JP4534702B2 - Cell culture container and medium storage method - Google Patents

Cell culture container and medium storage method Download PDF

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JP4534702B2
JP4534702B2 JP2004294822A JP2004294822A JP4534702B2 JP 4534702 B2 JP4534702 B2 JP 4534702B2 JP 2004294822 A JP2004294822 A JP 2004294822A JP 2004294822 A JP2004294822 A JP 2004294822A JP 4534702 B2 JP4534702 B2 JP 4534702B2
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義洋 吉川
秀明 村橋
一樹 石原
奈穂美 中谷
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Description

本発明は、炭酸塩を含有する培地の長期保存に優れた細胞培養用容器に関する。好ましくは培養中における培地への通気が十分である細胞培養用容器に関する。   The present invention relates to a cell culture container excellent in long-term storage of a medium containing carbonate. Preferably, the present invention relates to a cell culture vessel in which ventilation to the medium during culture is sufficient.

近年、機能障害や機能不全に陥った生体組織および臓器に対して、細胞を積極的に利用して、その機能の再生をはかることを目的とした再生医療の分野が発展してきた。哺乳動物から健常な細胞を取り出し、該細胞を生体外で培養した後、再び生体に戻す技術もこの一環である。このような細胞の培養に用いる培地は、エネルギー源、栄養素、補酵素、無機イオン、ｐＨ緩衝液を培養すべき細胞に応じてそれぞれの配合量で調製する。必要に応じて、血清や細胞成長因子も添加する。エネルギー源としては、グルコースなどが用いられ、細胞の生理活動に必要な燃料となる。栄養素は核酸、アミノ酸などの細胞の構成原料が選択される。補酵素は、ビタミン類や微量金属などの細胞内酵素活性の増強と安定化する役割のものが選択される。無機イオンはナトリウムイオン、カリウムイオンなどの生体内に存在するイオンであり、培地の浸透圧の調整を行う役割を果たす。そして、ｐＨ緩衝剤液は以上の成分を配合した培地のｐＨを調整する役割を果たし、リン酸緩衝液、重曹、Ｎ−２−ヒドロキシエチルピペラジン−Ｎ’−２−エタンスルホン酸（ＨＥＰＥＳ）などから適切なものが使用される。   In recent years, the field of regenerative medicine has been developed in which cells are actively used for biological tissues and organs that have become dysfunctional or dysfunctional to regenerate their functions. Part of this is a technique in which healthy cells are removed from mammals, cultured in vitro, and then returned to the living body. The medium used for culturing such cells is prepared in an appropriate amount according to the cells to be cultured with the energy source, nutrients, coenzyme, inorganic ions, and pH buffer solution. If necessary, serum and cell growth factors are also added. As an energy source, glucose or the like is used and becomes a fuel necessary for the physiological activities of cells. Nutrients are selected from cell constituents such as nucleic acids and amino acids. As the coenzyme, one having a role of enhancing and stabilizing intracellular enzyme activities such as vitamins and trace metals is selected. Inorganic ions are ions existing in the living body such as sodium ions and potassium ions, and play a role of adjusting the osmotic pressure of the medium. And the pH buffer solution plays the role which adjusts the pH of the culture medium which mix | blended the above component, phosphate buffer solution, baking soda, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES), etc. The appropriate one is used.

従来、哺乳動物の懸濁細胞を生体外で行う培養は、ポリスチレン製のシャーレの中で行われてきた。しかし、このような容器はガス透過性が低いため、細胞増殖に必要な酸素や二酸化炭素の交換を十分行うために、培養液の液厚を３ｍｍ程度とし、容器中に空気層を設けなくては、十分な細胞増殖が得られなかった。従って、細胞を大量に培養するときには、無駄な空間があるため、多くのスペースを必要としていた。   Conventionally, culturing of mammalian suspension cells in vitro has been performed in a petri dish made of polystyrene. However, since such a container has low gas permeability, in order to sufficiently exchange oxygen and carbon dioxide necessary for cell growth, the thickness of the culture solution is about 3 mm, and an air layer is not provided in the container. Did not provide sufficient cell growth. Therefore, when culturing a large amount of cells, a lot of space is required because there is a useless space.

