JP2008061609A - Method for producing cell-culturing container and cell culturing container - Google Patents

Method for producing cell-culturing container and cell culturing container Download PDF

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JP2008061609A
JP2008061609A JP2006244608A JP2006244608A JP2008061609A JP 2008061609 A JP2008061609 A JP 2008061609A JP 2006244608 A JP2006244608 A JP 2006244608A JP 2006244608 A JP2006244608 A JP 2006244608A JP 2008061609 A JP2008061609 A JP 2008061609A
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Hayao Tanaka
速雄 田中
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Sumitomo Bakelite Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a cell-culturing container capable of obtaining a perfect floating state and also good three-dimensional cell aggregate lump for preventing the irritation given to the cells from the surface of the cell-culturing container, and to provide the cell-culturing container. <P>SOLUTION: This method for producing the cell-culturing container includes a process of preparing the container, a process of forming a water soluble covering layer on the surface of the container by attaching the water soluble resin on the inner surface of the container and a process of curing the covering layer to make a cured water-insoluble film, and also the above process of forming the cured film is characterized by comprising a process of introducing an aqueous solution containing the water soluble resin into the container. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、細胞培養容器の製造方法および細胞培養容器に関する。   The present invention relates to a method for producing a cell culture container and a cell culture container.

近年、細胞を完全な浮遊状態、すなわちより生体内に近い環境で培養することで非常に幅広い研究に応用できる事が明らかになってきている。様々な分野用途において完全な浮遊状態での培養が重要な細胞培養の手法となりつつある。   In recent years, it has been clarified that cells can be applied to a very wide range of research by culturing cells in a completely floating state, that is, in an environment closer to a living body. Cultivation in a completely suspended state is becoming an important cell culture technique for various fields of use.

例えば、肝細胞、腎細胞、乳腺上皮細胞等の機能性細胞を、三次元の立体構造である細胞凝集体の形態に培養する方法は、それら細胞の機能発現を維持することで、バイオリアクターの生成または薬効や毒物の評価、人工臓器の開発研究等様々な分野用途で応用されている。
また、再生医療分野の基礎研究における幹細胞の分化誘導研究においては、未分化の状態で増殖させるためには基材からの細胞に対する刺激が無い浮遊状態での培養が必要である。
更にマクロファージや樹状細胞の刺激応答を調べる際や細胞間相互作用の研究においても如何に完全な浮遊状態・培養容器からの刺激が無い状態で培養出来ているかが実験結果を左右する重要な要因になる。 For example, a method of culturing functional cells such as hepatocytes, kidney cells, mammary epithelial cells, etc. in the form of cell aggregates that are three-dimensional structures is a bioreactor by maintaining the functional expression of these cells. It is applied in various fields such as production or evaluation of medicinal properties and toxic substances, and development research on artificial organs.
In addition, in stem cell differentiation induction research in basic research in the field of regenerative medicine, in order to proliferate in an undifferentiated state, culturing in a floating state without stimulation of cells from the substrate is necessary.
In addition, when investigating the stimulation response of macrophages and dendritic cells and in the study of cell-cell interaction, it is an important factor that determines the experimental results on how the cells can be cultured in a completely floating state and without stimulation from the culture vessel. become.

例えば、三次元培養をおこなう場合、ローラーボトル等で物理的な回転力を与えて細胞凝集塊を作る方法や、使用する培養容器の表面状態をコントロールして細胞と培養容器表面との相互作用を無くす方法があり、培養容器の表面状態をコントロールする方法としてはこれまでに培養容器の表面にコラーゲンゲルをコーティングする方法や親水性のポリマーをコーティングする技術が開示されている（例えば特許文献１）。   For example, when three-dimensional culture is performed, a method of forming a cell aggregate by applying a physical rotational force with a roller bottle or the like, or controlling the surface state of the culture vessel to be used, the interaction between the cell and the surface of the culture vessel As a method for controlling the surface state of the culture vessel, a method for coating the surface of the culture vessel with a collagen gel and a technique for coating a hydrophilic polymer have been disclosed (for example, Patent Document 1). .

しかしながら、ポリヒドロキシエチルメタクリレート共重合体またはポリイソプロピルアクリルアミドやプロピオネートといった親水性化合物を培養容器表面にコーティングする方法においては、コーティングした親水性化合物が培地水溶液中に溶出し、細胞の機能・形態に影響を与える可能性があった。
更にポリヒドロキシエチルメタアクリレート共重合体等を使用した場合は親水性化合物が放射線に対する耐性を有していないため、コーティング後に放射線で滅菌することが出来ないものが多く、無菌的な生産を余儀なくされていた。 However, in the method in which a hydrophilic compound such as polyhydroxyethyl methacrylate copolymer or polyisopropylacrylamide or propionate is coated on the surface of the culture vessel, the coated hydrophilic compound elutes in the aqueous medium solution, affecting the function and morphology of the cells. Could give.
Furthermore, when polyhydroxyethyl methacrylate copolymers are used, the hydrophilic compounds are not resistant to radiation, so many of them cannot be sterilized by radiation after coating, which necessitates aseptic production. It was.

