JP2011177046A - Method and apparatus for culturing cell - Google Patents

Method and apparatus for culturing cell Download PDF

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JP2011177046A
JP2011177046A JP2010042076A JP2010042076A JP2011177046A JP 2011177046 A JP2011177046 A JP 2011177046A JP 2010042076 A JP2010042076 A JP 2010042076A JP 2010042076 A JP2010042076 A JP 2010042076A JP 2011177046 A JP2011177046 A JP 2011177046A
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cylindrical member
culture
cells
cell
cell culture
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Ryoichi Haga
良一 芳賀
Keisuke Shibuya
啓介 渋谷
Masaru Nanba
勝 難波
Ken Amano
研 天野
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Hitachi Plant Technologies Ltd
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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
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture method for efficiently culturing cells by preventing the attachment of the cells or the like to the surface of a cylindrical member, and to provide a culture apparatus. <P>SOLUTION: The method for culturing the cells in a medium for culturing the cells, stored in a culture vessel 1 so that one end of the cylindrical member 2 may be exposed to the exterior, includes feeding an oxygen-containing gas as bubbles for dividing the interior of the cylindrical member in the vertical direction. The culture apparatus is also provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は、培養液に筒状部材を配置し、当該筒状部材内部に酸素含有ガスを供給しながら当該筒状部材外部において細胞を培養する細胞培養方法及び細胞培養装置に関する。   The present invention relates to a cell culturing method and a cell culturing apparatus in which a cylindrical member is disposed in a culture solution and cells are cultured outside the cylindrical member while supplying an oxygen-containing gas inside the cylindrical member.

一般に、動物細胞や、植物細胞、細菌などを対象として培養槽内に張り込んだ培養液で培養することで細胞自体或いは細胞が産生する物質を製造することが行われており、特に培養規模を大きくしてより生産性を高めることが望まれている。このような細胞培養においては、嫌気性の細胞を対象とする以外、培地中に酸素を供給する必要がある。例えば、特許文献１には、コイル状に整形した多孔質チューブの表面から酸素供給を行うことを特徴とする培養装置に関する記載がある。また、特許文献２には，撹拌翼にガス透過膜を固定して回転させる培養槽に関する記載がある。   In general, it is practiced to produce animal cells, plant cells, bacteria, etc., by culturing them in a culture medium that has been placed in a culture tank, and in particular the production scale of the cells. It is desired to increase the productivity by increasing the size. In such cell culture, it is necessary to supply oxygen into the medium except for anaerobic cells. For example, Patent Document 1 describes a culture apparatus characterized in that oxygen is supplied from the surface of a porous tube shaped into a coil shape. Patent Document 2 describes a culture tank in which a gas permeable membrane is fixed to a stirring blade and rotated.

生体細胞の培養では、目的生産物の生産性向上のために生体細胞を高密度で培養することが望まれている。しかし、高密度で細胞を培養した場合など、細胞や細胞塊が酸素供給を行うガス透過膜や上記多孔質チューブ等に付着してしまい、効率的な酸素供給を行えないといった問題が生じることがある。   In the culture of biological cells, it is desired to culture biological cells at a high density in order to improve the productivity of the target product. However, when cells are cultured at a high density, the cells and cell clumps may adhere to the gas permeable membrane that supplies oxygen, the porous tube, and the like, resulting in a problem that efficient oxygen supply cannot be performed. is there.

細胞のなかでも、付着依存性を有する細胞の場合には、表面または内部に細胞が付着して増殖することが可能な培養面を有するマイクロキャリアと称される微細な粒子が使用される。非特許文献１には、マイクロキャリア培養において、培養に好適なマイクロキャリア密度があること、及び過剰な撹拌は大きな増殖阻害を引き起こすことが記載されている。   Among the cells, in the case of cells having adhesion dependence, fine particles called microcarriers having a culture surface on which the cells can adhere and grow can be used. Non-Patent Document 1 describes that, in microcarrier culture, there is a microcarrier density suitable for culture, and that excessive agitation causes great growth inhibition.

マイクロキャリア培養において培養液に酸素を供給するシステムとしては以下のような特許文献３〜６が挙げられる。特許文献３には、培養槽外に酸素供給槽を設け、培養槽内に設置した振動スクリーンによってマイクロキャリアを除いた培養液を酸素供給槽に導き、酸素供給槽内で液中通気法によって酸素を溶解させた後、培養槽に循環させることを特徴とする培養装置に関する記載がある。特許文献４には、培養槽外に酸素供給用の循環管路を設け，培養槽内に設置したスクリーンによって生体細胞を含む固形粒子を除いた培養液を循環させ、循環管路の途中に酸素含有ガスを注入して酸素を溶解させることを特徴とする生体触媒反応装置に関する記載がある。特許文献５には、細胞増殖用の充填ボデイを充填した円筒カラム型培養装置において、培養装置底部に接する部分に前記の充填ボデイの侵入を防止しかつ培養液を通過せしめる穴を設けた空気拡散管を配し、該空気拡散菅内部に空気入り口を設けて酸素供給と培養液の循環を行わせることを特徴とする培養装置に関する記載がある。特許文献６には、培養槽内にマイクロキャリアの通過を阻止し培養液を通過せしめる微細な孔を有する部材で囲まれ、撹拌軸の回転によって振動する酸素供給ゾーンを設け、マイクロキャリアの存在しない酸素供給ゾーンに酸素含有ガスを液中通気することを特徴とする培養装置に関する記載がある。   Examples of systems for supplying oxygen to the culture medium in microcarrier culture include the following Patent Documents 3 to 6. In Patent Document 3, an oxygen supply tank is provided outside the culture tank, and a culture solution from which microcarriers have been removed is guided to the oxygen supply tank by a vibrating screen installed in the culture tank, and oxygen is supplied by submerged aeration in the oxygen supply tank. There is a description of a culture apparatus characterized in that after being dissolved, it is circulated in a culture tank. In Patent Document 4, a circulation pipe for supplying oxygen is provided outside the culture tank, and a culture solution excluding solid particles including biological cells is circulated by a screen installed in the culture tank. There is a description of a biocatalytic reactor characterized in that oxygen is dissolved by injecting a contained gas. Patent Document 5 discloses that in a cylindrical column type culture apparatus filled with a cell growth packing body, an air diffusion having a hole that prevents the packed body from entering and allows a culture solution to pass through a portion in contact with the bottom of the culture apparatus. There is a description of a culture apparatus characterized in that a tube is provided and an air inlet is provided inside the air diffusion chamber to supply oxygen and circulate the culture solution. In Patent Literature 6, an oxygen supply zone that is surrounded by a member having fine holes that prevent the passage of the microcarrier and allow the culture medium to pass therethrough is provided in the culture tank, and vibrates by the rotation of the stirring shaft. There is a description of a culture apparatus characterized in that an oxygen-containing gas is passed through the oxygen supply zone.

特開平3-272678公報JP-A-3-272678 特開平5-304943公報JP-A-5-304943 特開平6-269274公報JP-A-6-269274 特開平3-43070公報JP-A-3-43070 特開平5-276926公報Japanese Patent Application Laid-Open No. 5-76926 特開平5-252933公報Japanese Patent Application Laid-Open No. H5-252933

バイオテクノロジー アンド バイオエンジニアリング（Biotechnology and Bioengineering）第32巻975頁〜982頁（1988年発行）Biotechnology and Bioengineering, Vol. 32, pages 975-982 (issued in 1988)

ところが、上述した従来の手法は、細胞やマイクロキャリア等の密度を高密度化するために必要となる酸素の供給方法に主眼がおかれて開発されたものであるが、酸素供給手段の表面や、酸素供給ゾーンを区画する微細な孔を有する部材の表面に細胞やマイクロキャリア等が付着してしまうといった問題があった。このような問題が生じると、培養液中の溶存酸素濃度を適切に制御することが困難となり、効率的な細胞培養を達成できないといった問題が生じる。   However, the above-described conventional method has been developed with a focus on a method for supplying oxygen necessary for increasing the density of cells, microcarriers, and the like. There has been a problem that cells, microcarriers, and the like adhere to the surface of a member having fine pores that define the oxygen supply zone. When such a problem arises, it becomes difficult to appropriately control the dissolved oxygen concentration in the culture solution, resulting in a problem that efficient cell culture cannot be achieved.