かかる問題を解決すべく、酸素透過性を有するアイオノマー樹脂からなる容器が細胞培養用容器として開示されている（特許文献１）。また、気体透過性材料で作製された第１の容器の内部に、液体透過性材料で作製された第２の容器で形成された細胞培養用容器が開示されており、外部と第１の容器との間で、十分な酸素付加を可能にし、内部の第２の容器にて高密度で細胞を培養することを可能にした細胞培養用培養容器が開示されている（特許文献２、３）。さらに、容器の一面が気体透過性であるシリコン誘導スチレンポリマーで形成され、強度に優れた培養容器が開示されている（特許文献４）。加えて、ポリ−４−メチルペンテン−１樹脂とポリ−４−メチルペンテン−１樹脂以外のポリオレフィン樹脂からなる組成物で形成され、酸素透過性および光透過性に優れ、植物細胞の培養に適した細胞培養用容器が開示されている（特許文献５）。   In order to solve this problem, a container made of ionomer resin having oxygen permeability is disclosed as a cell culture container (Patent Document 1). Also disclosed is a cell culture container formed of a second container made of a liquid permeable material inside the first container made of a gas permeable material, the outside and the first container. A culture vessel for cell culture that enables sufficient oxygenation and enables high-density culture of cells in a second vessel inside (Patent Documents 2 and 3). . Furthermore, a culture vessel is disclosed in which one side of the vessel is formed of a silicon-derived styrene polymer that is gas permeable and has excellent strength (Patent Document 4). In addition, it is formed of a composition comprising a poly-4-methylpentene-1 resin and a polyolefin resin other than poly-4-methylpentene-1 resin, and is excellent in oxygen permeability and light permeability and suitable for plant cell culture. A cell culture vessel has been disclosed (Patent Document 5).

しかし、これらの細胞培養用容器は、培養時における培地への通気性は十分であるが、緩衝液として炭酸塩を含有する培地を用いた場合、二酸化炭素の拡散による培地のｐＨが変化し、細胞培養用培地の長期保存が困難であった。   However, these cell culture containers have sufficient air permeability to the medium during the culture, but when a medium containing carbonate is used as a buffer, the pH of the medium changes due to the diffusion of carbon dioxide, Long-term storage of cell culture media was difficult.

特開昭６０−１６０８８１号公報JP 60-160881 A 特開平３−１０６７５号公報Japanese Patent Laid-Open No. 3-10675 特開平３−１０６７６号公報Japanese Patent Laid-Open No. 3-10676 特開平１１−２８０８３号公報JP-A-11-28083 特開２００１−１９０２６７号公報JP 2001-190267 A

したがって、炭酸塩を含有する培地の長期保存を可能にし、好ましくは培養中における培地への通気が十分である細胞培養用容器を提供することが求められている。   Accordingly, there is a need to provide a cell culture vessel that enables long-term storage of a medium containing carbonate and that is preferably well ventilated to the medium during culture.

本発明は、
（１） 少なくとも炭酸塩を含有する培地成分を収容し、非通気性材料からなり、細胞導入口を設けた第１の容器と、該炭酸塩以外の培地成分を含有し、通気性材料からなる第２の容器と、該第１の容器と該第２の容器とを連通する連通手段からなる細胞培養用容器、
（２） 非通気性材料の二酸化炭素透過係数が約１０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ以下である（１）に記載の細胞培養用容器、
（３） 非通気性材料の水蒸気透過係数が約１０００ｇ／ｍ２・２４ｈｒ・ａｔｍ以下である（１）に記載の細胞培養用容器、
（４） 通気性材料の酸素透過係数が約１００〜５０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍである（１）に記載の細胞培養用容器、
（５） 通気性材料の二酸化炭素透過係数が約１００１〜２００００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍである（１）に記載の細胞培養用容器、
（６） 炭酸塩が炭酸水素ナトリウムである（１）に記載の細胞培養用容器、
および（７） 少なくとも炭酸塩を含有する培地成分を収容し、非通気性材料からなり、細胞導入口を設けた第１の容器と、該炭酸塩以外の培地成分を含有し、通気性材料からなる第２の容器と、該第１の容器と該第２の容器とを連通する連通手段からなる細胞培養用容器を用い、以下の行程よりなる細胞培養方法；
(i) 第１の容器に収容した、少なくとも炭酸塩を含有する培地成分を、連通手段を通じて第２の容器に移動し、少なくとも炭酸塩を含有する培地成分と、該炭酸塩以外の培地成分とを混合する培地調製工程：
(ii) 細胞導入口から、第１の容器、連通手段を経て、第２の容器へ細胞を導入する細胞導入工程：
(iii) 細胞を培養する細胞培養工程に関する。 The present invention
(1) A medium component containing at least a carbonate is contained, made of a non-breathable material, a first container provided with a cell inlet, a medium component other than the carbonate, and made of a breathable material A cell culture container comprising a second container and communication means for communicating the first container and the second container;
(2) The cell culture vessel according to (1), wherein the non-breathable material has a carbon dioxide permeability coefficient of about 1000 cm 3 / m 2 · 24 hr · atm or less,
(3) The cell culture vessel according to (1), wherein the water vapor permeability coefficient of the non-breathable material is about 1000 g / m 2 · 24 hr · atm or less,
(4) The cell culture vessel according to (1), wherein the breathable material has an oxygen permeability coefficient of about 100 to 5000 cm 3 / m 2 · 24 hr · atm.
(5) The container for cell culture according to (1), wherein the carbon dioxide permeability coefficient of the breathable material is about 1001 to 20000 cm 3 / m 2 · 24 hr · atm.
(6) The cell culture vessel according to (1), wherein the carbonate is sodium hydrogen carbonate,
And (7) containing at least a medium component containing carbonate, made of a non-breathable material, having a cell introduction port, containing medium components other than the carbonate, and made from a breathable material A cell culture method comprising the following steps using a second container and a container for cell culture comprising communication means for communicating the first container and the second container;
(i) The medium component containing at least carbonate contained in the first container is transferred to the second container through the communication means, and the medium component containing at least carbonate, and medium components other than the carbonate, Medium preparation process for mixing:
(ii) Cell introduction step of introducing cells from the cell introduction port into the second container through the first container and the communication means:
(iii) It relates to a cell culture process for culturing cells.

