JP5657744B2 - Method for producing cell culture support - Google Patents
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- JP5657744B2 JP5657744B2 JP2013101159A JP2013101159A JP5657744B2 JP 5657744 B2 JP5657744 B2 JP 5657744B2 JP 2013101159 A JP2013101159 A JP 2013101159A JP 2013101159 A JP2013101159 A JP 2013101159A JP 5657744 B2 JP5657744 B2 JP 5657744B2
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Images
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Description
本発明は細胞培養支持体の製造方法に関する。 The present invention relates to a method for producing a cell culture support.
本発明者らは既に放射線を用いて温度応答性ポリマーを基材表面にグラフト化させる製造方法において、表面に共有結合させるモノマーを基材に均一にコートし、均質で安定的なグラフト表面を得るために、重合前のモノマーを含有する塗布用組成物にオリゴマー又はプレポリマーを混入させることが良いということを発明し、特許出願済みである（特願2007-066700号）。 In the production method in which a temperature-responsive polymer is already grafted onto a substrate surface using radiation, the present inventors uniformly coat the substrate with a monomer to be covalently bonded to the surface to obtain a homogeneous and stable graft surface. Therefore, it has been invented that an oligomer or a prepolymer is preferably mixed into a coating composition containing a monomer before polymerization, and a patent application has been filed (Japanese Patent Application No. 2007-066700).
従来、放射線を用いて温度応答性ポリマーを基材表面にグラフト化させる製造方法において作製された細胞培養支持体は、通常細胞培養に用いられるポリスチレンの細胞培養支持体より少し細胞接着が弱い傾向がある。このため、接着性の細胞においても通常の細胞培養支持体であるポリスチレン支持体と比較すると接着細胞数が少なかったり、そのために、細胞増殖が遅れたり、所望の細胞を増殖させるために、不要な細胞が増えたり、異なる細胞への分化が進んだり、細胞種によって所望の細胞シートを得るために不都合なことがあった。そのため、基材表面にゼラチンやコラーゲン、フィブロネクチン等のプレコートをして、未処理の支持体と細胞接着性と表面接着たんぱく質の構成を代えて細胞培養を行う場合があった。一部の強い接着性を持った細胞ではポリマーの下限臨界溶解温度以下でも細胞が剥がれ難く、ピペッティングやタッピングなどの物理的な刺激を加えてみたり、希釈したトリプシンやEDTAなどと温度処理の併用をしても細胞シートが得られないことがあった。 Conventionally, cell culture supports prepared in a production method in which a temperature-responsive polymer is grafted onto a substrate surface using radiation tend to have a slightly weaker cell adhesion than polystyrene cell culture supports usually used for cell culture. is there. For this reason, even in the case of adherent cells, the number of adherent cells is small compared to a polystyrene support, which is a normal cell culture support, and therefore, cell growth is delayed or unnecessary for growing desired cells. In some cases, the number of cells increases, differentiation into different cells proceeds, and it is inconvenient to obtain a desired cell sheet depending on the cell type. For this reason, there are cases where the substrate surface is precoated with gelatin, collagen, fibronectin or the like, and cell culture is carried out by changing the constitution of the untreated support, cell adhesion and surface adhesion protein. Some cells with strong adhesiveness are difficult to peel off even below the lower critical lysis temperature of the polymer, so try to apply physical stimuli such as pipetting or tapping, or use temperature treatment with diluted trypsin or EDTA. Even when used in combination, a cell sheet may not be obtained.
また、細胞シートを再生医療の組織として用いる際は、培養に用いる培地には自己血清を入れるか無血清のものを用いる。さらに、各種細胞の分化・誘導を目的としたES細胞、間様系細胞、iPS細胞、前駆体細胞等では無血清培地を用いた培養を行うことが多い。こうした無血清培地を用いた細胞培養では細胞の支持体接着性が弱まる傾向があり、前述の天然物由来のプレコート等で複雑な接着条件設定、煩雑な操作、再現性の犠牲等が問題となった。 When the cell sheet is used as a tissue for regenerative medicine, the medium used for the culture contains autoserum or is serum-free. Furthermore, ES cells, mesenchymal cells, iPS cells, precursor cells, and the like for the purpose of differentiation / induction of various cells are often cultured using a serum-free medium. In cell culture using such a serum-free medium, cell support adhesion tends to be weakened, and complicated adhesion condition setting, complicated operation, sacrifice of reproducibility, etc. are caused by the above-mentioned natural product-derived precoat. It was.
このため、細胞培養支持体の細胞接着性は支持体へのプレコートで所望の細胞を接着・増殖させる上で適当な状態が求められ、温度応答性ポリマーを基材表面にグラフト化させる製造方法においても、プレコートに置換わる接着性・剥離性の強弱を制御できる方法が望まれた。 For this reason, the cell adhesion of the cell culture support is required to be in an appropriate state for adhering / proliferating desired cells by pre-coating on the support, and in the production method in which the temperature-responsive polymer is grafted to the substrate surface. However, a method capable of controlling the strength of adhesiveness and peelability to replace the precoat is desired.