そこで、本発明は、上述したような実情に鑑み、培養液に筒状部材を配置し、当該筒状部材内部に酸素含有ガスを供給しながら当該筒状部材外部において細胞を培養するに際して、当該筒状部材の表面に対する細胞等の付着を防止することで、効率的に細胞を培養することができる細胞の培養方法及び細胞培養装置を提供することを目的とする。   Therefore, in view of the above situation, the present invention is arranged when a cell is cultured outside the cylindrical member while arranging the cylindrical member in the culture solution and supplying an oxygen-containing gas inside the cylindrical member. An object of the present invention is to provide a cell culture method and a cell culture apparatus that can efficiently culture cells by preventing adhesion of cells and the like to the surface of a cylindrical member.

上記の目的を達成する本発明は、以下の内容を包含する。   The present invention for achieving the above object includes the following contents.

すなわち、本発明に係る細胞の培養方法は、側面に細孔を有し、長手方向の一方端部が開放されるとともに他方端部が封止された筒状部材と、上記他方端部側から上記筒状部材の内部に酸素含有ガスを供給する散気手段と、上記筒状部材の長手方向が鉛直下方向と略平行かつ、上記他方端部が下方になるように配設した培養槽とを備える細胞培養装置を使用する。また、本発明に係る細胞培養方法は、上記培養槽内に対して、上記筒状部材の一方端部が液外方へ露出するように細胞を培養するための培地が張り込められ、細胞を培養する方法であって、上記酸素含有ガスを、上記筒状部材の内部を上下方向に分割する気泡として上記散気手段から供給することを特徴とするものである。   That is, the method for culturing cells according to the present invention includes a cylindrical member having pores on the side surface, one end in the longitudinal direction being opened and the other end sealed, and the other end side. An aeration means for supplying an oxygen-containing gas into the cylindrical member, and a culture tank disposed such that the longitudinal direction of the cylindrical member is substantially parallel to the vertical downward direction and the other end is downward. Is used. In the cell culture method according to the present invention, a medium for culturing the cells is embedded in the culture tank so that one end of the cylindrical member is exposed to the outside of the liquid. A method for culturing is characterized in that the oxygen-containing gas is supplied from the aeration means as bubbles that divide the inside of the cylindrical member in the vertical direction.

本発明に係る細胞培養法では、上記散気手段から供給された気泡が上記筒状部材の内部を上方向に移動することによって、当該気泡より下側の部分が陰圧となり細孔を介して筒状部材の内部に培養液が流入することとなる。また、当該気泡より上側の部分では、圧力が高まり、筒状内部の培養液が細孔を介して外側に流出する。このように、本発明に係る細胞培養方法においては、筒状部材の内部と外部との間で細孔を介して培養液の流入及び流出が繰り返されることとなる。これにより、筒状部材の側面に対する細胞等の付着を防止することができる。   In the cell culture method according to the present invention, the air bubbles supplied from the air diffuser move upward in the cylindrical member, so that the portion below the air bubbles becomes a negative pressure and passes through the pores. The culture solution will flow into the cylindrical member. In addition, in the portion above the bubbles, the pressure increases, and the culture medium inside the cylinder flows out through the pores. Thus, in the cell culture method according to the present invention, the inflow and outflow of the culture solution are repeated through the pores between the inside and the outside of the cylindrical member. Thereby, adhesion of the cell etc. with respect to the side surface of a cylindrical member can be prevented.

特に、本発明に係る細胞培養法においては、上記散気手段は、上記気泡が上記筒状部材の内部を通って上記一方端部側で消泡したタイミングで次の気泡を供給することが好ましい。   In particular, in the cell culture method according to the present invention, it is preferable that the air diffuser supplies the next bubble at a timing when the bubble passes through the inside of the cylindrical member and disappears on the one end side. .

また、本発明に係る細胞培養法においては、培養対象の細胞が付着依存性を有する生体細胞であって、当該生体細胞の足場となる培養面を有するマイクロキャリアとともに当該生体細胞を培養する方法にも適用することができる。   Further, in the cell culture method according to the present invention, the cell to be cultured is a living cell having adhesion dependency, and the living cell is cultured together with a microcarrier having a culture surface as a scaffold for the living cell. Can also be applied.

さらに、本発明に係る細胞培養方法においては、上記筒状部材の側面における細孔は、培養対象の細胞又は当該細胞の足場となるマイクロキャリアよりも小径であることが好ましく、上記筒状部材の側面における細孔を50〜150μmの直径を有する貫通孔とすることがより好ましい。   Furthermore, in the cell culture method according to the present invention, the pores on the side surface of the cylindrical member preferably have a smaller diameter than the cell to be cultured or the microcarrier serving as a scaffold for the cell. More preferably, the side pores are through-holes having a diameter of 50 to 150 μm.

さらにまた、上記筒状部材の少なくとも側面は可撓性を有する材質から形成されることが好ましく、上記気泡の通過に伴って当該側面が変形することが好ましい。   Furthermore, at least the side surface of the cylindrical member is preferably formed of a flexible material, and it is preferable that the side surface is deformed as the bubbles pass.

なお、本発明に係る細胞培養方法において、上記培養装置は、培養槽内に複数の上記筒状部材を配設したものを使用することがより好ましい。   In the cell culture method according to the present invention, it is more preferable to use a culture apparatus in which a plurality of cylindrical members are arranged in a culture tank.

また、本発明に係る細胞培養装置は、側面に細孔を有し、長手方向の一方端部が開放されるとともに他方端部が封止された筒状部材と、上記他方端部側から上記筒状部材の内部に酸素含有ガスを供給する散気手段と、上記筒状部材の長手方向が鉛直下方向と略平行かつ、上記他方端部が下方になるように配設した培養槽とを備える。本発明に係る細胞培養装置において、上記散気手段は、上記筒状部材の内部に培養液が張り込まれた状態において、上記酸素含有ガスを、上記筒状部材の内部を上下方向に分割する気泡として供給するものである。   Further, the cell culture device according to the present invention has a cylindrical member having pores on the side surface, one end portion in the longitudinal direction being opened and the other end portion being sealed, and the above-mentioned from the other end portion side. An aeration means for supplying an oxygen-containing gas to the inside of the cylindrical member, and a culture tank disposed so that the longitudinal direction of the cylindrical member is substantially parallel to the vertical downward direction and the other end portion is downward. Prepare. In the cell culture device according to the present invention, the aeration means divides the oxygen-containing gas in the vertical direction inside the cylindrical member in a state where the culture solution is stuck inside the cylindrical member. It is supplied as bubbles.

本発明に係る細胞培養装置では、上記散気手段から供給された気泡が上記筒状部材の内部を上方向に移動することによって、当該気泡より下側の部分が陰圧となり細孔を介して筒状部材の内部に培養液が流入することとなる。また、当該気泡より上側の部分では、圧力が高まり、筒状内部の培養液が細孔を介して外側に流出する。このように、本発明に係る細胞培養装置においては、筒状部材の内部と外部との間で細孔を介して培養液の流入及び流出が繰り返されることとなる。これにより、筒状部材の側面に対する細胞等の付着を防止することができる。   In the cell culture device according to the present invention, the bubbles supplied from the aeration means move upward in the cylindrical member, so that the portion below the bubbles becomes negative pressure and passes through the pores. The culture solution will flow into the cylindrical member. In addition, in the portion above the bubbles, the pressure increases, and the culture medium inside the cylinder flows out through the pores. Thus, in the cell culture apparatus according to the present invention, the inflow and outflow of the culture solution are repeated between the inside and the outside of the cylindrical member via the pores. Thereby, adhesion of the cell etc. with respect to the side surface of a cylindrical member can be prevented.

また、本発明に係る細胞培養装置は、上記気泡が上記筒状部材の内部を通って上記一方端部側で消泡したタイミングで次の気泡を供給するように、上記散気手段を制御する制御装置を更に備えることが好ましい。   Moreover, the cell culture apparatus according to the present invention controls the aeration means so that the next bubble is supplied at a timing when the bubble passes through the inside of the cylindrical member and disappears on the one end side. It is preferable to further comprise a control device.