本発明の細胞培養用容器は、炭酸塩を含有する培地の長期保存、および十分に通気される雰囲気下での培養を可能にする。さらに、細胞導入口を連通ピース等で閉鎖された第１の容器に設けているため、培地の調製を行ってからではないと培養することができないため、使用者の誤った操作を防止することができる。   The container for cell culture of the present invention enables long-term storage of a medium containing carbonate and culture under a well-ventilated atmosphere. Furthermore, since the cell introduction port is provided in the first container closed with a communication piece or the like, the culture can be performed only after the medium is prepared. Can do.

本発明における細胞培養用容器は、培地成分として、少なくとも炭酸塩を含有する培地成分（以下、培地成分Ａともいう）を収容し、非通気性材料からなり、細胞導入口を設けた第１の容器と、該炭酸塩以外の培地成分（以下、培地成分Ｂともいう）を含有し、通気性材料からなる第２の容器と、該第１の容器と該第２の容器とを連通する連通手段からなることを特徴としている。第１の容器及び第２の容器の形状は特に限定することはなく、バッグ、ボトルなどが挙げられるが、量産が容易であり、軽量である可撓性のシートで形成された袋状であることが好ましい。   The cell culture container in the present invention contains a medium component containing at least carbonate (hereinafter also referred to as a medium component A) as a medium component, is made of a non-breathable material, and is provided with a cell inlet. A container, a medium component other than the carbonate (hereinafter also referred to as medium component B), a second container made of a breathable material, and a communication for communicating the first container and the second container It is characterized by comprising means. The shape of the first container and the second container is not particularly limited, and examples thereof include bags, bottles, etc., but they are bag-shaped formed of a flexible sheet that is easy to mass-produce and lightweight. It is preferable.

本発明の第１の容器は、非通気性材料であることを特徴としている。非通気性材料は、二酸化炭素が透過しにくい材料を意味し、具体的には、温度２５℃、湿度５０％、大気圧下における二酸化炭素透過係数が約１０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ以下、好ましくは約１００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ以下であり、さらに好ましくは約１ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ以下であり、水蒸気透過係数が約１０００ｇ／ｍ２・２４ｈｒ・ａｔｍ以下、好ましくは約１００ｇ／ｍ２・２４ｈｒ・ａｔｍ以下であり、さらに好ましくは約１０ｇ／ｍ２・２４ｈｒ・ａｔｍ以下である。さらに工業的に成形加工性に優れたものであり、ガンマ線滅菌に耐えうるものであることがより好ましい。選択すべき適切な材料としては、延伸ナイロン、ポリエステル、ポリ塩化ビニリデン、ポリ塩化ビニリデンコート延伸ナイロン、ポリ塩化ビニリデンコートポリエステル、ポリ塩化ビニルコートポリプロピレン、ポリビニルアルコール、ポリ（エチレン−ビニルアルコール）コポリマー、アルミ蒸着ポリエステル、シリカコートポリエステルなどが挙げられる。 The first container of the present invention is a non-breathable material. The non-breathable material means a material that does not easily transmit carbon dioxide. Specifically, the carbon dioxide permeability coefficient at a temperature of 25 ° C., a humidity of 50%, and atmospheric pressure is about 1000 cm 3 / m 2 · 24 hr · atm or less. Preferably about 100 cm 3 / m 2 · 24 hr · atm or less, more preferably about 1 cm 3 / m 2 · 24 hr · atm or less, and a water vapor transmission coefficient of about 1000 g / m 2 · 24 hr · atm or less, preferably Is about 100 g / m 2 · 24 hr · atm or less, more preferably about 10 g / m 2 · 24 hr · atm or less. Furthermore, it is more preferable that it is industrially excellent in moldability and can withstand gamma ray sterilization. Suitable materials to select include oriented nylon, polyester, polyvinylidene chloride, polyvinylidene chloride coated oriented nylon, polyvinylidene chloride coated polyester, polyvinyl chloride coated polypropylene, polyvinyl alcohol, poly (ethylene-vinyl alcohol) copolymer, aluminum Examples include vapor-deposited polyester and silica-coated polyester.