また、本発明者らは特願2007-111757号で従来プラスチックシャーレ素材のポリスチレンや一部スライドガラス等のガラスにシラン処理したものでしか、実施されなかった温度応答性のグラフトについて、各種プラスチック素材へ展開する方法を示したが、ベース基材が異なると温度応答性を示すものの、細胞接着力、細胞剥離力が変化し、短期間で細胞シートを作製し、細胞シート剥離出来るものと、温度応答性細胞接着・脱離は観察されるが、細胞シートが得にくいもの等が観察された。その中で、適材適所の素材上に最適な温度応答性ポリマーを基材表面にグラフト化させる製造方法が求められた。 In addition, in the Japanese Patent Application No. 2007-111757, the present inventors used various plastic materials for temperature-responsive grafts that were only implemented using silane-treated glass, such as polystyrene and some glass slides. Although it shows temperature responsiveness when the base substrate is different, the cell adhesion force and cell detachment force change, and a cell sheet can be produced in a short period of time. Although responsive cell adhesion / detachment was observed, it was difficult to obtain a cell sheet. Among them, a production method for grafting an optimal temperature-responsive polymer onto a base material surface on a material at an appropriate position was required.
本発明は、温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種の刺激応答性高分子が共有結合を介して表面に固定化された細胞培養支持体における、細胞接着性、細胞シートの剥離性、又は単一細胞の剥離性を基材の種類に応じて最適化する技術を提供することを目的とする。 The present invention provides a cell culture support in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on a surface via a covalent bond. It is an object of the present invention to provide a technique for optimizing cell adhesion, cell sheet peelability, or single cell peelability according to the type of substrate.
本発明者は、刺激応答性高分子が共有結合を介して表面に固定化された細胞培養支持体の製造において、前記刺激応答性高分子を形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物中の前記モノマーの配合量を基材の種類に応じて選択することにより、細胞接着性、細胞シートの剥離性又は単一細胞の剥離性を最適化することができることを見出した。すなわち本発明は以下の発明を包含する。 In the production of a cell culture support in which a stimulus-responsive polymer is immobilized on a surface through a covalent bond, the present inventor is formed by polymerizing the monomer capable of forming the stimulus-responsive polymer and the monomer. By selecting the amount of the monomer in the coating composition containing an oligomer or prepolymer and an organic solvent according to the type of substrate, cell adhesion, cell sheet release or single cell release We found that sex could be optimized. That is, the present invention includes the following inventions.
（１）温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種の刺激応答性高分子が共有結合により表面に固定化された細胞培養支持体の製造方法であって、放射線照射により重合して前記刺激応答性高分子を形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物を、放射線照射により前記刺激応答性高分子が共有結合を介して導入され得る材料を含む表面を備えた基材に塗布して、前記基材の表面上に塗膜を形成する塗布工程と、前記塗膜に放射線を照射して、基材表面上における前記刺激応答性高分子の形成反応を進行させる放射線照射工程と、前記塗膜を乾燥させる乾燥工程とを含み、前記塗布用組成物中の前記モノマーの配合量が、製造される細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つが所定の程度となるように決定された配合量である、前記方法。
（２）前記基材が、表面が接着処理されたポリエチレンテレフタレート、表面にウレタンアクリレートを被覆したポリエチレンテレフタレート、又は表面にウレタンアクリレートを被覆したポリカーボネートであり、前記塗布用組成物中の前記モノマーの配合量が３〜２０重量％である、（１）の方法。
（３）前記基材が、表面がプラズマ処理されたポリカーボネート、又は表面化プラズマ処理されたポリカーボネートとＡＢＳ樹脂のブレンド若しくはポリマーアロイであり、前記塗布用組成物中の前記モノマーの配合量が２〜８重量％である、（１）の方法。
（４）前記基材が、表面がプラズマ処理された多孔質のポリカーボネート、又は表面がプラズマ処理された多孔質のポリカーボネートとＡＢＳ樹脂のブレンド若しくはポリマーアロイであるであり、前記塗布用組成物中の前記モノマーの配合量が１〜４重量％である、（１）の方法。
（５）前記塗布用組成物中の前記モノマーの配合量と前記オリゴマー又はプレポリマーの配合量との比が、製造される細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つが所定の程度となるように決定された比である、（１）〜（４）のいずれかの方法。
（６）（１）〜（５）のいずれかの方法により製造された細胞培養支持体。
（７）温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種の刺激応答性高分子が共有結合により表面に固定化された細胞培養支持体の製造工程であって、放射線照射により重合して前記刺激応答性高分子を形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物を、放射線照射により前記刺激応答性高分子が共有結合を介して導入され得る材料を含む表面を備えた基材に塗布して、前記基材の表面上に塗膜を形成する塗布工程と、前記塗膜に放射線を照射して、基材表面上における前記刺激応答性高分子の形成反応を進行させる放射線照射工程と、前記塗膜を乾燥させる乾燥工程とを含む前記製造工程において、製造される細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つを調節する方法であって、
前記塗布用組成物中の前記モノマーの配合量を、前記基材の種類に応じて調節することを含む方法。
（８）前記塗布用組成物中の前記モノマーの配合量と前記オリゴマー又はプレポリマーの配合量との比を、前記基材の種類に応じて調節することを含む（７）の方法。
(1) A method for producing a cell culture support in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on the surface by a covalent bond A composition for coating comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is irradiated with radiation. An application step of applying the stimulus-responsive polymer to a substrate having a surface including a material capable of being introduced via a covalent bond to form a coating film on the surface of the substrate; and applying radiation to the coating film Irradiating the surface of the substrate with a radiation irradiation step for causing the formation reaction of the stimuli-responsive polymer, and a drying step for drying the coating film. The blending amount of the mer is a blending amount determined so that at least one of the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the peelability of a single cell has a predetermined degree, Method.