さらに、本発明に係る細胞培養装置においては、培養対象の細胞が付着依存性を有する生体細胞であって、当該生体細胞の足場となる培養面を有するマイクロキャリアとともに当該生体細胞を培養することができる。   Furthermore, in the cell culture device according to the present invention, the cell to be cultured is a living cell having adhesion dependency, and the living cell is cultured together with a microcarrier having a culture surface as a scaffold for the living cell. it can.

さらにまた、本発明に係る細胞培養装置において、上記筒状部材の側面における細孔は、培養対象の細胞又は当該細胞の足場となるマイクロキャリアよりも小径であることが好ましく、上記筒状部材の側面における細孔は、50〜150μmの直径を有する貫通孔であることがより好ましい。   Furthermore, in the cell culture device according to the present invention, the pores on the side surface of the cylindrical member preferably have a smaller diameter than the cell to be cultured or the microcarrier serving as a scaffold for the cell. The pores on the side surface are more preferably through-holes having a diameter of 50 to 150 μm.

さらにまた、本発明に係る細胞培養装置において、上記筒状部材の少なくとも側面は可撓性を有する材質から形成されることが好ましく、上記気泡の通過に伴って当該側面が変形することが好ましい。   Furthermore, in the cell culture device according to the present invention, it is preferable that at least a side surface of the cylindrical member is formed of a flexible material, and that the side surface is deformed as the bubbles pass.

なお、本発明に係る細胞培養装置は、上記培養槽内に複数の上記筒状部材を配設したもとすることが好ましい。   In the cell culture device according to the present invention, it is preferable that a plurality of the cylindrical members are arranged in the culture tank.

本発明に係る細胞培養方法及び細胞培養装置によれば、筒状部材の内部を分割する気泡を当該筒状部材内部に通過させることで、筒状部材の側面に対する細胞やマイクロキャリア等の付着を防止することができ、より効率的に細胞を培養することができる。したがって、本発明に係る細胞培養方法及び細胞培養装置によれば、細胞自体或いは細胞が産生する物質を製造する際の生産性を大幅に向上させることができる。   According to the cell culture method and the cell culture apparatus according to the present invention, the bubbles that divide the inside of the cylindrical member are allowed to pass through the inside of the cylindrical member, thereby attaching the cells, microcarriers, and the like to the side surface of the cylindrical member. Can be prevented, and cells can be cultured more efficiently. Therefore, according to the cell culturing method and the cell culturing apparatus according to the present invention, the productivity in producing the cell itself or the substance produced by the cell can be greatly improved.

本発明を適用した細胞培養装置の一例を示す概略構成図である。It is a schematic block diagram which shows an example of the cell culture apparatus to which this invention is applied. 本発明の酸素供給方法の作用メカニズムを説明する概要図である。It is a schematic diagram explaining the action mechanism of the oxygen supply method of the present invention. 本発明を適用した細胞培養装置における酸素供給能力を示す特性図である。It is a characteristic view which shows the oxygen supply capability in the cell culture apparatus to which this invention is applied. 本発明を適用した細胞培養装置における培養特性を示す特性図である。It is a characteristic view which shows the culture | cultivation characteristic in the cell culture apparatus to which this invention is applied. 本発明を適用した細胞培養装置の他の例を示す概略構成図である。It is a schematic block diagram which shows the other example of the cell culture apparatus to which this invention is applied.

以下、本発明に係る細胞培養方法及び細胞培養装置について、図面を用いて詳細に説明する。   Hereinafter, a cell culture method and a cell culture apparatus according to the present invention will be described in detail with reference to the drawings.

本発明において、培養対象の細胞としては、特に限定されず、例えば医薬品等の主原料となる物質を生産する細胞の培養に適用することができる。生産対象の物質としては、培養した細胞自体、細胞が産生する抗体や酵素等のタンパク質、低分子化合物若しくは高分子化合物等の生理活性物質及びウイルスを挙げることができる。また、培養対象の細胞としては、動物細胞、植物細胞、昆虫細胞、細菌、酵母、真菌及び藻類等を挙げることができる。特に、抗体や酵素等のタンパク質、ウイルスを生産する動物細胞を培養対象とすることが好ましい。また、培養対象の細胞は、表面及び/又は内部に細胞が付着して増殖することが可能な培養面を有するマイクロキャリアを使用して培養するものであっても良い。マイクロキャリアを用いた培養は、特に、付着依存性を有する動物培養に適用することが好ましい。   In the present invention, the cells to be cultured are not particularly limited, and can be applied to, for example, culturing cells that produce substances that are main raw materials such as pharmaceuticals. Examples of substances to be produced include cultured cells themselves, proteins such as antibodies and enzymes produced by the cells, physiologically active substances such as low molecular compounds or high molecular compounds, and viruses. Examples of cells to be cultured include animal cells, plant cells, insect cells, bacteria, yeasts, fungi and algae. In particular, it is preferable to culture animal cells that produce proteins such as antibodies and enzymes, and viruses. Further, the cells to be cultured may be cultured using a microcarrier having a culture surface on which cells can adhere and grow on the surface and / or inside. The culture using a microcarrier is particularly preferably applied to animal culture having adhesion dependency.

本発明を適用した細胞培養装置の一例を図１に示す。細胞培養装置は、培養液を張り込むことができる培養槽１と、側面に細孔を有し、長手方向の一方端部が開放されるとともに他方端部が封止された筒状部材２とを備えている。筒状部材２は、培養槽１の内部に配設されている。筒状部材２は、培養槽１の内部において、その長手方向が鉛直下方向と略平行かつ、封止されている上記他方端部が下方になるように配設されている。ここで、筒状部材２は、その長手方向が鉛直下方向に対して厳密に平行な方向となるように配設しても良い。また、詳細を後述するように、筒状部材２の内部に供給した気泡が上昇することが可能であれば、筒状部材２は、その長手方向が鉛直下方向に対してやや傾斜するように配設しても良い。上述した「鉛直下方向と略平行」とは、このように、筒状部材２の長手方向が鉛直下方向に対してやや傾斜した形態を包含する意味である。   An example of a cell culture apparatus to which the present invention is applied is shown in FIG. The cell culture apparatus includes a culture tank 1 in which a culture solution can be placed, a cylindrical member 2 having a pore on a side surface, one end in the longitudinal direction being opened and the other end sealed. It has. The cylindrical member 2 is disposed inside the culture tank 1. The tubular member 2 is disposed inside the culture tank 1 so that the longitudinal direction thereof is substantially parallel to the vertically downward direction and the other end portion that is sealed is positioned downward. Here, the cylindrical member 2 may be disposed so that the longitudinal direction thereof is strictly parallel to the vertical downward direction. In addition, as will be described in detail later, if the bubbles supplied to the inside of the cylindrical member 2 can rise, the cylindrical member 2 has a longitudinal direction slightly inclined with respect to the vertical downward direction. It may be arranged. As described above, “substantially parallel to the vertically downward direction” means that the longitudinal direction of the tubular member 2 is slightly inclined with respect to the vertically downward direction.

また、細胞培養装置は、培養槽１内の培養液に含まれる溶存酸素を測定するためのセンサー３と、センサー３からのシグナルを入力し溶存酸素濃度を算出する溶存酸素濃度計測手段４とを備えていても良い。センサー３は、培養槽１の底部近傍（例えば、底部から50ｍｍの位置）に配置されていることが望ましい。   Further, the cell culture apparatus includes a sensor 3 for measuring dissolved oxygen contained in the culture solution in the culture tank 1 and a dissolved oxygen concentration measuring means 4 for inputting a signal from the sensor 3 and calculating a dissolved oxygen concentration. You may have. The sensor 3 is desirably disposed near the bottom of the culture tank 1 (for example, at a position 50 mm from the bottom).