また、第２の容器の材料は、通気性材料であることを特徴としている。通気性材料は、温度２５℃における酸素の透過係数が約１００〜５０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、好ましくは約１１００〜３０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、さらに好ましくは約１２５０〜２７５０ｃｍ３／ｍ２・２４ｈｒ・ａｔｍであり、二酸化炭素透過係数が約１００１〜２００００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、好ましくは約３０００〜１１５００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍであり、さらに好ましくは約５０００〜９０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍである。さらに工業的に成形加工性に優れたものが好ましく、ガンマ線滅菌に耐えうるものであり、かつ内部の培地の様子を観察することができる透明性の高い材料であることが好ましい。選択すべき適切な材料としては、ポリエチレン、ポリ塩化ビニル、ポリ（エチレン−ビニルアセテート）コポリマー、ポリ（エチレン−エチルアクリレート）コポリマー、ポリ（エチレン−メタアクリレート）コポリマーなどが挙げられる。 The material of the second container is a breathable material. The breathable material has an oxygen permeability coefficient at a temperature of 25 ° C. of about 100 to 5000 cm 3 / m 2 · 24 hr · atm, preferably about 1100 to 3000 cm 3 / m 2 · 24 hr · atm, more preferably about 1250 to 2750 cm 3. / M 2 · 24 hr · atm and a carbon dioxide permeability coefficient of about 1001 to 20000 cm 3 / m 2 · 24 hr · atm, preferably about 3000 to 11500 cm 3 / m 2 · 24 hr · atm, more preferably about 5000. It is -9000cm < 3 > / m < 2 > * 24hr * atm. Furthermore, those excellent in moldability are preferred industrially, preferably materials that can withstand gamma ray sterilization and that can observe the state of the medium inside. Suitable materials to select include polyethylene, polyvinyl chloride, poly (ethylene-vinyl acetate) copolymer, poly (ethylene-ethyl acrylate) copolymer, poly (ethylene-methacrylate) copolymer, and the like.

本発明における培地は基本的に、エネルギー源、栄養素、補酵素、無機イオン、血清およびｐＨ緩衝液から構成される汎用のものを意味する。これらの培地成分は、培養する細胞に応じてそれぞれの配合量で調製する。エネルギー源は細胞の生理活動に必要な燃料となり、グルコースなどが挙げられる。栄養素として核酸、アミノ酸などが添加され、細胞構成原料となる。アミノ酸はバリン、ロイシン、リジンなどの必須アミノ酸だけでなく、非必須アミノ酸なども添加される。補酵素は、ビタミン類や微量金属などの細胞内酵素活性の増強と安定化させる役割がある。無機イオンは生体内に存在するイオンであり、培地の浸透圧の調整を行う役割を果たす。例としては、ナトリウムイオン、カリウムイオン、マグネシウムイオンおよびカルシウムイオンなどが挙げられる。また必要に応じて、血清や細胞成長因子も添加する。そして、ｐＨ緩衝液は以上の成分を配合した培地のｐＨを調整する役割を果たし、リン酸緩衝液、炭酸塩（重曹）、Ｎ−２−ヒドロキシエチルピペラジン−Ｎ’−２−エタンスルホン酸（ＨＥＰＥＳ）などが挙げられる。   The medium in the present invention basically means a general-purpose medium composed of an energy source, nutrients, coenzymes, inorganic ions, serum and pH buffer solution. These medium components are prepared in respective amounts according to the cells to be cultured. The energy source becomes a fuel necessary for the physiological activity of the cell, and glucose and the like can be mentioned. Nucleic acids, amino acids, and the like are added as nutrients to become cell constituent materials. Amino acids include not only essential amino acids such as valine, leucine, and lysine, but also non-essential amino acids. Coenzymes have the role of enhancing and stabilizing intracellular enzyme activities such as vitamins and trace metals. Inorganic ions are ions present in the living body and play a role of adjusting the osmotic pressure of the medium. Examples include sodium ion, potassium ion, magnesium ion and calcium ion. If necessary, serum and cell growth factor are also added. The pH buffer serves to adjust the pH of the medium containing the above components, and is phosphate buffer, carbonate (bicarbonate), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid ( HEPES).

また、これらの培地には上記成分以外の組成物または天然物を適時加えてもよい。これらの添加物としては、インスリン、ホルボールエステル、ヒドロコルチゾンおよび動物由来脳下垂体抽出物などが挙げられる。   In addition, compositions or natural products other than the above components may be added to these media as appropriate. These additives include insulin, phorbol ester, hydrocortisone and animal-derived pituitary extracts.

本発明の炭酸塩を含有する培地とは、上記培地における培地成分の一つであるｐＨ緩衝液として炭酸塩を選択した培地を意味する。これ以外の培地成分は、特に限定することはなく、培養すべき細胞に対して適時選択される。   The medium containing the carbonate of the present invention means a medium in which carbonate is selected as a pH buffer that is one of the medium components in the medium. Other medium components are not particularly limited and are appropriately selected for the cells to be cultured.

