need to control the conditions of the gas such as humidity, and management may be limited to the atmosphere of the gas exchange means.
［循環工程］
本発明の細胞培養方法は、細胞培養容器に取り付けられた培地を循環するための循環ライン内に設けられた送液ポンプにより循環ライン内の培地を送液することで循環ライン内の培地を循環する循環工程を有する。この循環工程は、細胞培養容器内で細胞が接着して安定したころから、適宜、連続的や断続的に循環させながら、培養工程と併行して行われる。
[Circulation process]
The cell culture method of the present invention circulates the medium in the circulation line by feeding the medium in the circulation line with a liquid feed pump provided in the circulation line for circulating the medium attached to the cell culture vessel. It has a circulation process to. This circulation step is carried out in parallel with the culture step while appropriately or continuously circulating the cells after the cells are adhered and stabilized in the cell culture container.
［ガス交換工程］
本発明の細胞培養方法は、前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段によりガス交換するガス交換工程とを有する。このガス交換は、限られた場でのガス交換であり、培養条件の管理が行いやすい。一方で、ガス交換手段内では、培地と、調整用ガスとの接触面積を中空糸膜の表面積に応じて広いものとすることができるため、調整用ガスを制御すると優れた応答性でガス交換させて培地のガス濃度を制御することもできる。
[Gas exchange process]
The cell culture method of the present invention comprises a gas exchange means having a hollow fiber membrane for performing gas exchange between a gas contained in the medium and a regulating gas provided in the circulation line and circulated in the circulation line. And a gas exchange step of exchanging gas. This gas exchange is a gas exchange in a limited place, and it is easy to manage the culture conditions. On the other hand, in the gas exchange means, the contact area between the culture medium and the adjusting gas can be widened according to the surface area of the hollow fiber membrane, so that controlling the adjusting gas allows gas exchange with excellent responsiveness. It is also possible to control the gas concentration of the medium.
さらに、本発明の培養では、培地が循環されるため、細胞培養容器に連続的に供給されながら培養される。このため、細胞培養容器内に仮に空気が一時的に含まれても、その空気は容器外に押し出されるように培養することもできる。押し出された空気はガス交換手段の中空糸膜に補足されたり、ガス交換されて調整用ガス側に吐き出されながら培養できる。このため、細胞培養容器内等の系内は空気層が実質的にない状態での培養ができる。 Furthermore, in the culture of the present invention, since the medium is circulated, it is cultured while being continuously supplied to the cell culture vessel. Therefore, even if air is temporarily contained in the cell culture container, the air can be cultured so as to be pushed out of the container. The extruded air can be supplemented by the hollow fiber membrane of the gas exchanging means, or can be cultured while exchanging the gas and being exhaled to the adjusting gas side. Therefore, the culture can be performed in the system such as the cell culture container without the air layer substantially.
本発明の細胞培養装置および細胞培養方法は、細胞培養容器内の培地のガス濃度の制御に適している。さらには、培養条件や、培養装置の構成を適宜調整することで、種々の利点を有する培養が可能となる。例えば、湿度制御する場を限定的なものとして周辺環境や装置負荷が低く自由度が高い培養を行うこともできる。また、培養中の培地交換も容易に行うこともできる。また、コンタミを低減した閉鎖系の培養を行うこともできる。また、小型化に適しており、省スペースで大量培養することもできる。さらに、閉鎖系で、大量培養を安定して行うことができるため、自動化にも適している。 INDUSTRIAL APPLICABILITY The cell culture device and cell culture method of the present invention are suitable for controlling the gas concentration of the medium in the cell culture vessel. Furthermore, by appropriately adjusting the culture conditions and the configuration of the culture device, it becomes possible to perform culture with various advantages. For example, it is possible to perform a culture with a low degree of freedom in the surrounding environment or a device by limiting the place where humidity is controlled. Also, the medium exchange during the culture can be easily performed. It is also possible to perform a closed system culture with reduced contamination. Further, it is suitable for downsizing, and it is possible to carry out mass culture in a space-saving manner. Furthermore, it is suitable for automation because it enables stable large-scale culture in a closed system.