さらに、細胞培養装置は、溶存酸素濃度計測手段４で測定した溶存酸素濃度値を入力する第１の制御装置５、第２の制御装置６及び第３の制御装置７と、第１の制御装置５により動作制御されるガス分圧調節装置８と、第２の制御装置６により動作制御される撹拌機駆動モータ９と、第３の制御装置７により動作制御される通気ガス調節装置１０と、撹拌機駆動モータ９に接続された撹拌軸１１、撹拌翼１２ａ及び１２ｂとを備えていてもよい。なお、この場合、細胞培養装置は、図示しないが、ガス分圧調節装置８及び通気ガス調節装置１０に接続され、これらガス分圧調節装置８及び通気ガス調節装置１０に酸素含有ガス（通気ガス）を供給するガス供給装置を備えている。なお、撹拌翼１２ａ及び１２ｂとしては、例えば回転直径100ｍｍのパドル形のものを使用することができる。   Further, the cell culture device includes a first control device 5, a second control device 6, a third control device 7 for inputting the dissolved oxygen concentration value measured by the dissolved oxygen concentration measuring means 4, and a first control device. A gas partial pressure adjusting device 8 whose operation is controlled by 5, a stirrer driving motor 9 whose operation is controlled by the second control device 6, a ventilation gas adjusting device 10 whose operation is controlled by the third control device 7, You may provide the stirring shaft 11 connected to the stirrer drive motor 9, and the stirring blades 12a and 12b. In this case, although not shown, the cell culture device is connected to the gas partial pressure adjusting device 8 and the aeration gas adjusting device 10, and an oxygen-containing gas (aeration gas) is connected to the gas partial pressure adjusting device 8 and the aeration gas adjusting device 10. ) Is provided. In addition, as the stirring blades 12a and 12b, for example, paddles having a rotation diameter of 100 mm can be used.

さらにまた、細胞培養装置は、通気ガス調節装置１０が筒状部材２の封止された他方端部に接続されている。通気ガス調節装置１０は、酸素含有ガス（通気ガス）を排出するノズル（図示せず）を有している。このノズルは、封止された他方端部を介して筒状部材２の内部に臨んでいる。   Furthermore, in the cell culture device, the aeration gas regulating device 10 is connected to the other end portion of the cylindrical member 2 that is sealed. The ventilation gas adjusting device 10 has a nozzle (not shown) for discharging an oxygen-containing gas (aeration gas). This nozzle faces the inside of the cylindrical member 2 through the sealed other end.

特に、筒状部材２は、側面に細孔が形成されており、細孔を介して培養液が内部へ流入したり、内部の培養液が外部へ流出したりすることができる。ここで、細孔としては、培養液の流出入が可能であり、且つ、細胞又はマイクロキャリアの通過が阻止できる大きさ及び形状であればよい。例えば、培養対象の細胞をマイクロキャリアに担持させて培養する場合、筒状部材２の側面における細孔は50〜150μmの直径を有する貫通孔とすることが好ましい。   In particular, the cylindrical member 2 has pores formed on the side surfaces, and the culture solution can flow into the inside or the inside culture solution can flow out to the outside through the pores. Here, the pores may be of any size and shape that allows the culture medium to flow out and in and prevents passage of cells or microcarriers. For example, when culturing cells to be cultured on a microcarrier, the pores on the side surface of the cylindrical member 2 are preferably through-holes having a diameter of 50 to 150 μm.

また、筒状部材２の少なくとも側面は可撓性を有する材質から形成されることが好ましい。可塑性を有する材質としては、例えばポリプロピレン等の樹脂を挙げることができる。このような樹脂を使用したメッシュシートを筒状に加工することで、筒状部材２の側面を形成することができる。また、筒状部材２は、メッシュシートを筒状に加工した後、一方の端部を封止部材２ｓによって封止することで作製することができる。例えば、ガラス製で直径240ｍｍ、培養液張り込み高さ230ｍｍ、培養容積10Lの培養槽１を使用してマイクロキャリアを使用した細胞培養を行う場合、100×100μｍの細孔を有するポリプロピレン製メッシュシートで構成された内径30ｍｍ、高さ250ｍｍの形状を有する筒状部材２を使用することができる。この筒状部材２を、その長手方向を鉛直下方向に対して略平行とし、且つ、封止された端部を培養槽１の底部近傍に位置するように、培養槽１に設置することで、筒状部材における開放された一方端部が培養液入り込み高さを超える位置となる。   Moreover, it is preferable that at least the side surface of the cylindrical member 2 is formed of a flexible material. Examples of the plastic material include resins such as polypropylene. The side surface of the cylindrical member 2 can be formed by processing a mesh sheet using such a resin into a cylindrical shape. Moreover, the cylindrical member 2 can be produced by processing one end of the mesh sheet into a cylinder and then sealing one end with the sealing member 2s. For example, when cell culture using a microcarrier is carried out using a culture tank 1 made of glass having a diameter of 240 mm, a culture medium embedding height of 230 mm, and a culture volume of 10 L, a polypropylene mesh sheet having 100 × 100 μm pores is used. The cylindrical member 2 having a configured shape having an inner diameter of 30 mm and a height of 250 mm can be used. By installing the tubular member 2 in the culture tank 1 so that the longitudinal direction thereof is substantially parallel to the vertical downward direction and the sealed end portion is located in the vicinity of the bottom of the culture tank 1. The open one end of the cylindrical member is at a position exceeding the height of the culture solution entering.

また、筒状部材２の材質としては、ポリプロピレン樹脂のほかに、ステンレス材、四フッ化エチレン樹脂、ポリエチレンテレフタレート樹脂など、スチーム殺菌に耐え、酸及びアルカリ性の洗浄剤を用いた洗浄操作に安定で、細胞への毒性を示さない材料からなるメッシュシートを使用することができる。   In addition to polypropylene resin, the cylindrical member 2 is resistant to steam sterilization, such as stainless steel, tetrafluoroethylene resin, and polyethylene terephthalate resin, and is stable in cleaning operations using acid and alkaline cleaning agents. A mesh sheet made of a material that does not show toxicity to cells can be used.

以上のように構成された本発明に係る細胞培養装置を用いることで、上述した細胞を培養することができる。以下では、マイクロキャリアを使用して動物細胞を培養する形態について説明するが、本発明の技術的範囲はこの形態に限定されるものではない。   By using the cell culture apparatus according to the present invention configured as described above, the above-described cells can be cultured. Below, although the form which culture | cultivates an animal cell using a microcarrier is demonstrated, the technical scope of this invention is not limited to this form.

培養槽１内に張り込まれた培養液１３中には、微細粒子であるマイクロキャリア１４が浮遊している。マイクロキャリア１４には、付着依存性を有する細胞が増殖するための培養面が設けられている。細胞はマイクロキャリア１４に接触することにより培養面に付着し、増殖を開始する。マイクロキャリア１４は、培養液１３よりもわずかに比重が大きく、細胞が増殖するとさらに比重が大きくなる。このため、マイクロキャリア１４は、静置状態では培養槽１の底部に沈降する。細胞培養装置では、第２の制御装置６が撹拌機駆動モータ９の回転速度を制御して、撹拌翼１２ａ及び１２ｂを回転させて培養液１３を流動させることにより、マイクロキャリア１４を浮遊させている。特に、培養期間が長くなるほど、細胞の増殖によってマイクロキャリアを含む微粒子の比重が大きくなり、より沈降しやすくなる。この場合、撹拌回転数を増加させるように制御することで、マイクロキャリアを良好に培養液１３中に浮遊させることができる。   A microcarrier 14 that is fine particles floats in the culture solution 13 that is stuck in the culture tank 1. The microcarrier 14 is provided with a culture surface for growing cells having adhesion dependency. When the cells come into contact with the microcarrier 14, they adhere to the culture surface and start to grow. The specific gravity of the microcarrier 14 is slightly larger than that of the culture solution 13, and the specific gravity further increases as the cells grow. For this reason, the microcarrier 14 settles on the bottom of the culture tank 1 in a stationary state. In the cell culture device, the second control device 6 controls the rotational speed of the stirrer drive motor 9 to rotate the stirring blades 12a and 12b to cause the culture solution 13 to flow, thereby floating the microcarrier 14. Yes. In particular, the longer the culture period, the greater the specific gravity of the microparticles containing microcarriers due to cell growth, and the easier it is to settle. In this case, the microcarrier can be satisfactorily suspended in the culture solution 13 by controlling to increase the number of rotations of stirring.