本発明における炭酸塩とは、溶媒に溶解したときに二酸化炭素を発生するものであり、炭酸イオンとして遊離しうるもの全てを含む。炭酸塩の例としては、炭酸水素ナトリウム、炭酸水素カリウム、炭酸マグネシウムなどが挙げられるが、炭酸水素ナトリウムが一般に使用される。   The carbonate in the present invention generates carbon dioxide when dissolved in a solvent, and includes all that can be liberated as carbonate ions. Examples of carbonates include sodium bicarbonate, potassium bicarbonate, magnesium carbonate, etc., but sodium bicarbonate is generally used.

本発明における少なくとも炭酸塩を含有する培地成分（培地成分Ａ）とは、炭酸塩を含有する培地を構成している培地成分のうち少なくとも炭酸塩を含有していればよく、炭酸塩のみ、炭酸塩と補酵素、炭酸塩と無機イオンなどその組み合わせにとらわれるものではなく、炭酸塩を含有する培地自体である場合も含まれる。   The medium component (medium component A) containing at least carbonate in the present invention is only required to contain at least carbonate among the medium components constituting the medium containing carbonate. It is not limited to the combination of salts and coenzymes, carbonates and inorganic ions, and includes a medium containing carbonate itself.

この際、第２の容器には培地成分Ａ以外の培地成分（培地成分Ｂ）を収容する。培地成分Ｂは、炭酸塩を含まなければ特に限定することはないが、第１の容器に収容した培地成分Ａと、第２の容器に収容した培地成分Ｂを混合した時に、目的の培地が調製されるようにすることが好ましい。例えば、第１の容器に培地成分Ａとして炭酸塩を収容した場合、第２の容器には培地成分Ｂとしてエネルギー源、栄養素、補酵素、無機イオンが収容され、第１の容器に培地成分Ａとして炭酸塩及びエネルギー源を収容した場合、第２の容器には培地成分Ｂとして栄養素、補酵素、無機イオンが収容される。   At this time, a medium component (medium component B) other than the medium component A is accommodated in the second container. The medium component B is not particularly limited as long as it does not contain carbonate, but when the medium component A stored in the first container and the medium component B stored in the second container are mixed, the target medium is It is preferable to be prepared. For example, when carbonate is contained as the medium component A in the first container, the second container contains the energy source, nutrients, coenzyme, and inorganic ions as the medium component B, and the medium component A is stored in the first container. When a carbonate and an energy source are stored, nutrients, coenzymes, and inorganic ions are stored as the medium component B in the second container.

また、上記のような無機イオン、栄養素などのカテゴリーによって分ける必要はなく、例えば、培地成分Ａとして炭酸塩、カルシウムイオン、グルコースおよびグルタミン酸などを第１の容器に収容し、培地成分Ｂとしてそれ以外の培地成分を第２の容器に収容することもできる。   Moreover, it is not necessary to divide according to the above categories such as inorganic ions and nutrients. For example, carbonate component, calcium ion, glucose, glutamic acid and the like are accommodated in the first container as the medium component A, and other components as the medium component B. The medium component can be accommodated in the second container.

本発明の第１の容器と、第２の容器には連通手段を設ける。該連通手段はチューブなどが一般的であるが、第１の容器と第２の容器を組み合わせた２室型の形態も含む。該チューブは、塩化ビニルまたはシリコンなどの汎用の材料を使用することができる。   Communication means is provided in the first container and the second container of the present invention. The communication means is generally a tube or the like, but includes a two-chamber type in which the first container and the second container are combined. A general-purpose material such as vinyl chloride or silicon can be used for the tube.

また、搬送時または保存時などにおいて、培地成分Ａと培地成分Ｂとが混合しないように、連通手段には再度開通を可能にする閉鎖手段を設けることが好ましい。再度開通を可能にする閉鎖手段としては、クリップなどの狭持体、連通ピース（折れ棒）または２室型の場合は弱シールなどが挙げられる。これらの再度開通を可能にする閉鎖手段は、除去または連通させることにより、培地成分Ａと培地成分Ｂとを混合することができる。   Moreover, it is preferable to provide the communication means with a closing means that allows the communication means to be opened again so that the culture medium component A and the culture medium component B are not mixed during transportation or storage. Examples of the closing means that can be opened again include a holding member such as a clip, a communication piece (folding bar), or a weak seal in the case of a two-chamber type. The closing means that enables the reopening can be removed or communicated to mix the medium component A and the medium component B.

さらに、第１の容器には細胞導入口を設ける。該細胞導入口はある程度可撓性を有する材料であり、第１の容器の材料（非通気性材料）と熱溶着性のよいものを使用することが好ましく、例えば超低密度ポリエチレンなどで作製したものが挙げられる。また、細胞導入口を取り付ける位置は特に限定されるものではないが、取り扱いの上で連通手段と対向する位置に配置することが好ましい。   Further, the first container is provided with a cell introduction port. The cell inlet is a material having a certain degree of flexibility, and it is preferable to use a material having good heat-weldability with the material of the first container (non-breathable material), for example, made of ultra-low density polyethylene. Things. Further, the position where the cell introduction port is attached is not particularly limited, but it is preferably disposed at a position facing the communication means in handling.