容器表面にポリエチレンオキシドやプロピルイソシアネート等の親水性材料をグラフト重合する事により、表面に親水性を付与する方法もあるが、親水性材料をグラフト重合する方法においてはグラフト鎖長を均一に制御する事が難しく、更にグラフト鎖の導入密度を上げる事が困難である事から、改質のばらつきが大きく、充分な改質効果を得る事が難しいという問題点を有していた。
また、いずれの方法も細胞の接着を完全に防止する事は難しく、培養容器からの刺激を受けていない状態での培養とは言えず、研究目的や細胞の種類が限定されるという問題点を有していた。
特開平６−１５３９０５号公報 There is also a method of imparting hydrophilicity to the surface by graft polymerizing a hydrophilic material such as polyethylene oxide or propyl isocyanate on the surface of the container, but in the method of graft polymerizing the hydrophilic material, the graft chain length is uniformly controlled. This is difficult, and it is difficult to increase the density of graft chain introduction. Therefore, there is a problem that variation in modification is large and it is difficult to obtain a sufficient modification effect.
In addition, it is difficult to completely prevent cell adhesion by either method, and it cannot be said that the cells are not cultivated in the state of being stimulated from the culture vessel, and the problem is that the research purpose and the type of cells are limited. Had.
JP-A-6-153905

本発明は、上記事情に鑑みてなされたものであり、細胞培養容器の表面から細胞に与える刺激を防止するために、完全な浮遊状態でなおかつ良好な三次元細胞凝集塊を得る事が出来る細胞培養容器の製造方法および細胞培養容器を提供することにある。   The present invention has been made in view of the above circumstances, and in order to prevent stimulation given to the cells from the surface of the cell culture container, cells that can obtain a good three-dimensional cell aggregate in a completely floating state It is in providing the manufacturing method of a culture container, and a cell culture container.

本発明による細胞培養容器の製造方法は、容器を用意する工程と、水溶性樹脂を前記容器内面に付着させ該容器の表面に水溶性被覆層を形成する工程と、前記被覆層を硬化させて水不溶性の硬化皮膜にする工程と、を含むことを特徴とする。   The method for producing a cell culture container according to the present invention includes a step of preparing a container, a step of attaching a water-soluble resin to the inner surface of the container to form a water-soluble coating layer on the surface of the container, and curing the coating layer. And a step of forming a water-insoluble cured film.

本発明の細胞培養容器の製造方法においては、水不溶性の硬化皮膜が形成されている。これにより、コーティングした親水性皮膜が培地水溶液中に溶出し、細胞の機能・形態に影響を与えることのない細胞培養容器の製造方法を提供することができる。また、細胞培養容器の表面に対する細胞の接着・伸展増殖を防止することができる細胞培養容器の製造方法が実現される。また、これらの方法で製造された細胞培養容器とすることができる。   In the method for producing a cell culture container of the present invention, a water-insoluble cured film is formed. Thereby, the manufacturing method of the cell culture container which the coated hydrophilic membrane | film | coat elutes in culture medium aqueous solution and does not affect the function and form of a cell can be provided. Moreover, the manufacturing method of the cell culture container which can prevent the adhesion | attachment and extension proliferation of the cell with respect to the surface of a cell culture container is implement | achieved. Moreover, it can be set as the cell culture container manufactured by these methods.

本発明によれば、完全な浮遊状態でなおかつ良好な三次元細胞凝集塊を得る事が出来る細胞培養容器の製造方法および細胞培養容器を提供することができる。   ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of a cell culture container and a cell culture container which can obtain a favorable three-dimensional cell aggregate in a completely floating state can be provided.

以下、本発明について詳細に説明する。   Hereinafter, the present invention will be described in detail.

細胞低接着表面及び細胞低接着容器で重要な事は、基材から細胞に対する刺激を抑える事であり、本発明においては超親水性表面による疎水性相互作用の低減、及びハイドロゲル層の最適な厚みを構築する事による物理的な刺激の低減、更に水不溶性の硬化皮膜とする事で溶出物による刺激の低減、の３つの効果によって細胞がより完全な浮遊状態で培養できる細胞低接着表面を達成している。   What is important for cell low adhesion surfaces and cell low adhesion containers is to suppress irritation to cells from the substrate. In the present invention, the hydrophobic interaction is reduced by the superhydrophilic surface, and the optimal hydrogel layer is used. A low cell adhesion surface that allows cells to be cultured in a more complete floating state by reducing the physical stimulus by constructing the thickness and reducing the stimulus by the eluate by using a water-insoluble cured film. Have achieved.

本発明の細胞培養容器の製造方法は、容器を用意する工程と、水溶性樹脂を前記容器内面に付着させ該容器の表面に水溶性被覆層を形成する工程と、前記被覆層を硬化させて水不溶性の硬化皮膜にする工程と、を含むことを特徴とするものである。
水溶性樹脂は、被覆層を形成し、さらに硬化させることにより水不溶性皮膜が形成出来る物であれば良く、硬化のために感光性の反応基、放射線反応性の反応基、感熱性の反応基を分子構造に含むことが好ましい。 The method for producing a cell culture container of the present invention comprises a step of preparing a container, a step of attaching a water-soluble resin to the inner surface of the container to form a water-soluble coating layer on the surface of the container, and curing the coating layer. And a step of forming a water-insoluble cured film.
The water-soluble resin may be any material that can form a water-insoluble film by forming a coating layer and further curing it. For curing, a photosensitive reactive group, a radiation-reactive reactive group, a heat-sensitive reactive group. Is preferably included in the molecular structure.