(2) The base material is polyethylene terephthalate whose surface is subjected to adhesion treatment, polyethylene terephthalate whose surface is coated with urethane acrylate, or polycarbonate whose surface is coated with urethane acrylate, and blending of the monomer in the coating composition The method of (1), wherein the amount is from 3 to 20% by weight.
(3) The base material is a polycarbonate whose surface is plasma-treated, or a blend or polymer alloy of polycarbonate and ABS resin which has been surface-treated with plasma treatment, and the blending amount of the monomer in the coating composition is 2-8. The method according to (1), wherein the method is by weight.
(4) The base material is a porous polycarbonate whose surface has been plasma-treated, or a blend or polymer alloy of a porous polycarbonate and ABS resin whose surface has been plasma-treated, in the coating composition. The method of (1), wherein the amount of the monomer is 1 to 4% by weight.
(5) The ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition is such that the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the single cell The method according to any one of (1) to (4), wherein the ratio is determined so that at least one of the peelability is a predetermined level.
(6) A cell culture support produced by the method according to any one of (1) to (5).
(7) A process for producing a cell culture support in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on the surface by a covalent bond A composition for coating comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is irradiated with radiation. An application step of applying the stimulus-responsive polymer to a substrate having a surface including a material capable of being introduced via a covalent bond to form a coating film on the surface of the substrate; and applying radiation to the coating film In the manufacturing process, including a radiation irradiation process for promoting the formation reaction of the stimulus-responsive polymer on the surface of the substrate and a drying process for drying the coating film, That cell adhesiveness of the cell culture support, a method of modulating at least one of the peeling of the release property and a single cell of the cell sheet,
The method including adjusting the compounding quantity of the said monomer in the said composition for application | coating according to the kind of said base material.
(8) The method according to (7), comprising adjusting the ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition according to the type of the substrate.
本発明の一態様によれば、従来の技術では刺激応答性高分子を基材表面にグラフト化させると細胞接着性が低くなり、細胞シート形成が難しかった、親水性の高い基材、濡れ性の高い基材、または空隙率の高い（比重の低い）基材においても、ポリスチレンを基材とした場合と同等の細胞シートを安定的に量産作成することが可能な細胞培養支持体を製造することができる。 According to one aspect of the present invention, when a stimulus-responsive polymer is grafted to the surface of a substrate in the prior art, the cell adhesiveness is low, and it is difficult to form a cell sheet. Cell culture supports capable of stably mass-producing and producing cell sheets equivalent to those using polystyrene as a base material even for high-base materials or substrates with high porosity (low specific gravity) be able to.
本発明の他の一態様によれば、基材への細胞接着力の弱い細胞や、細胞間接着が弱く基材上でコンフレントを作製し難い細胞種の培養に適合した基材表面を形成することができる。 According to another aspect of the present invention, a substrate surface suitable for culturing cells with weak cell adhesion to the substrate or cell types with weak intercellular adhesion and difficult to produce confluent on the substrate is formed. be able to.
本発明の更に他の一態様によれば、細胞接着力が強く、低温処理によって細胞外マトリクスと基材の接着力を弱めても細胞シート剥離し難かった細胞種の培養に適合した基材表面を形成することができる。 According to yet another aspect of the present invention, the substrate surface is suitable for culturing a cell type that has strong cell adhesion and is difficult to peel off even if the adhesion between the extracellular matrix and the substrate is weakened by low-temperature treatment. Can be formed.
（刺激応答性高分子）
本発明は、温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種の刺激応答性高分子が共有結合により表面に固定化（すなわちグラフト化）された細胞培養支持体の製造方法に関する。
刺激応答性高分子としては特に温度応答性ポリマーが好ましいがこれには限定されない。
(Stimulus responsive polymer)
The present invention relates to a cell in which at least one stimulus-responsive polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on a surface (that is, grafted) by a covalent bond. The present invention relates to a method for producing a culture support.