以下、実施例により本発明を更に詳細に説明するが、本発明は、その要旨を変更しない限り以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is changed.
［評価項目］
・細胞数
細胞計数装置ＮＣ−１００（株式会社エステムテクノシステムズ社）を用いて細胞数を測定した。
・溶存酸素
ＤＯ計（Ｓｃｉｅｎｔｉｆｉｃ Ｂｉｏｐｒｏｃｅｓｓｉｎｇ社製）により、溶存酸素濃度を測定した。なお、培養温度（３７℃）における水の飽和溶存酸素濃度に対する比率で、溶存酸素濃度（％）を求めた。
・顕微鏡観察
倒立顕微鏡（オリンパス社製）により、培養された細胞の状態を顕微観察した像を撮像した。
[Evaluation item]
-Cell number The cell number was measured using the cell counter NC-100 (Estem Techno Systems Co., Ltd.).
-Dissolved oxygen The dissolved oxygen concentration was measured with a DO meter (manufactured by Scientific Bioprocessing). The dissolved oxygen concentration (%) was determined by the ratio to the saturated dissolved oxygen concentration of water at the culture temperature (37 ° C).
-Microscopic observation An image obtained by microscopically observing the state of the cultured cells was taken with an inverted microscope (manufactured by Olympus).
［細胞培養装置の構成］
・細胞培養器
板厚約３ｍｍのポリスチレン製の板を上下の板として用いて、幅２６０ｍｍ、長さ２１０ｍｍ、上下の板間隔８ｍｍの薄い略直方体状の細胞培養容器として使用した。下側に用いる板の内面側は、プラズマ処理を行い接着性が向上するものとした。側壁部分は、ガス透過性のシリコーン膜を四方に配置し、四隅に剛性が高いポリスチレン製の柱を設けて高さを保持するものとした（特開２０１８−３３３８７号公報参照）。さらに、上面の板に幅方向の両端付近に２か所貫通孔を設け循環ライン用チューブの接続部とした。
・循環ライン
シリコーン製チューブにより、循環ラインを設けた。
・送液ポンプ
送液量約２０ｍＬ／ｍｉｎまで送液可能な送液ポンプ（ウエルコ社製）を、循環ラインのチューブに接続して送液した。培養開始時は約１〜５ｍＬ／ｍｉｎの送液とし、培養状態を観察しながら培養後期は５〜１０ｍＬ／ｍｉｎの送液を行った。
・シリコーン中空糸ガス分離膜カラム
シリコーン製の中空糸膜を内蔵するシリコーン中空糸ガス分離膜（永柳工業株式会社製「Ｍ６０（膜本数：４５００本）」）を、ガス交換手段として用いた。
[Configuration of cell culture device]
Cell culture device A polystyrene plate having a plate thickness of about 3 mm was used as the upper and lower plates, and used as a thin rectangular cell culture container having a width of 260 mm, a length of 210 mm, and an upper and lower plate interval of 8 mm. The inner surface of the plate used for the lower side was treated with plasma to improve the adhesiveness. In the side wall portion, a gas permeable silicone film was arranged in four directions, and polystyrene columns having high rigidity were provided in the four corners to maintain the height (see JP-A-2018-33387). Further, two through holes were provided in the upper plate near both ends in the width direction to serve as a connecting portion for the circulation line tube.
-Circulation line A circulation line was provided using a silicone tube.
-Liquid-feeding pump A liquid-feeding pump (manufactured by Welco) capable of feeding up to a liquid-feeding amount of about 20 mL / min was connected to the tube of the circulation line to feed the liquid. At the start of the culture, the solution was delivered at about 1 to 5 mL / min, and at the latter stage of the culture, the solution was delivered at 5 to 10 mL / min while observing the culture state.