細胞培養装置を使用した細胞培養においては、培養液１３を所望の溶存酸素濃度となるように適宜、調節を行いながら培養を継続する。細胞培養装置においては、溶存酸素濃度計測手段４を備えており、センサー３及び溶存酸素濃度計測手段４で測定した溶存酸素濃度値を基に、撹拌機の回転数を増減する方法、気相部の酸素濃度を増減する方法、及び筒状部材２へ酸素含有ガスを吹き込む方法のいずれか又はこれらの３つの方法を併用することによって、培養液１３中の溶存酸素濃度を制御することができる。   In the cell culture using the cell culture device, the culture is continued while appropriately adjusting the culture solution 13 to have a desired dissolved oxygen concentration. The cell culture device includes a dissolved oxygen concentration measuring means 4, a method for increasing or decreasing the rotational speed of the stirrer based on the dissolved oxygen concentration values measured by the sensor 3 and the dissolved oxygen concentration measuring means 4, and a gas phase part The dissolved oxygen concentration in the culture solution 13 can be controlled by using either one of the method for increasing or decreasing the oxygen concentration and the method for blowing the oxygen-containing gas into the cylindrical member 2 or using these three methods in combination.

撹拌機の回転数を増減する方法は、溶存酸素濃度計測手段４で測定した培養液１３中の溶存酸素濃度値に基づいて、上述したように、第２の制御装置６が撹拌機駆動モータ９の回転速度を制御する方法である。撹拌回転数が増加すると混合が強化され、酸素溶解量が増加する。   The method of increasing / decreasing the rotation speed of the stirrer is based on the dissolved oxygen concentration value in the culture solution 13 measured by the dissolved oxygen concentration measuring means 4, as described above, the second controller 6 uses the stirrer drive motor 9. This is a method for controlling the rotation speed of the. As the stirring speed increases, mixing is strengthened and the amount of dissolved oxygen increases.

また、気相部の酸素濃度を増減する方法は、第１の制御装置５がガス分圧調節装置８を制御し、培養液１３上方の気相部に対する酸素含有ガス（通気ガス）の供給量を調整する方法である。また、第１の制御装置５及びガス分圧調節装置８により、培養液１３のｐＨの変化に対応して炭酸ガス分圧の増減を行うようにしてもよい。なお、第１の制御装置５及びガス分圧調節装置８による溶存酸素濃度のコントロールは、細胞播種後の低細胞密度の時期に主に使用することが好ましい。   In addition, the method for increasing or decreasing the oxygen concentration in the gas phase portion is such that the first control device 5 controls the gas partial pressure adjusting device 8 to supply the oxygen-containing gas (aeration gas) to the gas phase portion above the culture solution 13. It is a method of adjusting. Further, the first control device 5 and the gas partial pressure adjusting device 8 may increase or decrease the carbon dioxide partial pressure in response to a change in the pH of the culture solution 13. In addition, it is preferable to mainly use the control of the dissolved oxygen concentration by the first control device 5 and the gas partial pressure adjusting device 8 at the time of low cell density after cell seeding.

さらに、筒状部材２へ酸素含有ガスを吹き込む方法は、筒状部材２、第３の制御装置７及び通気ガス調節装置１０により、培養液１３の溶存酸素濃度を調整する方法である。具体的には、図２に模式的に示すように、封止部材２ｓに設けられたノズルより通気ガスが筒状部材２の内部に供給されると気泡２ｂが形成され、気泡２ｂより培養液１３へ酸素が供給される。このとき、気泡２ｂは、筒状部材２の内部に張り込んだ培養液１３を上下に分割することとなる。また、気泡２ｂは液面に向かって上昇する。気泡２ｂは、いわばプラグとして機能することとなる。気泡２ｂが筒状部材２内部を上昇するとき、筒状部材２に細孔が無いと仮定すると、内部では気泡２ｂが入ることによって見かけの培養液１３の比重が減少して液面が上昇し、ΔPに相当する負の圧力差が生じる。このときの筒状部材２内部の圧力は外部に比べて、気泡２ｂより下では−ΔP、気泡２ｂより上ではΔPとなる。細孔を有する筒状部材２においても同様な関係が生じるため、気泡２ｂより下では培養液１３が内部に流入し、気泡２ｂより上では内部の培養液１３が外部に流出する。また、筒状部材２の側面において、気泡２ｂの上昇に伴って、培養液１３が流入する部分と流出する部分が変化する。   Further, the method of blowing the oxygen-containing gas into the cylindrical member 2 is a method of adjusting the dissolved oxygen concentration of the culture solution 13 by the cylindrical member 2, the third control device 7 and the aeration gas adjusting device 10. Specifically, as schematically shown in FIG. 2, when the aeration gas is supplied into the cylindrical member 2 from the nozzle provided in the sealing member 2s, bubbles 2b are formed, and the culture solution is formed from the bubbles 2b. 13 is supplied with oxygen. At this time, the bubble 2b divides the culture solution 13 stuck inside the cylindrical member 2 vertically. Moreover, the bubble 2b rises toward the liquid level. The bubble 2b functions as a plug. Assuming that there are no pores in the cylindrical member 2 when the bubble 2b rises inside the cylindrical member 2, the apparent density of the culture medium 13 decreases due to the bubble 2b entering inside and the liquid level rises. , A negative pressure difference corresponding to ΔP occurs. The pressure inside the cylindrical member 2 at this time is -ΔP below the bubble 2b and ΔP above the bubble 2b, compared to the outside. Since the same relationship also occurs in the cylindrical member 2 having pores, the culture solution 13 flows into the inside below the bubbles 2b, and the inside culture solution 13 flows out to the outside above the bubbles 2b. In addition, on the side surface of the cylindrical member 2, as the bubble 2b rises, the portion into which the culture solution 13 flows and the portion from which it flows out change.

これによって、筒状部材２の細孔部分に付着したマイクロキャリア等を剥離することができる。以上のように、本発明に係る細胞培養装置において、筒状部材２の内部を上下方向に分割するような気泡２ｂを筒状部材２に通過させることによって、筒状部材２の側面に対して細胞やマイクロキャリア等が付着することを防止できる。このため、筒状部材２内部において、培養槽１に張り込まれた培養液１３の溶存酸素濃度を所期の値に制御することが可能となる。   As a result, the microcarriers attached to the pores of the cylindrical member 2 can be peeled off. As described above, in the cell culture device according to the present invention, the air bubble 2b that divides the inside of the cylindrical member 2 in the vertical direction is allowed to pass through the cylindrical member 2, thereby allowing the side surface of the cylindrical member 2 to pass. It is possible to prevent cells and microcarriers from adhering. For this reason, it becomes possible to control the dissolved oxygen concentration of the culture solution 13 stuck in the culture tank 1 to an intended value inside the cylindrical member 2.

特に、上述したように筒状部材２内部に対して培養液を流出入させるには、気泡２ｂを介して上記圧力差を形成することが必要であるため通気ガスは間歇的に供給される。なお、1回あたりの通気時間は０．５〜１．５ｓの短時間とすることで、上述したようにプラグ状の気泡２ｂを形成することができる。また、１回あたりの通気ガス吹き込み量Ｖ(ｍｌ)は下記の式１によって決定される。なお、式１は筒状部材２が円筒状である場合に適用される。   In particular, as described above, in order to allow the culture solution to flow into and out of the cylindrical member 2, it is necessary to form the pressure difference via the bubble 2 b, so the aeration gas is supplied intermittently. In addition, the plug-like bubble 2b can be formed as described above by setting the ventilation time per time to a short time of 0.5 to 1.5 s. Further, the ventilation gas blowing amount V (ml) per one time is determined by the following formula 1. In addition, Formula 1 is applied when the cylindrical member 2 is cylindrical.