The stimulus-responsive polymer is particularly preferably a temperature-responsive polymer, but is not limited thereto.
本発明に好適に使用できる温度応答性ポリマーは細胞培養温度下（通常、３７℃程度）において疎水性を示し、培養した細胞シートの回収時の温度下において親水性を示すものである。なお、温度応答性ポリマーが、疎水性から親水性に変化する温度（水に対する臨界溶解温度（Ｔ））としては、特に限定されないが、培養後の細胞シートの回収の容易さの観点からは、細胞培養温度よりも低い温度であることが好ましい。このような温度応答性ポリマー成分を含むことで、細胞培養時においては、細胞の足場（細胞接着面）が充分に確保されるため細胞培養を効率よく行うことができる。その一方、培養後の細胞シートの回収時においては、疎水性部分を親水性に変化させ、培養された細胞シートを細胞培養基材から分離させることで、細胞シートの回収をより一層容易にすることができる。特に所定の臨界溶解温度未満の温度で親水性を示し、同温度以上の温度で疎水性を示す温度応答性ポリマーが好ましい。このような温度応答性ポリマーにおける臨界溶解温度を特に下限臨界溶解温度と呼ぶ。 The temperature-responsive polymer that can be suitably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at the time of recovering the cultured cell sheet. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (critical solution temperature in water (T)) is not particularly limited, but from the viewpoint of ease of recovery of the cell sheet after culture, The temperature is preferably lower than the cell culture temperature. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of collecting the cell sheet after culturing, the hydrophobic part is changed to hydrophilic, and the cultured cell sheet is separated from the cell culture substrate, thereby making it easier to collect the cell sheet. be able to. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature.
本発明に好適に使用できる温度応答性ポリマーは具体的には下限臨界溶解温度Ｔが０〜８０℃、好ましくは０〜５０℃であるポリマーが好ましい。Ｔが８０℃を越えると細胞が死滅する可能性があるので好ましくない。またＴが０℃より低いと、一般に細胞増殖速度が極度に低下するか、または細胞が死滅してしまうため好ましくない。そのような好適なポリマーとしてはアクリル系ポリマー又はメタクリル系ポリマーが挙げられる。好適なポリマーは例えば特許文献３にも記載されている。具体的に適当なポリマーとしては、例えばポリ−Ｎ−イソプロピルアクリルアミド(Ｔ＝３２℃)、ポリ−Ｎ−ｎ−プロピルアクリルアミド(Ｔ＝２１℃)、ポリ−Ｎ−ｎ−プロピルメタクリルアミド(Ｔ＝３２℃)、ポリ−Ｎ−エトキシエチルアクリルアミド(Ｔ＝約３５℃)、ポリ−Ｎ−テトラヒドロフルフリルアクリルアミド(Ｔ＝約２８℃)、ポリ−Ｎ−テトラヒドロフルフリルメタクリルアミド（Ｔ＝約３５℃）、及びポリ−Ｎ,Ｎ−ジエチルアクリルアミド(Ｔ＝３２℃)等が挙げられる。その他のポリマーとしては、例えばポリ−Ｎ−エチルアクリルアミド、ポリ−Ｎ−イソプロピルメタクリルアミド、ポリ−Ｎ−シクロプロピルアクリルアミド、ポリ−Ｎ−シクロプロピルメタクリルアミド、ポリ−Ｎ−アクリロイルピロリジン、ポリ−Ｎ−アクリロイルピペリジン、ポリメチルビニルエーテル、メチルセルロース、エチルセルロース、ヒドロキシプロピルセルロース等のアルキル置換セルロース誘導体や、ポリポリプロピレンオキサイドとポリエチレンオキサイドとのブロック共重合体等に代表されるポリアルキレンオキサイドブロック共重合体や、ポリアルキレンオキサイドブロック共重合体が挙げられる。 Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower critical solution temperature T of 0 to 80 ° C, preferably 0 to 50 ° C. If T exceeds 80 ° C., the cells may die, which is not preferable. If T is lower than 0 ° C., the cell growth rate is generally extremely reduced, or the cells are killed, which is not preferable. Such suitable polymers include acrylic or methacrylic polymers. Suitable polymers are also described, for example, in US Pat. Specific suitable polymers include, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.) ), And poly-N, N-diethylacrylamide (T = 32 ° C.). Examples of other polymers include poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N- Polyalkylene oxide block copolymers represented by alkyl-substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, and polyalkylenes An oxide block copolymer is mentioned.