その中でも感光性の反応基を含むことが特に好ましい。これにより、簡易な製造設備で短時間に効率的に前記側鎖に第一の官能基を有する水溶性樹脂を架橋反応する事が出来る。
このような反応基としては、窒素原子を含む官能基、硫黄原子を含む官能基、臭素原子を含む官能基、塩素原子を含む官能基またはそれらいずれの原子も含まない官能基等が挙げられる。これらの中でも窒素原子を含む官能基が好ましい。 Among these, it is particularly preferable to contain a photosensitive reactive group. Thereby, the water-soluble resin which has a 1st functional group in the said side chain can be bridge | crosslinked efficiently in a short time with simple manufacturing equipment.
Examples of such a reactive group include a functional group containing a nitrogen atom, a functional group containing a sulfur atom, a functional group containing a bromine atom, a functional group containing a chlorine atom, or a functional group containing neither of these atoms. Among these, a functional group containing a nitrogen atom is preferable.

具体的にはアジド基を含む官能基、ジアゾ基を含む官能基、ジアジド基を含む官能基等が挙げられる。これらの中でもアジド基を含む官能基が好ましい。これにより、実用的な２３０〜５００ｎｍの波長で反応させる事が出来、更に優れた解像性により皮膜の形成性を向上することができる。   Specific examples include a functional group containing an azide group, a functional group containing a diazo group, and a functional group containing a diazide group. Among these, a functional group containing an azide group is preferable. Thereby, it is possible to react at a practical wavelength of 230 to 500 nm, and it is possible to improve the film formability with excellent resolution.

水溶性樹脂としては、例えばポリ酢酸ビニルのけん化物、ポリビニルピロリドン、ポリエチレングリコール、ポリアクリルアミド、ポリメタアクリルアミド、ポリヒドロキシエチルメタアクリレート、ポリペンタエリスリトールトリアクリレート、ポリペンタエリスリトールテトラアクリレート、ポリジエチレングリコールジアクリレート、およびそれらを構成するモノマー同士の共重合体、また２−メタクリロイルオキシエチルホスホリルコリンと他のモノマー（例えばブチルメタクリレート等）との共重合体等が挙げられる。これらの中でもポリ酢酸ビニルのけん化物、ポリビニルピロリドン、ポリエチレングリコールの中から選ばれる１種以上と前記反応基からなる構造が好ましい。これにより、細胞の接着量を低減し、細胞凝集塊形成効果を向上することができる。   Examples of water-soluble resins include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate, And a copolymer of monomers constituting them, and a copolymer of 2-methacryloyloxyethyl phosphorylcholine and another monomer (for example, butyl methacrylate). Among these, a structure comprising at least one selected from saponified products of polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol and the reactive group is preferable. Thereby, the adhesion amount of a cell can be reduced and the cell aggregate formation effect can be improved.

ここで、ポリ酢酸ビニルのけん化物とは、ポリビニルアルコールまたはビニルアルコールと他の化合物との共重合体をいう。さらには、ビニルアルコールと、親水基変性、疎水基変性、アニオン変性、カチオン変性、アミド基変性またはアセトアセチル基のような反応基変性等の変性酢酸ビニルのけん化物等も含まれる。
なお、ここで水溶性樹脂とは、２５℃の水１００ｇに対して１．０ｇ以上溶解可能なものをいう。 Here, the saponified product of polyvinyl acetate means polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. Further, vinyl alcohol and modified vinyl acetate saponified products such as hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or reactive group modification such as acetoacetyl group are included.
In addition, water-soluble resin means what can melt | dissolve 1.0g or more with respect to 100g of 25 degreeC water here.

前記水溶性樹脂の平均重合度は、特に限定されないが、１００以上、１００００以下が好ましく、特に２００以上、５，０００以下が好ましい。平均重合度が１００以上であると、細胞培養容器の表面に均一に皮膜を成形することができ、また、平均重合度が１００００以下であれば作業性に適した水溶性の粘度とすることができる。   The average degree of polymerization of the water-soluble resin is not particularly limited, but is preferably 100 or more and 10,000 or less, particularly preferably 200 or more and 5,000 or less. When the average degree of polymerization is 100 or more, a film can be uniformly formed on the surface of the cell culture vessel. When the average degree of polymerization is 10,000 or less, the water-soluble viscosity is suitable for workability. it can.