本発明の細胞培養用容器を用いて細胞を培養方法とは、本発明の第１の容器に収容された培地成分Ａと第２の容器に収容された培地成分Ｂとを混合して調製された炭酸塩を含有する培地を用いて、十分に通気された雰囲気下で細胞を培養することができる。十分に通気された雰囲気下とは、一般的には約５％二酸化炭素含有気体での雰囲気下を意味する。   The method for culturing cells using the cell culture container of the present invention is prepared by mixing the medium component A accommodated in the first container of the present invention and the medium component B accommodated in the second container. Cells can be cultured in a well-ventilated atmosphere using a medium containing carbonate. A well-ventilated atmosphere generally means an atmosphere with a gas containing about 5% carbon dioxide.

図１は、本発明の細胞培養用容器の一実施形態である。非通気性材料で形成した第１の容器１には、細胞導入口３が設けられており、培地成分として重曹（炭酸水素ナトリウム水溶液）４１が収容されている。第２の容器２には、重曹以外の培地成分４２が収容されている。第１の容器と第２の容器は組み合わせることによって連通されており（連通手段５）、該連通手段には、再度開通を可能にする閉鎖手段として弱シール部６を形成している。弱シール部は、第１の容器または第２の容器を押圧することにより連通が可能となる。   FIG. 1 is an embodiment of the cell culture container of the present invention. The first container 1 formed of a non-breathable material is provided with a cell inlet 3 and contains sodium bicarbonate (sodium bicarbonate aqueous solution) 41 as a medium component. The second container 2 contains a medium component 42 other than baking soda. The first container and the second container are communicated by being combined (communication means 5), and a weak seal portion 6 is formed in the communication means as a closing means that enables reopening. The weak seal part can be communicated by pressing the first container or the second container.

さらに図２は、本発明の他の一実施形態である。非通気性材料で形成した第１の容器１には、細胞導入口３が設けられており、培地成分として重曹４１が収容されている。第２の容器２には、重曹以外の培地成分４２が収容されている。第１の容器と第２の容器とを塩化ビニル製チューブ５で接続し、該チューブには再度開通を可能にする閉鎖手段として連通ピース（折れ棒）６を設けている。連通ピース（折れ棒）は、内部の部材を折ることにより再連通が可能となる。   FIG. 2 shows another embodiment of the present invention. The first container 1 formed of a non-breathable material is provided with a cell inlet 3 and contains baking soda 41 as a medium component. The second container 2 contains a medium component 42 other than baking soda. The first container and the second container are connected by a tube 5 made of vinyl chloride, and a communication piece (folding bar) 6 is provided in the tube as a closing means for enabling opening again. The communication piece (folding bar) can be reconnected by folding an internal member.

以下に本発明を、実施例を用いて詳細に説明するが、本発明はこれらの実施例に限定されるものではない。   Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to these examples.

実施例１：図１の細胞培養用容器の作製
第１の容器は、内層が低密度ポリエチレン、中間層がポリ（エチレン−ビニルアルコール）コポリマー、外層に低密度ポリエチレンの２枚のシートを用意し、２枚のシートの間にポリエチレン−ポリプロピレン高分子ブレンド（混合比３：７）製の弱シール形成用シートを所定長分埋没するように配置した。次いで、２枚のシートの側縁および弱シール用シートを含んだ３枚のシートが重なる部分を通る帯状部分を熱溶着して、口部を有する袋状の容器を作製した。この袋状の容器に、炭酸水素ナトリウム水溶液３０ｍｌを収容し、袋状の容器の口部に、低密度ポリエチレン製の細胞導入口を設け、熱溶着して作製した。 Example 1 Production of Cell Culture Container in FIG. 1 A first container is prepared by preparing two sheets of low density polyethylene for the inner layer, poly (ethylene-vinyl alcohol) copolymer for the intermediate layer, and low density polyethylene for the outer layer. A sheet for weak seal formation made of polyethylene-polypropylene polymer blend (mixing ratio 3: 7) was placed between the two sheets so as to be buried for a predetermined length. Next, a band-shaped portion passing through a portion where three sheets including the side edges of the two sheets and the weak sealing sheet overlap each other was thermally welded to produce a bag-like container having a mouth portion. This bag-shaped container accommodated 30 ml of a sodium hydrogen carbonate aqueous solution, and a cell introduction port made of low-density polyethylene was provided at the mouth of the bag-shaped container and heat-welded.

さらに別途で、線状低密度ポリエチレンをインフレーション法によりチューブ状のフィルムを作製した後、片方の口部に弱シール形成用シートが所定長突出するように配置し、弱シール用シートを含んだ３枚のシートが重なる部分を通る帯状部分を溶着して、口部を有する袋状の容器を作製した。この袋状の容器に、炭酸水素ナトリウム水溶液以外の培地成分すべてを収容し、袋状の容器の口部を熱溶着し、第２の容器（容量１００ｍｌ）２を作製した。   In addition, a tube-shaped film was produced from linear low-density polyethylene by an inflation method, and then a weak seal-forming sheet was arranged so that it protruded for a predetermined length at one mouth portion. A band-shaped portion passing through the overlapping portion of the sheets was welded to prepare a bag-like container having a mouth portion. All the medium components other than the sodium hydrogen carbonate aqueous solution were accommodated in this bag-shaped container, and the mouth of the bag-shaped container was thermally welded to produce a second container (capacity 100 ml) 2.