これらのポリマーを形成するためのモノマーとしては、例えばモノマーの単独重合体がＴ＝０〜８０℃を有するようなモノマーであって、放射線照射によって重合し得るモノマーが挙げられる。モノマーとしては例えば、(メタ)アクリルアミド化合物、Ｎ−(若しくはＮ,Ｎ−ジ)アルキル置換(メタ)アクリルアミド誘導体、環状基を有する(メタ)アクリルアミド誘導体、及びビニルエーテル誘導体等が挙げられ、これらの１種以上を使用してよい。モノマーが一種類単独で使用された場合、基材上に形成されるポリマーはホモポリマーとなり、モノマーが複数種一緒に使用された場合、基材上に形成されるポリマーはコポリマーとなるが、どちらの形態も本発明に包含される。また、増殖細胞の種類によってＴを調節する必要がある場合や、被覆物質と細胞培養支持体との相互作用を高める必要が生じた場合や、細胞支持体の親水・疎水性のバランスを調整する必要がある場合などには、上記以外の他のモノマー類を更に加えて共重合してよい。更に本発明に使用する上記ポリマーとその他のポリマーとのグラフトまたはブロック共重合体、あるいは本発明のポリマーと他のポリマーとの混合物を用いてもよい。また、ポリマー本来の性質が損なわれない範囲で架橋することも可能である。 Examples of the monomer for forming these polymers include monomers having a monomer homopolymer having T = 0 to 80 ° C. and capable of being polymerized by irradiation. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds may be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a copolymer. These forms are also encompassed by the present invention. In addition, when it is necessary to adjust T depending on the type of proliferating cell, when it is necessary to enhance the interaction between the coating substance and the cell culture support, and the balance between the hydrophilicity and hydrophobicity of the cell support is adjusted. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above-mentioned polymer used in the present invention and another polymer, or a mixture of the polymer of the present invention and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.
ｐＨ応答性ポリマーおよびイオン応答性ポリマーは作製しようとする細胞シートに適したものを適宜選択することができる。 As the pH responsive polymer and the ion responsive polymer, those suitable for the cell sheet to be prepared can be appropriately selected.
（基材）
塗布用組成物が塗布される基材は、その表面が、放射線照射により前記刺激応答性高分子が共有結合を介して導入され得る材料を含むものである限り特に限定されない。表面のみが、前記応答性ポリマーと放射線照射により共有結合し得る材料を含むものであってもよいし、基材の全部がそのような材料を含むものであってもよい。このような基材の材料は、通常細胞培養に用いられるガラス類、プラスチック類、セラミックス、金属等が挙げられるが、細胞培養が可能な材料であれば特に限定されない。基材の表面または中間層に本発明の目的を妨げない限り任意の層を設けてもよいし、任意の処理を施してもよい。例えば、支持体表面にオゾン処理、プラズマ処理、スパッタリング等の処理技術を用いて親水化を施すことができる。
(Base material)
The base material to which the coating composition is applied is not particularly limited as long as the surface thereof includes a material into which the stimulus-responsive polymer can be introduced through covalent bonding by irradiation with radiation. Only the surface may contain a material that can be covalently bonded to the responsive polymer by irradiation, or the entire substrate may contain such a material. Examples of the material for the base material include glasses, plastics, ceramics, metals, and the like that are usually used for cell culture, but are not particularly limited as long as the material can be used for cell culture. An arbitrary layer may be provided on the surface of the substrate or the intermediate layer as long as the object of the present invention is not hindered, and an arbitrary treatment may be performed. For example, the support surface can be hydrophilized using a treatment technique such as ozone treatment, plasma treatment, or sputtering.
基材を構成する材料であって、それ自体が上記応答性ポリマーと共有結合を形成し得るものとしては、ポリスチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリウレタン、ウレタンアクリレート、ポリメチルメタクリレート等のアクリル系樹脂、ポリアミド(ナイロン)、ポリカーボネート、共役結合を持つ天然ゴム、共役結合を持つ合成ゴム、ポリシリコンを含有するシリコンゴム等が挙げられる。基材はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。 Materials that constitute the substrate and can themselves form a covalent bond with the responsive polymer include polystyrene, low-density polyethylene, medium-density polyethylene, high-density polyethylene, polyurethane, urethane acrylate, and polymethyl methacrylate. Examples thereof include acrylic resins such as polyamide (nylon), polycarbonate, natural rubber having a conjugated bond, synthetic rubber having a conjugated bond, and silicon rubber containing polysilicon. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials.
上記応答性ポリマーと共有結合するように表面処理された基材としては、表面が易接着処理されたポリエチレンテレフタレート、表面がコロナ処理またはプラズマ処理された合成樹脂、表面がウレタンアクリレート等のアクリル系樹脂により被覆された合成樹脂等が挙げられる。基材はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。合成樹脂としてはナイロン、低密度ポリエチレン、中密度ポリエチレン、ポリプロピレン又はポリエチレンテレフタレート、ポリカーボネート、ポリスチレン等が挙げられる。合成樹脂はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。 Examples of the base material surface-treated so as to be covalently bonded to the responsive polymer include polyethylene terephthalate whose surface is easily bonded, synthetic resin whose surface is corona-treated or plasma-treated, and acrylic resin such as urethane acrylate. And synthetic resins coated with the above. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials. Examples of the synthetic resin include nylon, low density polyethylene, medium density polyethylene, polypropylene or polyethylene terephthalate, polycarbonate, and polystyrene. The synthetic resin may be composed of a blend polymer or polymer alloy containing two or more of these materials.