また、前記ポリ酢酸ビニルのけん化物を用いる場合、前記ポリ酢酸ビニルのけん化物のけん化度は特に限定されないが、該ポリ酢酸ビニル全体の２０ｍｏｌ％以上、１００ｍｏｌ％以下が好ましく、特に５０ｍｏｌ％以上、９５ｍｏｌ％以下が好ましい。前記ポリ酢酸ビニルのけん化度が前記範囲内であると、細胞の接着量の低減、細胞凝集塊形成効果が特に優れる。
前記水溶性樹脂は、２０℃における粘度が１ｍＰａ・ｓ以上、１０ｍＰａ・ｓ以下に、好ましくは２ｍＰａ・ｓ以上、７ｍＰａ・ｓ以下となるよう溶媒を用いて調製されたものを使用する事が好ましい。その際に使用する溶媒は水もしくは溶解度を高めるために水と有機溶媒の混合物を使用することができる。
水溶性樹脂の粘度が前記範囲内であると、細胞の接着量が少なく、細胞凝集塊形成効果が特に優れる。充分な細胞の接着低減効果により良好な細胞凝集塊形成性が得られる被覆層の厚みとしては、１００ｎｍないし５，０００ｎｍが好ましく、１５０ないし１，０００ｎｍがより好ましい。
このように、細胞培養容器の表面に予め被覆層を形成する工程により細胞の接着低減効果が得られ、良好な細胞凝集塊形成に適した厚みの被覆層を得ることができる。
前記被覆層を硬化させて水不溶性の硬化皮膜にする工程の後に、硬化皮膜を洗浄する工程を含める事で、反応が不充分で硬化していない水溶性樹脂を除去する事で、溶出物による細胞への刺激をより効果的に抑える事が出来る。 Further, when the saponified product of polyvinyl acetate is used, the saponification degree of the saponified product of polyvinyl acetate is not particularly limited, but is preferably 20 mol% or more and 100 mol% or less of the whole polyvinyl acetate, particularly 50 mol% or more, 95 mol% or less is preferable. When the degree of saponification of the polyvinyl acetate is within the above range, the reduction of cell adhesion and the effect of forming cell aggregates are particularly excellent.
It is preferable to use the water-soluble resin prepared using a solvent so that the viscosity at 20 ° C. is 1 mPa · s or more and 10 mPa · s or less, preferably 2 mPa · s or more and 7 mPa · s or less. . The solvent used in that case can be water or a mixture of water and an organic solvent in order to increase solubility.
When the viscosity of the water-soluble resin is within the above range, the cell adhesion amount is small, and the cell aggregate formation effect is particularly excellent. The thickness of the coating layer capable of obtaining good cell aggregate formation due to sufficient cell adhesion reduction effect is preferably from 100 nm to 5,000 nm, more preferably from 150 to 1,000 nm.
As described above, the effect of reducing cell adhesion is obtained by the step of previously forming the coating layer on the surface of the cell culture container, and a coating layer having a thickness suitable for forming a good cell aggregate can be obtained.
By including a step of washing the cured film after the step of curing the coating layer to form a water-insoluble cured film, by removing the water-soluble resin that is insufficiently cured and uncured, Stimulation of cells can be suppressed more effectively.

培養容器の必須条件である滅菌に関しては、エチレンオキサイドガス滅菌、感熱滅菌、蒸気滅菌、放射線滅菌等が挙げられるが、γ線あるいは電子線を用いた放射線滅菌が好ましく、大量生産をおこなう場合は放射線透過性の点でγ線滅菌が特に好ましい。
放射線の吸収線量については特に限定するものではないが、当然の事ながら吸収線量が低すぎると滅菌性は確保されず、高すぎると細胞培養容器および被覆層が劣化してしまう。本発明における放射線の吸収線量としては１ｋＧｙ以上、５０ｋＧｙ以下が好ましく、５ｋＧｙ以上、３０ｋＧｙ以下が特に好ましい。これによって本発明の培養容器の特性を充分に保持したまま滅菌性を付与する事ができる。 For sterilization, which is an essential condition for culture vessels, ethylene oxide gas sterilization, heat-sensitive sterilization, steam sterilization, radiation sterilization, etc. can be mentioned, but radiation sterilization using γ rays or electron beams is preferable, and radiation is required for mass production. Gamma sterilization is particularly preferred from the viewpoint of permeability.
The absorbed dose of radiation is not particularly limited, but naturally, if the absorbed dose is too low, sterility is not ensured, and if it is too high, the cell culture container and the coating layer are deteriorated. The absorbed dose of radiation in the present invention is preferably 1 kGy or more and 50 kGy or less, particularly preferably 5 kGy or more and 30 kGy or less. As a result, sterility can be imparted while sufficiently maintaining the characteristics of the culture container of the present invention.

次に前記被覆層を硬化する工程について詳細に説明する。   Next, the step of curing the coating layer will be described in detail.