〔式１〕 Ｖ＝β・Ｄ３・π／４
ここで、β：通気計数（−）
Ｄ：円筒直径（ｃｍ） [Formula 1] V = β · D 3 · π / 4
Where β: aeration rate (−)
D: Cylindrical diameter (cm)

通気計数βの値は、筒状部材２の長さによって経験的に決定されるもので、長さ０．２ｍ〜１．５ｍの範囲では１〜５となる。   The value of the air flow coefficient β is determined empirically by the length of the tubular member 2 and is 1 to 5 in the range of 0.2 m to 1.5 m in length.

そして、筒状部材２内部を通過した気泡２ｂは、筒状部材２における開放された一方端部付近で破裂し、消泡する。このとき、筒状部材２における一方端部は、培養液１３の液面より外方に露出しているため、気泡２ｂは筒状部材２の内部で破裂することとなる。このため、気泡２ｂの破裂に伴う衝撃により、培養液１３に浮遊する細胞やマイクロキャリアが損傷されることを防止できる。   And the bubble 2b which passed through the inside of the cylindrical member 2 is ruptured and defoamed in the vicinity of one open end of the cylindrical member 2. At this time, since one end of the cylindrical member 2 is exposed outward from the liquid surface of the culture solution 13, the bubble 2 b is ruptured inside the cylindrical member 2. For this reason, it is possible to prevent the cells and microcarriers floating in the culture solution 13 from being damaged by the impact accompanying the burst of the bubbles 2b.

また、筒状部材２内部に通気ガスを吹き込むタイミングとしては、前回の吹き込みで生じた気泡２ｂが液面上に達した後とすることが好ましい。若しくは、筒状部材２内部に通気ガスを吹き込むタイミングとしては、前回の吹き込みで生じた気泡２ｂが液面付近で消泡した後とすることが好ましい。筒状部材２の内部において、上下方向に複数の気泡２ｂを生じさせた場合、気泡と気泡の間に上述したような圧力差が形成されず、培養液１３の流出入が達成できないからである。   Moreover, it is preferable that the timing of blowing the aeration gas into the cylindrical member 2 is after the bubbles 2b generated by the previous blowing reach the liquid level. Alternatively, it is preferable that the timing of blowing the aeration gas into the cylindrical member 2 is after the bubbles 2b generated by the previous blowing are defoamed near the liquid surface. This is because, when a plurality of bubbles 2b are generated in the vertical direction inside the cylindrical member 2, the pressure difference as described above is not formed between the bubbles and the inflow / outflow of the culture solution 13 cannot be achieved. .

さらに、細胞培養装置において筒状部材２を可撓性の材質で構成した場合には、気泡２ｂの移動によって筒状部材２の側面に揺動と伸縮する動きが生じ、これらの動きがマイクロキャリア等を剥離させる作用として働く。このため、筒状部材２を可撓性の材質で構成した場合には、筒状部材２の側面に対するマイクロキャリア等の付着をより効果的に除去することができる。   Furthermore, when the cylindrical member 2 is made of a flexible material in the cell culture apparatus, the movement of the bubble 2b causes the side surface of the cylindrical member 2 to swing and expand and contract, and these movements are microcarriers. It works as an action to peel off. For this reason, when the cylindrical member 2 is comprised with a flexible material, adhesion of the microcarrier etc. with respect to the side surface of the cylindrical member 2 can be removed more effectively.

また、図１や図２に示すように、単一の気泡２ｂが筒状部材２内部をプラグフロー状態で上昇することがもっとも好ましい。筒状部材２の内径を大きくすることによって単一気泡によるプラグフローを実現し得ない場合は、前記の式１にて決定した容積のガスを１〜１．５秒の短時間に供給することで得られる気泡群によって筒状部材２の内部を分割しても良い。   Further, as shown in FIGS. 1 and 2, it is most preferable that the single bubble 2b rises in the tubular member 2 in a plug flow state. When plug flow with a single bubble cannot be realized by increasing the inner diameter of the cylindrical member 2, the gas having the volume determined by the above equation 1 is supplied in a short time of 1 to 1.5 seconds. The inside of the cylindrical member 2 may be divided by the bubble group obtained in the above.

図１及び２に示した細胞培養装置を用いて実際に計測した酸素溶解経過を図３に示す。図１に示す培養槽１に、マイクロキャリア（サイトデックス１、GEヘルスケア製）を乾燥重量で5％の濃度になるよう分散させた生理的リン酸緩衝液2Ｌを張り込み、30rpmで撹拌しながら37℃に加温した。ついで、生理的リン酸緩衝液中に窒素を注入して溶存酸素濃度がほぼ0％まで低下させた。つぎに筒状部材２内に10秒間隔で25ml/回の空気を1秒間で注入して溶存酸素濃度の変化を計測した。計測結果を図３中にＡで示す。比較として、図３中Ｂは筒状部材２から円筒部分を取り外して封止部材２ｓから空気を150ml/分で直接通気した場合の溶存酸素濃度の変化をしめす。また、図中Ｃは筒状部材２内に空気を１５０ｍｌ／分で連続して通気した場合の溶存酸素濃度の変化を示す。図３中のＡに示すように、筒状部材２を使用した場合、直接液中に通気したＢに比較して酸素溶解速度が劣ることは否めないが、気泡が培養細胞と接触しない状況下で酸素供給を行うことができることを示す。また筒状部材２を使用した場合においても、連続的に通気したＣに比べると、気泡２ｂを形成するように供給したＡのほうがより大きな酸素供給速度が得られることがわかる。この結果から、気泡２ｂを筒状部材２の内部に形成することによって、細孔の目詰まりが少なくなっていることが理解できる。   FIG. 3 shows the progress of oxygen dissolution actually measured using the cell culture apparatus shown in FIGS. Into the culture tank 1 shown in FIG. 1, 2 L of physiological phosphate buffer in which microcarriers (Cytodex 1, manufactured by GE Healthcare) are dispersed so as to have a concentration of 5% by dry weight is put and stirred at 30 rpm. Warmed to 37 ° C. Subsequently, nitrogen was injected into the physiological phosphate buffer to lower the dissolved oxygen concentration to almost 0%. Next, 25 ml / time of air was injected into the cylindrical member 2 at intervals of 10 seconds for 1 second, and the change in dissolved oxygen concentration was measured. The measurement result is indicated by A in FIG. As a comparison, B in FIG. 3 shows the change in dissolved oxygen concentration when the cylindrical portion is removed from the cylindrical member 2 and air is directly ventilated from the sealing member 2s at 150 ml / min. Further, C in the figure shows a change in dissolved oxygen concentration when air is continuously passed through the cylindrical member 2 at 150 ml / min. As shown in A of FIG. 3, when the cylindrical member 2 is used, it cannot be denied that the oxygen dissolution rate is inferior to that of B which is directly ventilated in the liquid, but under the situation where bubbles do not come into contact with the cultured cells. Shows that oxygen can be supplied. In addition, even when the cylindrical member 2 is used, it can be seen that a larger oxygen supply rate can be obtained when A is supplied so as to form the bubbles 2b than when C is continuously ventilated. From this result, it can be understood that the clogging of the pores is reduced by forming the bubbles 2b inside the cylindrical member 2.