次に第１の容器と第２の容器を接続した。このとき第２の容器において突出している弱シール形成用シール及び第２の容器の作製過程において形成した弱シール部を含む線状低密度ポリエチレン部の一部も第１の容器の熱溶着していない部分に挿入した。次に、第２の容器において突出していた弱シール形成用シートと第１の容器の熱溶着していない部分が重なっている箇所（３枚のシートが重なる部分）及び第２の容器の作製過程において形成した弱シール部を含む線状低密度ポリエチレンの一部と第１の容器の熱溶着していない部分が重なっている箇所（５枚のシートが重なる部分）を通る帯状部分を溶着して接続することで、図１に示す細胞培養用容器を作製した。   Next, the first container and the second container were connected. At this time, a part of the linear low density polyethylene part including the weak seal forming seal protruding in the second container and the weak seal part formed in the manufacturing process of the second container is also thermally welded to the first container. Inserted in no part. Next, a weak seal-forming sheet that protrudes in the second container overlaps with a portion of the first container that is not thermally welded (a portion where three sheets overlap), and a process for producing the second container A belt-shaped portion passing through a portion where a portion of the linear low density polyethylene including the weak seal portion formed in step 1 and a portion where the first container is not thermally welded overlaps (a portion where five sheets overlap) is welded. By connecting, the cell culture container shown in FIG. 1 was produced.

実施例２：図２の細胞培養用容器の作製
内層が低密度ポリエチレン、中間層がポリ（エチレン−ビニルアルコール）コポリマー、外層に低密度ポリエチレンの２枚のシートを用意し、２枚のシートを重ね合わせ、細胞導入口と、チューブを接続するポートを設置したうえで、シートの側縁を熱溶着して、第１の容器１を作成した。さらに、上記ポートから炭酸水素ナトリウム水溶液３０ｍｌを収容した。 Example 2 Preparation of Cell Culture Container in FIG. 2 Prepare two sheets of low density polyethylene for the inner layer, poly (ethylene-vinyl alcohol) copolymer for the intermediate layer, and low density polyethylene for the outer layer. The first container 1 was prepared by superimposing and installing a cell introduction port and a port for connecting the tube, and then thermally welding the side edges of the sheet. Further, 30 ml of an aqueous sodium hydrogen carbonate solution was accommodated from the port.

さらに別途で、線状低密度ポリエチレンをインフレーション法によりチューブ状のフィルムを作成した後、一方の口部にチューブを接続するポートを配置し、両方の口部を熱溶着して第２の容器１を作製した。さらに、上記ポートから炭酸水素ナトリウム水溶液以外の培地成分を収容した。
次に第１の容器と第２の容器を接続する。第１の容器及び第２の容器に設けたそれぞれチューブを接続するポートに、連通ピース（折れ棒）を設けたポリ塩化ビニル製チューブを接続して、図２に示す細胞培養用容器を作製した。 Separately, after forming a tube-like film of linear low-density polyethylene by an inflation method, a port for connecting the tube to one of the mouth portions is disposed, and both the mouth portions are thermally welded to form the second container 1. Was made. Furthermore, medium components other than the sodium hydrogen carbonate aqueous solution were accommodated from the port.
Next, the first container and the second container are connected. A tube for cell culture shown in FIG. 2 was prepared by connecting a tube made of polyvinyl chloride provided with a communication piece (folding bar) to each port connected to the tube provided in the first container and the second container. .

本発明の細胞培養用容器によれば、炭酸塩を含有する培地における二酸化炭素の拡散を抑制し、培地のｐＨ変化を抑制するため、培地の長期保存が可能である。さらに、通気された雰囲気下で細胞を培養することが可能である。また、細胞導入口を連通ピース等で閉鎖された非通気性の容器に設けているために、培地の混合をしないと細胞を培養することができない。そのため、培地の混合を忘れたまま培養するなどの使用者の操作ミスを防止することができる。   According to the cell culture vessel of the present invention, since the diffusion of carbon dioxide in the medium containing carbonate is suppressed and the pH change of the medium is suppressed, the medium can be stored for a long time. Furthermore, it is possible to culture the cells in an aerated atmosphere. In addition, since the cell inlet is provided in a non-breathable container closed with a communication piece or the like, the cells cannot be cultured unless the medium is mixed. Therefore, it is possible to prevent a user's operation mistake such as culturing while forgetting to mix the medium.

本発明の一実施形態である。It is one Embodiment of this invention. 本発明の一実施形態である。It is one Embodiment of this invention.