細胞培養容器の表面に構築された水溶性樹脂の被覆層は硬化することによって、耐水性を獲得し細胞培養容器として使用しうる基本性能が付与される。さらに本発明の特徴である細胞に刺激を与える溶出物の少ない表面を獲得する。
硬化の方法としては特に限定するものではなく、水溶性樹脂の側鎖に有する官能基を反応させることで達成できる、例えばジアゾ基、アジド基、シンモナイル基のような感光基であれば光照射により硬化させることが出来、ビニル基を有する場合は放射線により硬化させることが出来る。
光照射により硬化させる場合の光源は特に限定するものではなく、照度が５．０ｍＷ／ｃｍ2程度の超高圧水銀灯または０．１ｍＷ／ｃｍ2程度のＵＶランプを使用することも出来る。光照射による硬化は照度と照射時間で制御することが出来るため、照度の低い光源を用いる場合は照射時間を長くすればよく、反応性の高い感光基を選択した場合は蛍光灯下で硬化させることも可能である。
例えば５．０ｍＷ／ｃｍ2の超高圧水銀灯を使用した場合は１ないし１０秒の照射で、０．１ｍＷ／ｃｍ2のＵＶランプを使用した場合は３ないし１０分の照射で充分に硬化させることが出来る。 The water-soluble resin coating layer constructed on the surface of the cell culture container is cured to provide water resistance and basic performance that can be used as a cell culture container. Furthermore, a surface with less eluate that stimulates the cells, which is a feature of the present invention, is obtained.
The curing method is not particularly limited, and can be achieved by reacting a functional group having a side chain of the water-soluble resin. For example, a photosensitive group such as a diazo group, an azide group, or a simmonyl group can be irradiated by light irradiation. It can be cured, and when it has a vinyl group, it can be cured by radiation.
A light source in the case of curing by light irradiation is not particularly limited, illumination can also be used 5.0 mW / cm 2 of about ultra-high pressure mercury lamp or 0.1 mW / cm 2 about UV lamps. Curing by light irradiation can be controlled by illuminance and irradiation time, so when using a light source with low illuminance, the irradiation time can be lengthened, and when a highly reactive photosensitive group is selected, it is cured under a fluorescent lamp. It is also possible.
For example, when using an extra-high pressure mercury lamp with 5.0 mW / cm 2 irradiation 1 to 10 seconds, when using a UV lamp 0.1 mW / cm 2 be sufficiently cured by irradiation of 3 to 10 minutes I can do it.

次に細胞培養容器について説明する。   Next, the cell culture container will be described.

前記細胞培養容器は、樹脂製の材料で構成されている。この樹脂材料は、前記細胞培養容器をディスポーザルタイプにすることができるのに加え、種々の形状を容易に成形できるものである。
前記樹脂材料としては、例えばポリプロピレン樹脂、ポリエチレン樹脂、エチレン-プロピレン共重合体等のポリオレフィン系樹脂、ポリスチレン、アクリロニトリル−ブタジエン−スチレン系樹脂等のポリスチレン系樹脂、ポリカーボネート樹脂、ポリエチレンテレフタレート樹脂、ポリメチルメタクリレート樹脂等のメタクリル系樹脂、塩化ビニル樹脂、ポリブチレンテレフタレート樹脂、ポリアリレート樹脂、ポリサルホン樹脂、ポリエーテルサルホン樹脂、ポリエーテルエーテルケトン樹脂、ポリエーテルイミド樹脂、ポリテトラフルオロエチレン等のフッ素系樹脂、ポリメチルペンテン樹脂、ポリアクリロニトリル等のアクリル系樹脂、プロピオネート樹脂等の繊維素系樹脂等が挙げられる。これらの中でも細胞培養容器に求められる成形性、透明性、放射線耐性の点においてポリスチレン樹脂が特に好ましい。 The cell culture container is made of a resin material. In addition to making the cell culture container into a disposable type, this resin material can easily form various shapes.
Examples of the resin material include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers, polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene resins, polycarbonate resins, polyethylene terephthalate resins, and polymethyl methacrylate. Methacrylic resins such as resins, vinyl chloride resins, polybutylene terephthalate resins, polyarylate resins, polysulfone resins, polyethersulfone resins, polyetheretherketone resins, polyetherimide resins, fluorine resins such as polytetrafluoroethylene, Examples thereof include acrylic resins such as polymethylpentene resin and polyacrylonitrile, and fiber-based resins such as propionate resin. Among these, polystyrene resins are particularly preferable in terms of moldability, transparency, and radiation resistance required for cell culture containers.

前記樹脂材料の重量平均分子量は、特に限定されないが、１０，０００〜５００，０００が好ましく、特に２０，０００〜１００，０００が好ましい。重量平均分子量が前記範囲内であると、細胞培養容器の成形性に優れる。
前記重量平均分子量は、例えばＧ．Ｐ．Ｃ．を用いたスチレン換算で求めることができる。
前記樹脂材料には成形性向上、耐候性向上を目的として、本発明の目的を損なわない範囲で炭化水素系、脂肪酸アミド系の滑剤やフェノール系、アミン系の酸化防止剤等の添加剤を添加することができる。 The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 to 500,000, particularly preferably 20,000 to 100,000. When the weight average molecular weight is within the above range, the moldability of the cell culture container is excellent.
The weight average molecular weight is, for example, G.M. P. C. It can obtain | require in conversion of styrene using.
For the purpose of improving moldability and weather resistance, additives such as hydrocarbon-based, fatty acid amide-based lubricants, phenol-based and amine-based antioxidants are added to the resin material as long as the object of the present invention is not impaired. can do.