-Silicone hollow fiber gas separation membrane column A silicone hollow fiber gas separation membrane containing a hollow fiber membrane made of silicone ("M60 (number of membranes: 4500)" manufactured by Nagayanagi Industry Co., Ltd.) was used as a gas exchange means.
［試料等］
・細胞
株式会社アステック内で継代培養している、ＭＲＣ−５（人、肺組織、正常二倍体線維芽細胞, 胎児肺由来）を用いた。
・培地
ＭＥＭ培地に牛胎児血清（ＦＢＳ）を１０％添加したものを培養用培地として用いた。
・初期播種細胞数
前記細胞４×１０6ｃｅｌｌｓを、細胞培養容器内に播種して、培養を開始した。
[Samples, etc.]
-Cells MRC-5 (human, lung tissue, normal diploid fibroblast, fetal lung-derived) subcultured in Astec Co., Ltd. was used.
-Medium A MEM medium containing 10% fetal bovine serum (FBS) was used as a culture medium.
-Number of cells initially seeded 4x10 6 cells were seeded in a cell culture container to start culture.
［実施例１］
第一の実施形態に準じる構成で、前記細胞培養容器に、循環ライン、送液ポンプ、シリコーン中空糸ガス分離膜カラム（ガス交換手段）を取り付けて細胞培養を行った。培養温度は、温度３７℃、ＣＯ2濃度５％のＣＯ2インキュベーターで培養した。
[Example 1]
With the configuration according to the first embodiment, a cell line was cultivated by attaching a circulation line, a liquid feed pump, and a silicone hollow fiber gas separation membrane column (gas exchange means) to the cell culture container. The culture temperature was 37 ° C., and the culture was performed in a CO 2 incubator with a CO 2 concentration of 5%.
［比較例１］
細胞培養容器単独で、循環ライン、送液ポンプ、シリコーン中空糸ガス分離膜カラムを取り付けずに、細胞培養を行った。
[Comparative Example 1]
Cell culture was carried out using the cell culture container alone, without attaching a circulation line, a liquid feed pump, and a silicone hollow fiber gas separation membrane column.
［評価結果］
培養開始から、３日目および７日目の溶存酸度濃度（細胞培養容器の平面視方向（略長方形）の中央付近と、端部付近）と、７日培養後の細胞数を測定した。測定結果を表１に示す。また、７日時点での顕微鏡観察結果を図９、図１０に示す。図９は比較例１、図１０は実施例１の顕微観察結果である。
[Evaluation results]
On the 3rd and 7th days from the start of the culture, the dissolved acidity concentration (near the center and near the end in the plan view direction (substantially rectangular) of the cell culture container) and the number of cells after 7 days of culture were measured. The measurement results are shown in Table 1. The results of microscopic observation at 7 days are shown in FIGS. 9 and 10. 9 shows the results of microscopic observation of Comparative Example 1, and FIG. 10 shows the results of microscopic observation of Example 1.
実施例１、比較例１に用いた細胞培養容器は、側壁にシリコーン製のガス交換部を有するため、培養開始から３日目までは、いずれの培養も中央・端部ともに高い溶存酸素濃度を維持した培養ができた。しかしながら、培養が進行し、細胞数の増加等に伴い、酸素消費量が増加すると、比較例１はガス交換された酸素が中央まで到達できなくなったと考えられ、中央の溶存酸素濃度が低下した。一方、実施例１は、培地のガス交換を行いながら循環しているため、中央も溶存酸素濃度が高い状態が維持された。また、培養７日目の細胞数は、実施例１の方が顕著に多かった。顕微観察像からも、実施例１の方が、細胞密度が高いことが確認される。 Since the cell culture vessels used in Example 1 and Comparative Example 1 have a gas exchange section made of silicone on the side wall, from the start of the culture to the third day, both cultures had a high dissolved oxygen concentration in both the center and the end. Maintained culture was possible. However, when the oxygen consumption increased as the culture proceeded and the number of cells increased, in Comparative Example 1, it was considered that the gas-exchanged oxygen could not reach the center, and the dissolved oxygen concentration in the center decreased. On the other hand, in Example 1, since the medium was circulated while exchanging gas, the state where the dissolved oxygen concentration was high was maintained in the center as well. The number of cells on day 7 of culture was significantly higher in Example 1. From the microscopic observation image, it is confirmed that the cell density of Example 1 is higher.