基材の形状としては、ディッシュ形状や、フィルム形状などが挙げられる。フィルム形状基材を用いる場合、フィルム形状基材表面にグラフトポリマー層を形成した後、細胞培養に適した形状（例えばディッシュ形状）に加工することができる。加工の際は、必要に応じて他の材料からなる部材を前記基材と組み合わせて使用することもできる。ディッシュ形状基材を用いる場合、少なくとも細胞接着面となるディッシュ内底面部分がグラフトポリマー層により被覆されればよい。 Examples of the shape of the substrate include a dish shape and a film shape. When using a film-shaped base material, after forming a graft polymer layer on the film-shaped base material surface, it can be processed into a shape suitable for cell culture (for example, a dish shape). In processing, a member made of another material can be used in combination with the base material as necessary. In the case of using a dish-shaped substrate, it is sufficient that at least the inner bottom portion of the dish serving as the cell adhesion surface is covered with the graft polymer layer.
（塗布用組成物）
本発明の方法には、放射線照射により重合して前記ポリマーを形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物を用いる。この塗布用組成物はオリゴマー又はプレポリマーを含むことから、有機溶媒が少量の場合にも結晶化しにくい。このため、この塗布用組成物を基材表面に塗布し、放射線照射により重合を進行させると、基材表面の全面に亘り均一なポリマー層を形成することができる。
放射線重合成のモノマーについては上記の通りである。塗布用組成物にはモノマーが単独又は複数種含まれる。
(Coating composition)
In the method of the present invention, a coating composition comprising a monomer that can be polymerized by irradiation with radiation to form the polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is used. Since this coating composition contains an oligomer or a prepolymer, it is difficult to crystallize even when the amount of the organic solvent is small. For this reason, when this composition for application | coating is apply | coated to the base-material surface and superposition | polymerization is advanced by radiation irradiation, a uniform polymer layer can be formed over the whole surface of a base-material surface.
The radiation polysynthetic monomer is as described above. The coating composition contains one or more monomers.
塗布用組成物に含まれるオリゴマー又はプレポリマーの大きさはダイマー以上のものであれば特に限定されず、分子量約３，３００（典型的には２８分子ポリマー）より大きいものが好ましく、分子量５，７００以上のものがより好ましい。上限は特に限定されず、分子量１００万以上であってもよい。本発明ではこれらを総称して「オリゴマー又はプレポリマー」と呼ぶが、オリゴマーとプレポリマーとは特段区別されず、単に重合体と呼ぶこともできる。 The size of the oligomer or prepolymer contained in the coating composition is not particularly limited as long as it is a dimer or larger, and preferably has a molecular weight of more than about 3,300 (typically 28 molecular polymers). More than 700 are more preferable. The upper limit is not particularly limited, and may be a molecular weight of 1 million or more. In the present invention, these are collectively referred to as “oligomer or prepolymer”, but the oligomer and the prepolymer are not particularly distinguished, and may be simply referred to as a polymer.
有機溶媒としてはモノマー、オリゴマー又はプレポリマーを溶解しうるものであれば特に限定されないが、常圧下に於いて沸点１２０℃以下、特に６０〜１１０℃のものが好ましい。好ましい溶媒としては、具体的にはメタノール、エタノール、ｎ(若しくはｉ)−プロパノール、２(若しくはｎ)−ブタノール、及び水等が挙げられ、それらの１種以上使用してよい。その他の溶媒、例えば１−ペンタノール、２−エチル−１−ブタノール、２−ブトキシエタノール、及びエチレン(若しくはジエチレン)グリコール又はそのモノエチルエーテル等も１種以上使用してよい。上記溶液にはその他添加剤として、硫酸等で代表される酸類、モール塩等を配合してよい。
塗布用組成物の粘度は５×１０−３Ｐａ・ｓ〜１０Ｐａ・ｓであることが好ましい。
The organic solvent is not particularly limited as long as it can dissolve the monomer, oligomer or prepolymer, but those having a boiling point of 120 ° C. or less, particularly 60 to 110 ° C. under normal pressure are preferred. Preferable examples of the solvent include methanol, ethanol, n (or i) -propanol, 2 (or n) -butanol, and water, and one or more of them may be used. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof may be used. As other additives, acids such as sulfuric acid, Mole salt and the like may be added to the above solution.
The viscosity of the coating composition is preferably 5 × 10 −3 Pa · s to 10 Pa · s.