前記樹脂材料から前記細胞培養容器を製造する場合、例えば射出成形、ブロー成形、インジェクションブロー成形により前記細胞培養容器を製造することができる。
前記細胞培養容器としては、例えばマルチウェルプレートおよびシャーレ（ディッシュ）、フラスコ等の容器類が挙げられ、更にシート状の成形品であっても、容器底面等の細胞が培養できる環境下に設置して使用する事ができる。これらの中でも、、バイオリアクターの生成または薬効や毒物の評価、人工臓器の開発研究等で用いられる６〜３８４穴のマルチウェルプレートやシャーレとして用いられる事が好ましい。これにより、細胞凝集塊を用いた評価、研究の精度を向上させることが出来る。 When the cell culture container is manufactured from the resin material, the cell culture container can be manufactured by, for example, injection molding, blow molding, or injection blow molding.
Examples of the cell culture container include containers such as a multi-well plate, a petri dish (dish), and a flask. Further, even in the case of a sheet-like molded product, the cell culture container is installed in an environment where cells such as a container bottom can be cultured. Can be used. Among these, it is preferable to use as a 6-384-well multiwell plate or petri dish used for bioreactor generation or medicinal effects, toxicological evaluation, artificial organ development research, and the like. Thereby, the precision of evaluation and research using a cell aggregate can be improved.

更に特筆すべきは、本発明の細胞培養容器は細胞接着性が低く細胞凝集塊形成性が良好であるため、ラウンドボトムやＶボトムと呼ばれる底面が半球若しくは円錐状のマルチウェルプレートを使用すると１ウェルに１個の細胞凝集塊が均一な大きさで形成される為、評価・研究に好適に用いる事が出来る。   Further, it should be noted that the cell culture container of the present invention has low cell adhesion and good cell aggregate formation, and therefore, when a multiwell plate having a bottom surface called a round bottom or V bottom and having a hemisphere or conical shape is used, 1 Since one cell aggregate is formed in a well with a uniform size, it can be suitably used for evaluation and research.

以下、本発明を実施例および比較例に基づいて詳細に説明するが、本発明はこれに限定されるものではない。   EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

（実施例１）
樹脂材料としてポリスチレン樹脂（ＰＳジャパン社製、ＨＦ７７）を用いて、射出成形によりディッシュ（シャーレ）を形成した。得られたディッシュにプラズマ処理装置 （ＢＲＡＮＳＯＮ／ＩＰＣ社製 ＳＥＲＩＥＳ７０００）を用いてプラズマ処理（酸素プラズマ５分）を行い、ディッシュの表面に官能基を形成した。
なお、得られたチューブの形状は、高さ１３ｍｍ、内径３５ｍｍφのディッシュであった。 (Example 1)
A dish (petri dish) was formed by injection molding using a polystyrene resin (manufactured by PS Japan, HF77) as a resin material. The resulting dish was subjected to plasma treatment (oxygen plasma for 5 minutes) using a plasma treatment apparatus (SERIES7000, manufactured by BRANSON / IPC) to form functional groups on the surface of the dish.
In addition, the shape of the obtained tube was a dish having a height of 13 mm and an inner diameter of 35 mmφ.

次に、水溶性樹脂として側鎖にアジド基を有するポリビニルアルコール（東洋合成工業社製 ＡＷＰ、水溶性樹脂の平均重合度１８００、第１の官能基の変性率０．６ｍｏｌ％）をアルミ箔で遮光をしたガラス容器中で、２０容量％エタノール水溶液に溶解し、０．７重量％の溶液を調整した。溶液の粘度は、２．９ｍＰａ・ｓであった。
なお、水溶液の粘度は、日本薬局方第１４改正 ４５．粘度測定法 第２法回転粘度計法に従い測定した。
上述のディッシュを前記アルミ箔で遮光をしたガラス容器に１分間、浸漬した後、取り出し、ディッシュを裏返して溶液を充分廃棄し４０℃で６０分一次乾燥した後、ＵＶランプで２５０ｎｍのＵＶ光を０．１ｍＷ／ｃｍ2×３分間照射して水溶性樹脂を硬化した後純水で３回繰り返し洗浄し、乾燥後、γ線を吸収線量１０ｋＧｙで照射（ラジエ工業株式会社）して、本発明の細胞培養容器（ディッシュ）を得た。
得られたディッシュの表面には、前記水溶性樹脂で形成される層が厚さ１８０ｎｍで形成されていた。なお、層の厚さは液体窒素中で破断したディッシュの破断面を電子顕微鏡（ＦＥＩ社製 Ｑｕａｎｔａ４００Ｆ）を用いて測定した。 Next, polyvinyl alcohol having an azide group in the side chain as a water-soluble resin (AWP manufactured by Toyo Gosei Kogyo Co., Ltd., average polymerization degree of water-soluble resin 1800, first functional group modification rate 0.6 mol%) is made of aluminum foil. In a glass container protected from light, it was dissolved in a 20 vol% ethanol aqueous solution to prepare a 0.7 wt% solution. The viscosity of the solution was 2.9 mPa · s.
The viscosity of the aqueous solution is revised by the Japanese Pharmacopoeia 14th revision 45. Viscosity measurement method It measured according to the 2nd method rotational viscometer method.
After immersing the above-mentioned dish in a glass container shielded with aluminum foil for 1 minute, take it out, turn the dish upside down, discard the solution thoroughly and dry it primarily at 40 ° C. for 60 minutes, and then apply UV light at 250 nm with a UV lamp. Irradiated with 0.1 mW / cm 2 × 3 minutes to cure the water-soluble resin, washed repeatedly with pure water three times, dried, and then irradiated with γ-rays with an absorbed dose of 10 kGy (Radie Industries, Ltd.). Cell culture container (dish) was obtained.
On the surface of the obtained dish, a layer formed of the water-soluble resin was formed with a thickness of 180 nm. The thickness of the layer was measured using an electron microscope (Quanta 400F manufactured by FEI Co., Ltd.) on the fracture surface of the dish broken in liquid nitrogen.