符号の説明Explanation of symbols

１ 第１の容器
２ 第２の容器
３ 細胞導入口
４１ 培地成分として少なくとも炭酸塩を含有する培地成分（培地成分Ａ）
４２ 少なくとも炭酸塩を含有する培地成分以外の培地成分（培地成分Ｂ）
５ 連通手段
６ 再度開通を可能にする閉鎖手段
DESCRIPTION OF SYMBOLS 1 1st container 2 2nd container 3 Cell inlet 41 Medium component (medium component A) which contains at least carbonate as a medium component
42 Medium components other than medium components containing at least carbonate (medium component B)
5 Communicating means 6 Closing means enabling reopening

Claims (7)

少なくとも炭酸塩を含有する培地成分（培地成分Ａ）を収容し、非通気性材料からなり、細胞導入口を設けた第１の容器と、培地成分Ａ以外の培地成分（培地成分Ｂ）を収容し、通気性材料からなる第２の容器と、該第１の容器と該第２の容器とを連通する連通手段からなり、培地成分Ａと培地成分Ｂを混合することによって培地が調製される細胞培養用容器。 Containing a medium component containing at least carbonate salt (Medium components A), of a non-breathable material, housing a first container provided with a cell inlet, medium components other than the medium components A (medium components B) and, a second container made of breathable material, Ri Do from the communicating means communicating the vessel of the first container and the second, medium was prepared by mixing the medium components a and medium components B Cell culture container. 非通気性材料の二酸化炭素透過係数が１０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ以下である請求項１に記載の細胞培養用容器。 The container for cell culture according to claim 1, wherein the non-breathable material has a carbon dioxide permeability coefficient of 1000 cm 3 / m 2 · 24 hr · atm or less. 非通気性材料の水蒸気透過係数が１０００ｇ／ｍ２・２４ｈｒ・ａｔｍ以下である請求項１に記載の細胞培養用容器。 The vessel for cell culture according to claim 1, wherein the water vapor permeability coefficient of the non-breathable material is 1000 g / m 2 · 24 hr · atm or less. 通気性材料の酸素透過係数が１００〜５０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍである請求項１に記載の細胞培養用容器。 The container for cell culture according to claim 1, wherein the breathable material has an oxygen permeability coefficient of 100 to 5000 cm 3 / m 2 · 24 hr · atm. 通気性材料の二酸化炭素透過係数が１００１〜２００００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍである請求項１に記載の細胞培養用容器。 Cell culture vessel according to claim 1 carbon dioxide permeability coefficient of breathable material is 1001 ~20000cm 3 / m 2 · 24hr · atm. 炭酸塩が炭酸水素ナトリウムである請求項１に記載の細胞培養用容器。   The cell culture vessel according to claim 1, wherein the carbonate is sodium hydrogen carbonate. 少なくとも炭酸塩を含有する培地成分（培地成分Ａ）を収容し、非通気性材料からなり、細胞導入口を設けた第１の容器と、培地成分Ａ以外の培地成分（培地成分Ｂ）を収容し、通気性材料からなる第２の容器と、該第１の容器と該第２の容器とを連通する連通手段からなる細胞培養用容器を用い、以下の行程よりなる細胞培養方法；
(i) 第１の容器に収容した、培地成分Ａを、連通手段を通じて第２の容器に移動し、培地成分Ａと、培地成分Ｂとを混合する培地調製工程：
(ii) 細胞導入口から、第１の容器、連通手段を経て、第２の容器へ細胞を導入する細胞導入工程：
(iii) 細胞を培養する細胞培養工程。 Containing a medium component containing at least carbonate salt (Medium components A), of a non-breathable material, housing a first container provided with a cell inlet, medium components other than the medium components A (medium components B) A cell culture method comprising the following steps using a second container made of a breathable material and a container for cell culture comprising communication means for communicating the first container and the second container;
(i) is accommodated in the first container, the medium components A, moved to the second container through the communication unit, medium components A and, medium preparation step of mixing the media components B:
(ii) Cell introduction step of introducing cells from the cell introduction port into the second container through the first container and the communication means:
(iii) A cell culture process for culturing cells.
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JPH03172169A (en) * 1989-11-30 1991-07-25 Koojin Bio Kk Vessel for storage and tissue culture
JPH0428099U (en) * 1990-06-29 1992-03-05
JPH0471482A (en) * 1990-07-12 1992-03-06 Nissho Corp Production of liquid culture medium-containing culture bag
JP2005224145A (en) * 2004-02-12 2005-08-25 Nipro Corp Medium set for culture and method for culture preservation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03172169A (en) * 1989-11-30 1991-07-25 Koojin Bio Kk Vessel for storage and tissue culture
JPH0428099U (en) * 1990-06-29 1992-03-05
JPH0471482A (en) * 1990-07-12 1992-03-06 Nissho Corp Production of liquid culture medium-containing culture bag
JP2005224145A (en) * 2004-02-12 2005-08-25 Nipro Corp Medium set for culture and method for culture preservation

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