（塗布用組成物の配合を調整することにより、細胞培養支持体の機能を最適化する方法）
親水性の高い基材、濡れ性の高い基材、および空隙率の高い（比重の低い）基材は、刺激応答性分子が細胞表面にグラフト化により導入されたとき、細胞接着性が低く、細胞シート及び単一細胞の剥離性が劣る傾向がある。一方、疎水性の高い基材、濡れ性の低い基材、および空隙率の低い（比重の高い）基材は、刺激応答性分子が細胞表面にグラフト化により導入されたとき、細胞接着性が高く、細胞シート及び単一細胞の剥離性が優れる傾向がある。本発明の特徴は、基材の親／疎水性、濡れ性、空隙率等の性質に応じて、塗布用組成物中のモノマーの配合量を調整することにより、得られる細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つを所定の程度にすることにある。塗布用組成物中のモノマーの配合量とオリゴマー又はプレポリマーの配合量との比を、得られる細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つを所定の程度にするように設定することが更に好ましい。
(Method of optimizing the function of the cell culture support by adjusting the composition of the coating composition)
Highly hydrophilic substrates, highly wettable substrates, and substrates with high porosity (low specific gravity) have low cell adhesion when stimuli-responsive molecules are introduced onto the cell surface by grafting, There exists a tendency for the peelability of a cell sheet and a single cell to be inferior. On the other hand, a substrate with high hydrophobicity, a substrate with low wettability, and a substrate with low porosity (high specific gravity) have cell adhesion when a stimulus-responsive molecule is introduced onto the cell surface by grafting. It tends to be high and the cell sheet and single cell peelability tend to be excellent. The feature of the present invention is that the cell of the cell culture support obtained by adjusting the blending amount of the monomer in the coating composition according to the properties of the substrate such as hydrophilicity / hydrophobicity, wettability, porosity, etc. At least one of adhesiveness, cell sheet detachability, and single cell detachability is set to a predetermined level. The ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition is determined according to at least one of cell adhesion, cell sheet detachability and single cell detachability of the obtained cell culture support. Is more preferably set to a predetermined level.
基材の親／疎水性は表面の水接触角により評価することができる。例えば基材に使用できる材料の代表的なものの水接触角は以下の通りである。 The hydrophilicity / hydrophobicity of the substrate can be evaluated by the water contact angle of the surface. For example, water contact angles of typical materials that can be used for the substrate are as follows.
また実施例に使用した、易接着処理されたポリエチレンテレフタレートの水接触角は７８°であり、プラズマ処理されたポリカーボネートの水接触角は７２°であり、プラズマ処理された多孔質ポリカーボネート（孔径０．４μｍ、孔密度１．０ ｘ １０Ｅ８／ｃｍ２）は６５°である。 Also, the water contact angle of polyethylene terephthalate subjected to easy adhesion treatment used in the examples is 78 °, the water contact angle of polycarbonate treated with plasma is 72 °, and the porous polycarbonate treated with plasma (pore size 0. 4 μm, pore density 1.0 × 10E8 / cm 2 ) is 65 °.
実施例に示す実験結果によれば、水接触角が小さい（親水性が高い）基材には、塗布用組成物中のモノマー配合量を比較的少なくすることで、細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つを最適化することができる。一方、水接触角が大きい（親水性が低い）基材には、塗布用組成物中のモノマー配合量を比較的多くすることで、細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つを最適化することができる。 According to the experimental results shown in the examples, the substrate having a small water contact angle (high hydrophilicity) has a relatively small amount of monomer in the coating composition, so that cell adhesion, cell sheet At least one of detachability and single cell detachability can be optimized. On the other hand, for substrates with a large water contact angle (low hydrophilicity), cell adhesion, cell sheet detachment and single cell detachment can be achieved by using a relatively large amount of monomer in the coating composition. At least one of the sexes can be optimized.
具体的には、基材が、表面が接着処理されたポリエチレンテレフタレート、表面にウレタンアクリレートを被覆したポリエチレンテレフタレート、表面にウレタンアクリレートを被覆したポリカーボネートである場合には、塗布用組成物は、塗布用組成物全量に対してモノマーを３〜２０重量％含有することが好ましい。このとき、製造される細胞培養支持体は、その表面に固定化されたグラフトポリマー層の乾燥時の厚さが０．０５〜１０μｍであることが好ましい。この場合は更に、塗布用組成物に配合されるモノマーの、オリゴマーもしくはプレポリマーに対する重量比は０．６〜６．７が好ましい。 Specifically, when the base material is polyethylene terephthalate whose surface is bonded, polyethylene terephthalate whose surface is coated with urethane acrylate, and polycarbonate whose surface is coated with urethane acrylate, the coating composition is for coating It is preferable to contain 3 to 20% by weight of monomer with respect to the total amount of the composition. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.6 to 6.7.