（実施例２）
樹脂材料としてメチルペンテン（ＴＰＸ）樹脂（三井石油化学社製、ＲＴ−３１）を用いた以外は実施例１と同様にした。
得られたディッシュの表面には、前記側鎖に第１の官能基を有する水溶性樹脂で形成される層が厚さ１７０ｎｍで形成されていた。 (Example 2)
The same procedure as in Example 1 was performed except that methylpentene (TPX) resin (RT-31, manufactured by Mitsui Petrochemical Co., Ltd.) was used as the resin material.
On the surface of the obtained dish, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 170 nm.

（比較例１）
実施例１の工程から第２の官能基を生成する工程から水溶性樹脂への浸漬、及びＵＶランプによる硬化、洗浄、乾燥までの工程を除いたディッシュを比較例１とした。 (Comparative Example 1)
The dish excluding the steps from the step of Example 1 for generating the second functional group to the step of immersing in a water-soluble resin and curing, washing, and drying with a UV lamp was used as Comparative Example 1.

（比較例２）
水溶性樹脂として側鎖に官能基を有していないポリビニルアルコール：平均重合度約１，５００、けん化度８６〜９０ｍｏｌ％（和光純薬社製、１６０−０３０５５）を用いた以外は、実施例１と同様にした。 (Comparative Example 2)
Polyvinyl alcohol having no functional group in the side chain as a water-soluble resin: Examples except that an average polymerization degree of about 1,500 and a saponification degree of 86 to 90 mol% (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) were used Same as 1.

（比較例３）
水溶性樹脂への浸漬、及びＵＶランプによる硬化、洗浄、乾燥までの工程を除き、ディッシュをポリヒドロキシエチルメタクリレート共重合体（シグマアルドリッチ社製 ｐｏｌｙ−２ｈｙｄｒｏｘｙｅｔｈｌｍｅｔｈａｃｒｙｌａｔｅ）の３重量％エタノール溶液に浸漬し、一晩乾燥させた以外は実施例１と同様にした。 (Comparative Example 3)
Except for the steps of immersion in a water-soluble resin and curing, washing and drying with a UV lamp, the dish was immersed in a 3% by weight ethanol solution of a polyhydroxyethyl methacrylate copolymer (poly-2hydroxyethyl methacrylate) manufactured by Sigma-Aldrich, Same as Example 1 except dried overnight.

得られた容器について、以下の評価を行なった。評価項目を内容と共に示す。得られた結果を表１に示す。   The following evaluation was performed about the obtained container. The evaluation items are shown together with the contents. The obtained results are shown in Table 1.

１．Ｊ７７４Ａ．１（マウスマクロファージ）細胞を用いた接着性及び形態評価
Ｊ７７４Ａ．１細胞を１×１０4個／ｍＬの濃度で２ｍＬづつ播種（培地：ＤＭＥＭ＋ＦＢＳ１０％）し、３日後の形態を観察した。
２．ＨｅｐＧ２（ヒト肝癌）細胞を用いた接着性及び細胞凝集塊形成性評価
ＨｅｐＧ２細胞を１×１０4個／ｍＬの濃度で２ｍＬづつ播種（培地：ＭＥＭ＋ＦＢＳ１０％）し、３日後の形態を観察した。 1. J774A. 1 (Mouse Macrophage) Adhesive and Morphological Evaluation Using Cells J774A. One cell was seeded at a concentration of 1 × 10 4 cells / mL in 2 mL units (medium: DMEM + FBS 10%), and the morphology after 3 days was observed.
2. Evaluation of Adhesiveness and Cell Aggregate Formation Using HepG2 (Human Liver Cancer) Cells HepG2 cells were seeded in 2 mL units at a concentration of 1 × 10 4 cells / mL (medium: MEM + FBS 10%), and the morphology after 3 days was observed.