本発明の細胞培養装置および細胞培養方法は、各種細胞の細胞培養に利用することができ、細胞培養容器内に空気層を必要とせずに培養ができ、閉鎖系で、大量の細胞培養にも適しており、産業上有用である。 INDUSTRIAL APPLICABILITY The cell culture device and cell culture method of the present invention can be used for cell culture of various cells, can perform culture without requiring an air layer in the cell culture container, and can be used for large-scale cell culture in a closed system. Suitable and industrially useful.
１０、１０１、１１、１２ 細胞培養装置
２１ 細胞培養容器
２１１、２１２ 開口部
２１３、２１４ 多岐管
２１３１ 接続部
３１１〜３１３ 循環ライン
３２１、３２２ 開閉弁
４１ 送液ポンプ
５１ ガス交換手段
５１１〜５１４ 開口部
６１１、６１２ 配管
６２１、６２２ 開閉弁
７１ ガスポンプ
７２ フィルター
10, 101, 11, 12 Cell culture device 21 Cell culture vessel 211, 212 Opening part 213, 214 Manifold 2131 Connection part 311 to 313 Circulation line 321 and 322 Open / close valve 41 Liquid transfer pump 51 Gas exchange means 511 to 514 Opening part 611, 612 Piping 621, 622 Open / close valve 71 Gas pump 72 Filter
Claims (7)
前記細胞培養容器に取り付けられ、前記培地を循環するための循環ラインと、
前記循環ライン内に設けられ前記細胞培養容器に前記培地を送液する送液ポンプと、
前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段と、を有する細胞培養装置。 A cell culture vessel having a hollow inner layer for filling a medium and cells,
A circulation line attached to the cell culture vessel for circulating the medium,
A liquid feed pump that is provided in the circulation line and feeds the medium to the cell culture container,
A cell culture device comprising: a gas exchange unit having a hollow fiber membrane, which is provided in the circulation line and exchanges a gas contained in the culture medium circulated in the circulation line with a regulating gas.
前記細胞培養容器に充填された前記細胞を培養する培養工程と、
前記細胞培養容器に取り付けられた前記培地を循環するための循環ライン内に設けられた送液ポンプにより前記循環ライン内の前記培地を送液することで前記循環ライン内の前記培地を循環する循環工程と、
前記循環ライン内に設けられ前記循環ライン内で循環される前記培地に含まれるガスと調整用ガスとのガス交換をするための中空糸膜を有するガス交換手段によりガス交換するガス交換工程と、を有する細胞培養方法。 A filling step of filling a cell culture container having a hollow inner layer with a medium and cells,
A culturing step of culturing the cells filled in the cell culture container,
A circulation in which the medium in the circulation line is circulated by feeding the medium in the circulation line by a liquid feeding pump provided in a circulation line attached to the cell culture container for circulating the medium. Process,
A gas exchange step of performing gas exchange by a gas exchange means having a hollow fiber membrane for performing gas exchange between the gas contained in the medium and the adjusting gas provided in the circulation line and circulated in the circulation line, A method for culturing cells.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018207474A JP6890833B2 (en) | 2018-11-02 | 2018-11-02 | Cell culture device and cell culture method |
| PCT/JP2019/042323 WO2020090799A1 (en) | 2018-11-02 | 2019-10-29 | Cell culture device and cell culture method |
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