また、図１に示した細胞培養装置を用いて実際に動物細胞の培養を行った結果を図４に示す。図４に結果を示す実験例において、動物細胞としてはCHO-K1細胞を使用した。また、培地としては、CD-CHO培地（インビトロジェン製）を使用した。マイクロキャリアとしては、サイトデックス１（GEヘルスケア製）を乾燥重量で５％の比率で培地に加えた。培養中の酸素の通気は、溶存酸素濃度が制御値を下回ったときに筒状部材２中に酸素を断続的に通気した。通気は25ml/回の酸素を1秒間で噴出させるもので、これを10秒間隔で行った。培養を開始するに当たっては、あらかじめ別容器にて調製したCHO-K1細胞をトリプシン処理で剥離させ、細胞密度が2×10-5（個/ml）となるように播種して培養を開始した。なお、溶存酸素濃度計測手段４による溶存酸素濃度の制御値は3.0mg/Lに設定した。また、本培養装置の場合、撹拌回転数が20rpm以下ではマイクロキャリアの一部が培養槽底に沈積するため、培養開始持の回転数を30rpmとした。培養期間中、１日に１回の頻度で培養液を採取し、マイクロキャリア上で増殖した細胞量を計測した。なお、培養液採取の際は、マイクロキャリアが均一に培養液中に分散するよう、手動にて回転数を50rpmに増加させた。マイクロキャリア上の細胞の計測はトリプシン処理によって細胞を剥離させて計測した。結果、図４にＡで示すように最高細胞到達密度は、培養６日目の6.6×106（個/ml）であった。培養６日目のマイクロキャリアを光学顕微鏡で観察すると、その表面の全面が細胞によって覆われたいわゆるコンフルエントの状態であり、マイクロキャリアの損傷もほとんど認められなかった。 Moreover, the result of actually culturing animal cells using the cell culture apparatus shown in FIG. 1 is shown in FIG. In the experimental example whose results are shown in FIG. 4, CHO-K1 cells were used as animal cells. As the medium, CD-CHO medium (Invitrogen) was used. As a microcarrier, Cytodex 1 (manufactured by GE Healthcare) was added to the medium at a ratio of 5% by dry weight. As for the aeration of oxygen during the culture, oxygen was intermittently aerated through the cylindrical member 2 when the dissolved oxygen concentration fell below the control value. Aeration was performed by ejecting 25 ml / time of oxygen in 1 second, and this was performed at 10 second intervals. In starting the culture, CHO-K1 cells prepared in a separate container in advance were detached by trypsin treatment, seeded so that the cell density was 2 × 10 −5 (cells / ml), and the culture was started. In addition, the control value of the dissolved oxygen concentration by the dissolved oxygen concentration measuring means 4 was set to 3.0 mg / L. Further, in the case of the main culture apparatus, when the stirring rotation speed is 20 rpm or less, a part of the microcarriers is deposited on the bottom of the culture tank. During the culture period, the culture solution was collected once a day, and the amount of cells grown on the microcarriers was measured. When collecting the culture solution, the rotational speed was manually increased to 50 rpm so that the microcarriers were uniformly dispersed in the culture solution. The cells on the microcarrier were measured by detaching the cells by trypsin treatment. As a result, as indicated by A in FIG. 4, the maximum cell arrival density was 6.6 × 10 6 (cells / ml) on the 6th day of culture. When the microcarriers on the 6th day of culture were observed with an optical microscope, the entire surface was covered with cells, so that the microcarriers were hardly damaged.

比較として、筒状部材２の筒状部を取り外した封止部材２ｓから空気を直接通気した場合の培養結果を図４中のＢで示した。なお、通気は溶存酸素濃度が制御値を下回ったときに150ml/分で行った。実験装置としては前記の実験と同じ装置を使用し、培養手法も上記のとおりである。この場合、撹拌速度は全期間を通じて25rpmとした。最高細胞到達密度は、培養７日目の5.8×106（個/ml）であった。培養７日目のマイクロキャリアの表面には細胞の増殖していない部分が残されていた。 As a comparison, the culture result when air was directly ventilated from the sealing member 2s from which the cylindrical portion of the cylindrical member 2 was removed is shown by B in FIG. Aeration was performed at 150 ml / min when the dissolved oxygen concentration fell below the control value. As the experimental apparatus, the same apparatus as the above-mentioned experiment is used, and the culture technique is also as described above. In this case, the stirring speed was 25 rpm throughout the entire period. The highest cell arrival density was 5.8 × 10 6 (cells / ml) on the seventh day of culture. On the surface of the microcarrier on the 7th day of culture, a portion where cells did not grow was left.

図４に示した結果から、筒状部材２の内部に対して気泡２ｂを生じさせることで、筒状部材２の側面に対するマイクロキャリア等の付着を防止することができ、より効率的に細胞培養を行えることが明らかとなった。   From the results shown in FIG. 4, by generating bubbles 2b inside the cylindrical member 2, it is possible to prevent adhesion of microcarriers or the like to the side surface of the cylindrical member 2, and more efficiently cell culture. It became clear that can be done.

ところで、細胞培養装置は、図１に示したような構成に限定されず、例えば図５に示すように、複数の筒状部材２を培養槽１内に設置したような構成であってもよい。なお、図５に示した細胞培養装置は、追加供給用の培地１６を収容する培地槽１５と、使用済みの培養液１８を収容する廃液槽１７とを備え、培養中に培地の交換を実施できるタイプの培養装置である。培地槽１５は移送管路２１、廃液槽１７は移送管路２２によって培養槽１と連通している。移送管路２１には弁３１，移送管路２２には弁３２が設置されている。なお、図５中には図示していないが、図５に示す細胞培養装置は、空気、酸素、窒素及び炭酸ガス等のガス供給設備、温水冷水供給設備、蒸気供給設備、給排水設備及び各種の計測手段を具備している。   By the way, the cell culture device is not limited to the configuration as shown in FIG. 1, and may have a configuration in which a plurality of cylindrical members 2 are installed in the culture tank 1 as shown in FIG. 5, for example. . Note that the cell culture apparatus shown in FIG. 5 includes a medium tank 15 for storing the additional supply medium 16 and a waste liquid tank 17 for storing the used culture medium 18, and exchanging the medium during the culture. It is a type of culture device that can. The culture medium tank 15 communicates with the culture tank 1 through the transfer pipe line 21 and the waste liquid tank 17 communicates with the culture tank 1 through the transfer pipe line 22. A valve 31 is installed in the transfer pipeline 21, and a valve 32 is installed in the transfer pipeline 22. Although not shown in FIG. 5, the cell culture apparatus shown in FIG. 5 includes gas supply equipment such as air, oxygen, nitrogen and carbon dioxide, hot water / cold water supply equipment, steam supply equipment, water supply / drainage equipment, and various types of equipment. Measuring means are provided.

また、図５示す細胞培養装置は培養槽１内に４基の筒状部材２を備えている。これら４基の筒状部材２に対する通気ガスの供給は、それぞれ独立して制御しても良い、全て同時となるように制御しても良い。   Further, the cell culture apparatus shown in FIG. 5 includes four cylindrical members 2 in the culture tank 1. The supply of the aeration gas to these four cylindrical members 2 may be controlled independently, or may be controlled to be all at the same time.

さらに、細胞培養装置は、培養槽１の内圧を測定する圧力計２５を備え、圧力計２５による計測結果をもとに、圧力調整弁２６によって一定の圧力に保持することができる。通常、外部からの細菌等の侵入を防ぐため、培養槽１内部を0.01〜0.05MPaに加圧している。
なお、使用されるガス類はあらかじめ細菌等の微粒子を除去したものを使用する。 Furthermore, the cell culture device includes a pressure gauge 25 that measures the internal pressure of the culture tank 1, and can be held at a constant pressure by the pressure adjustment valve 26 based on the measurement result of the pressure gauge 25. Usually, in order to prevent invasion of bacteria and the like from the outside, the inside of the culture tank 1 is pressurized to 0.01 to 0.05 MPa.
In addition, the gas used removes particulates, such as bacteria, beforehand.

図５に示した細胞培養装置においても、図１に示した細胞培養装置と同様に、筒状部材２内部を上下方向に分割する気泡として通気ガスを供給することで、筒状部材２の側面に対する細胞やマイクロキャリア等の付着を防止して細胞培養、培地交換等の作業を行うことができる。また、細胞培養装置によれば、筒状部材２に形成された気泡が培養細胞と非接触の状態となる。このため、培養細胞が高濃度の酸素と接触することを防止するとともに、気泡の破裂の衝撃による損傷を防止することができる。したがって、培養細胞が直接高濃度の酸素ガスと接触することによる増殖阻害、気泡に同伴されたマイクロキャリアに付着した細胞が泡沫槽内に長時間閉じ込められることによる増殖阻害、及び気泡の破裂の衝撃による損傷などを原因とする培養への悪影響を防止でき、マイクロキャリアでの培養を良好に実施できる。   In the cell culture device shown in FIG. 5 as well, as in the cell culture device shown in FIG. 1, by supplying aeration gas as bubbles that divide the inside of the cylindrical member 2 in the vertical direction, Cell culture, medium exchange, etc. can be performed by preventing adhesion of cells, microcarriers, etc. Moreover, according to the cell culture apparatus, the air bubbles formed in the cylindrical member 2 are not in contact with the cultured cells. For this reason, it is possible to prevent the cultured cells from coming into contact with high-concentration oxygen and to prevent damage due to the burst burst impact. Therefore, growth inhibition due to direct contact of cultured cells with high-concentration oxygen gas, growth inhibition due to long-time trapping of cells attached to microcarriers entrained in bubbles in the foam tank, and impact of bubble rupture It is possible to prevent the adverse effect on the culture caused by damage caused by sucrose, and the culture with the microcarrier can be carried out satisfactorily.