Figure 2008061609
表１から明らかなように比較例ではマウスマクロファージ細胞は基材に接着し、一部の細胞では偽足の形成が確認された。これらの結果から比較例においては細胞がシャーレ表面からの刺激を受けている事、更にシャーレを異物認識している事が明らかである。
一方実施例においてマウスマクロファージ細胞は個々の細胞は単一の球状でありシャーレ表面への接着はなく、細胞はシャーレ表面からの刺激を受けず自然な状態で培養されている事が確認できる。
またＨｅｐＧ２細胞も比較例１では基材に接着し、凝集塊を形成しておらず、比較例２及び３ではシャーレ表面への接着はなく浮遊状態ではあるが、凝集塊が形成されていないかその大きさが不均一であることから、シャーレ表面からの刺激を受けているか、もしくは溶出物により細胞間相互作用が阻害されている事が考えられる。
実施例においては均一なサイズの凝集塊を形成している事から、細胞はシャーレ表面からの刺激もしくは溶出物からの刺激を受けず正常な細胞間相互作用が機能した状態で培養されている事が確認された。
Figure 2008061609
 As is clear from Table 1, in the comparative example, mouse macrophage cells adhered to the base material, and formation of pseudopods was confirmed in some cells. From these results, it is clear that in the comparative example, the cells are stimulated from the surface of the petri dish and the petri dish is recognized as a foreign substance.
On the other hand, in the examples, mouse macrophage cells each have a single spherical shape and do not adhere to the petri dish surface, and it can be confirmed that the cells are cultured in a natural state without being stimulated by the petri dish surface.
HepG2 cells also adhered to the substrate in Comparative Example 1 and did not form aggregates. In Comparative Examples 2 and 3, there was no adhesion to the surface of the petri dish and it was in a floating state, but aggregates were not formed. Since the size is non-uniform, it is considered that stimulation from the surface of the petri dish has been received, or cell-cell interaction is inhibited by the eluate.
In the examples, the cells are cultured in a state in which normal cell-cell interaction is functioning without being stimulated by the surface of the petri dish or stimulated by the eluate because an aggregate of uniform size is formed. Was confirmed.

本発明は表面への細胞の吸着を防ぎ、細胞培養容器の表面から細胞に与える刺激を防止し、完全な浮遊状態でなおかつ良好な三次元細胞凝集塊を得る事が出来る細胞低接着表面の構築方法および細胞低接着培養容器であり、肝細胞、腎細胞、乳腺上皮細胞等の機能性細胞を三次元の立体構造である細胞凝集体の形成による機能発現を維持、バイオリアクターの生成または薬効や毒物の評価、人工臓器の開発研究、及び、細胞に対する刺激が無い浮遊状態で幹細胞を未分化の状態で増殖させることで再生医療分野の基礎研究における幹細胞の分化誘導研究への応用、更にマクロファージや樹状細胞の刺激応答を調べる際や細胞間相互作用の研究において有効に使用することが出来る。

The present invention prevents the adsorption of cells to the surface, prevents irritation to the cells from the surface of the cell culture container, and constructs a low cell adhesion surface capable of obtaining a good three-dimensional cell aggregate in a completely floating state Method and cell low-adhesion culture container, and functional cells such as hepatocytes, kidney cells, and mammary epithelial cells maintain functional expression due to the formation of cell aggregates that are three-dimensional structures. Evaluation of toxic substances, research and development of artificial organs, and application to stem cell differentiation induction research in basic research in the field of regenerative medicine by proliferating stem cells in an undifferentiated state in a floating state where there is no stimulation to cells. It can be used effectively when investigating the stimulatory response of dendritic cells and in studying cell-cell interactions.

Claims (10)

容器を用意する工程と、
水溶性樹脂を前記容器内面に付着させ該容器の表面に水溶性被覆層を形成する工程と、
前記被覆層を硬化させて水不溶性の硬化皮膜にする工程と、
を含むことを特徴とする細胞培養容器の製造方法。 Preparing a container;
Attaching a water-soluble resin to the inner surface of the container to form a water-soluble coating layer on the surface of the container;
Curing the coating layer to form a water-insoluble cured film;
A method for producing a cell culture container, comprising: 前記硬化皮膜にする工程は、前記水溶性樹脂を含む水溶液を容器内に導入する工程を含む請求項１に記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to claim 1, wherein the step of forming the cured film includes a step of introducing an aqueous solution containing the water-soluble resin into the container. 前記水溶液の、２０℃における粘度が１ｍＰａ・ｓ以上、１０ｍＰａ・ｓ以下である請求項２に記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to claim 2, wherein the aqueous solution has a viscosity at 20 ° C of 1 mPa · s or more and 10 mPa · s or less. 前記水溶性樹脂は、側鎖に感光性の官能基を有するものである請求項１または３に記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to claim 1 or 3, wherein the water-soluble resin has a photosensitive functional group in a side chain. 前記官能基は、アジド基を有するものである請求項４に記載の細胞培養容器の製造方法   The method for producing a cell culture container according to claim 4, wherein the functional group has an azide group. 前記硬化皮膜にする工程は、光照射により行うものである請求項１ないし５のいずれかに記載の細胞培養容器の製造方法   The method for producing a cell culture container according to any one of claims 1 to 5, wherein the step of forming the cured film is performed by light irradiation. 前記硬化皮膜を、純水によって洗浄する工程をさらに含む請求項１ないし６のいずれかに記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to claim 1, further comprising a step of washing the cured film with pure water. 放射線を用いて滅菌する工程をさらに含む請求項１ないし７のいずれかに記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to any one of claims 1 to 7, further comprising a step of sterilization using radiation. 前記硬化皮膜の厚みは、１００ｎｍ以上、５０００ｎｍ以下である請求項１ないし８のいずれかに記載の細胞培養容器の製造方法。   The method for producing a cell culture container according to any one of claims 1 to 8, wherein the thickness of the cured film is 100 nm or more and 5000 nm or less. 請求項１ないし９のいずれかに記載の方法で製造されたことを特徴とする細胞培養容器。




A cell culture container produced by the method according to claim 1.




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