１…培養槽、２…筒状部材、４…溶存酸素濃度計測手段、５…第１の制御装置、６…第２の制御装置、７…第３の制御装置、１３…培養液、１４・・・マイクロキャリア DESCRIPTION OF SYMBOLS 1 ... Culture tank, 2 ... Cylindrical member, 4 ... Dissolved oxygen concentration measuring means, 5 ... 1st control apparatus, 6 ... 2nd control apparatus, 7 ... 3rd control apparatus, 13 ... Culture solution, 14 * ..Microcarrier

Claims (14)

側面に細孔を有し、長手方向の一方端部が開放されるとともに他方端部が封止された筒状部材と、
上記他方端部側から上記筒状部材の内部に酸素含有ガスを供給する散気手段と、
上記筒状部材の長手方向が鉛直下方向と略平行かつ、上記他方端部が下方になるように配設した培養槽とを備える細胞培養装置を使用し、上記培養槽内に対して、上記筒状部材の一方端部が液外方へ露出するように細胞を培養するための培地が張り込められ、細胞を培養する方法であって、
上記酸素含有ガスを、上記筒状部材の内部を上下方向に分割する気泡として上記散気手段から供給することを特徴とする細胞の培養方法。 A cylindrical member having pores on the side surface and having one end in the longitudinal direction opened and the other end sealed,
Aeration means for supplying oxygen-containing gas into the cylindrical member from the other end side;
Using a cell culture device provided with a culture tank arranged such that the longitudinal direction of the cylindrical member is substantially parallel to the vertical downward direction and the other end portion is below, A medium for culturing cells so that one end of the cylindrical member is exposed to the outside of the liquid is embedded, and the cells are cultured,
A method for culturing cells, characterized in that the oxygen-containing gas is supplied from the aeration means as bubbles that divide the inside of the cylindrical member in the vertical direction. 上記散気手段は、上記気泡が上記筒状部材の内部を通って上記一方端部側で消泡したタイミングで次の気泡を供給することを特徴とする請求項１記載の細胞の培養方法。   2. The cell culturing method according to claim 1, wherein the aeration means supplies the next bubble at a timing when the bubble passes through the inside of the tubular member and disappears on the one end side. 培養対象の細胞が付着依存性を有する生体細胞であって、当該生体細胞の足場となる培養面を有するマイクロキャリアとともに当該生体細胞を培養することを特徴とする請求項１記載の細胞の培養方法。   The method for culturing cells according to claim 1, wherein the cells to be cultured are living cells having adhesion dependency, and the living cells are cultured together with a microcarrier having a culture surface as a scaffold for the living cells. . 上記筒状部材の側面における細孔は、培養対象の細胞又は当該細胞の足場となるマイクロキャリアよりも小径であることを特徴とする請求項１記載の細胞の培養方法。   The method for culturing cells according to claim 1, wherein the pores on the side surfaces of the cylindrical member have a smaller diameter than the cells to be cultured or the microcarriers serving as the scaffolds for the cells. 上記筒状部材の側面における細孔は、50〜150μmの直径を有する貫通孔であることを特徴とする請求項１記載の細胞の培養方法。   The method for culturing cells according to claim 1, wherein the pores on the side surface of the cylindrical member are through-holes having a diameter of 50 to 150 µm. 上記筒状部材の少なくとも側面は可撓性を有する材質から形成され、上記気泡の通過に伴って当該側面が変形することを特徴とする請求項１記載の細胞の培養方法。   The method for culturing cells according to claim 1, wherein at least a side surface of the cylindrical member is formed of a flexible material, and the side surface is deformed as the bubble passes. 上記培養装置は、培養槽内に複数の上記筒状部材を配設したものであることを特徴とする請求項１記載の細胞の培養方法。   The method for culturing cells according to claim 1, wherein the culture apparatus comprises a plurality of cylindrical members arranged in a culture tank. 側面に細孔を有し、長手方向の一方端部が開放されるとともに他方端部が封止された筒状部材と、
上記他方端部側から上記筒状部材の内部に酸素含有ガスを供給する散気手段と、
上記筒状部材の長手方向が鉛直下方向と略平行かつ、上記他方端部が下方になるように配設した培養槽とを備え、
上記散気手段は、上記筒状部材の内部に培養液が張り込まれた状態において、上記酸素含有ガスを、上記筒状部材の内部を上下方向に分割する気泡として供給するものであることを特徴とする細胞培養装置。 A cylindrical member having pores on the side surface and having one end in the longitudinal direction opened and the other end sealed,
Aeration means for supplying oxygen-containing gas into the cylindrical member from the other end side;
A culture tank disposed so that the longitudinal direction of the cylindrical member is substantially parallel to the vertical downward direction and the other end is downward,
The aeration means supplies the oxygen-containing gas as bubbles that divide the inside of the cylindrical member in the vertical direction in a state where the culture solution is stretched inside the cylindrical member. A cell culture device. 上記気泡が上記筒状部材の内部を通って上記一方端部側で消泡したタイミングで次の気泡を供給するように、上記散気手段を制御する制御装置を更に備えることを特徴とする請求項８記載の細胞培養装置。   The apparatus further comprises a control device for controlling the aeration means so as to supply the next bubble at a timing when the bubble passes through the inside of the cylindrical member and disappears on the one end side. Item 9. The cell culture device according to Item 8. 培養対象の細胞が付着依存性を有する生体細胞であって、当該生体細胞の足場となる培養面を有するマイクロキャリアとともに当該生体細胞を培養することを特徴とする請求項８記載の細胞培養装置。   The cell culture device according to claim 8, wherein the cell to be cultured is a living cell having adhesion dependency, and the living cell is cultured together with a microcarrier having a culture surface as a scaffold for the living cell. 上記筒状部材の側面における細孔は、培養対象の細胞又は当該細胞の足場となるマイクロキャリアよりも小径であることを特徴とする請求項８記載の細胞培養装置。   9. The cell culture device according to claim 8, wherein the pores on the side surface of the cylindrical member have a smaller diameter than a cell to be cultured or a microcarrier serving as a scaffold for the cell. 上記筒状部材の側面における細孔は、50〜150μmの直径を有する貫通孔であることを特徴とする請求項８記載の細胞培養装置。   The cell culture device according to claim 8, wherein the pores on the side surface of the cylindrical member are through-holes having a diameter of 50 to 150 µm. 上記筒状部材の少なくとも側面は可撓性を有する材質から形成され、上記気泡の通過に伴って当該側面が変形することを特徴とする請求項８記載の細胞培養装置。   9. The cell culture device according to claim 8, wherein at least a side surface of the cylindrical member is formed of a flexible material, and the side surface is deformed as the bubbles pass. 上記培養槽内に複数の上記筒状部材を配設したものであることを特徴とする請求項８記載の細胞の細胞培養装置。
9. The cell culture apparatus for cells according to claim 8, wherein a plurality of the cylindrical members are disposed in the culture tank.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105143430A (en) * 2013-04-22 2015-12-09 费尔曼塔格公司 Reactor with integrated illumination
CN103224879A (en) * 2013-05-13 2013-07-31 宁夏大学 Photobioreactor device for high-density culturing of microalgae
JP2017029095A (en) * 2015-08-04 2017-02-09 オリンパス株式会社 Cell culture apparatus
JP2019505240A (en) * 2016-02-23 2019-02-28 コーニング インコーポレイテッド Perfusion bioreactor and method of use for performing continuous cell culture
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