基材が、表面がプラズマ処理されたポリカーボネート、表面化プラズマ処理されたポリカーボネートとＡＢＳ樹脂のブレンドまたはポリマーアロイである場合には、塗布用組成物は、塗布用組成物全量に対してモノマーを２〜８重量％含有することが好ましい。このとき、製造される細胞培養支持体は、その表面に固定化されたグラフトポリマー層の乾燥時の厚さが０．０５〜１０μｍであることが好ましい。この場合は更に、塗布用組成物に配合されるモノマーの、オリゴマーもしくはプレポリマーに対する重量比は０．４〜２．０が好ましい。 When the substrate is a polycarbonate whose surface is plasma-treated, a blend of a surface-treated plasma-treated polycarbonate and an ABS resin, or a polymer alloy, the coating composition has a monomer content of 2 to 2 with respect to the total amount of the coating composition. It is preferable to contain 8% by weight. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.4 to 2.0.
基材が、表面がプラズマ処理された多孔質のポリカーボネート、表面がプラズマ処理された多孔質のポリカーボネートとＡＢＳ樹脂のブレンドまたはポリマーアロイである場合には、塗布用組成物は、塗布用組成物全量に対してモノマーを１〜４重量％含有することが好ましい。このとき、製造される細胞培養支持体は、その表面に固定化されたグラフトポリマー層の乾燥時の厚さが０．０５〜１０μｍであることが好ましい。この場合は更に、塗布用組成物に配合されるモノマーの、オリゴマーもしくはプレポリマーに対する重量比は０．２〜０．８が好ましい。 When the base material is a porous polycarbonate whose surface is plasma-treated, a porous polycarbonate whose surface is plasma-treated and a blend or polymer alloy of ABS resin, the coating composition is the total amount of the coating composition. The content of the monomer is preferably 1 to 4% by weight. At this time, it is preferable that the cell culture support produced has a dry thickness of 0.05 to 10 μm of the graft polymer layer immobilized on the surface thereof. In this case, the weight ratio of the monomer blended in the coating composition to the oligomer or prepolymer is preferably 0.2 to 0.8.
（塗布工程）
本発明の方法は、前記塗布用組成物を、前記基材の表面に塗布してその表面上に塗膜を形成する塗布工程を含む。
本工程で形成される塗膜の塗布量はグラフトポリマーが機能（例えば温度応答性）を発揮する必要な塗布量である５０ｍｇ／ｍ２以上あればよい。塗布量の上限は特にないが、４０ｇ／ｍ２未満が好ましく、１０ｇ／ｍ２以下がより好ましい。塗布量が４０ｇ／ｍ２以上である場合には、厚みが増して塗膜厚が安定しないこと、厚みが増して放射線の貫通・照射量が安定しないこと、並びに照射エネルギーに由来する膜内の対流によりグラフトポリマーの被覆量にムラが生じることが本実施例中で確認されている。また、グラフトされない遊離のポリマーを洗浄するための洗浄時間を短くするためには塗膜量は１０ｇ／ｍ２以下が望ましい。
(Coating process)
The method of this invention includes the application | coating process which apply | coats the said composition for application | coating to the surface of the said base material, and forms a coating film on the surface.
The coating amount of the coating film formed in this step may be 50 mg / m 2 or more which is a necessary coating amount for the graft polymer to exhibit a function (for example, temperature responsiveness). The upper limit of the coating amount is not particularly limited, but is preferably less than 40 g / m 2 and more preferably 10 g / m 2 or less. When the coating amount is 40 g / m 2 or more, the thickness is increased and the coating thickness is not stable, the thickness is increased and the radiation penetration / irradiation amount is not stable, and in the film derived from the irradiation energy It has been confirmed in this example that unevenness occurs in the coating amount of the graft polymer by convection. In order to shorten the washing time for washing the ungrafted free polymer, the coating amount is desirably 10 g / m 2 or less.
塗布用組成物の基材への小面積への塗布方法としては公知のいずれの方法でもよく、例えばスピンコーター、バーコーター等による塗布法、噴霧塗布法等が挙げられる。
大面積への塗布方法としてはブレードコーティング法、グラビアコーティング法、ロッドコーティング法、ナイフコーディング法、リバースロールコーティング法、オフセットグラビアコーティング法等が使用できる。
As a method for applying the coating composition to a substrate on a small area, any known method may be used, and examples thereof include a coating method using a spin coater, a bar coater and the like, and a spray coating method.
As a coating method for a large area, a blade coating method, a gravure coating method, a rod coating method, a knife coding method, a reverse roll coating method, an offset gravure coating method and the like can be used.
ベタ形成においては、グラビアコート法、ロールコート法、スロットコート法、キスコ−ト法、スプレーコート法、ファウンテンコーティング法等公知のコーティング法を用いて形成することが出来る。又、絵柄層のパターン形成においては、グラビア印刷法、スクリーン印刷法、オフセット印刷法等公知の印刷法を用いることが出来る。塗布用組成物の基材への塗布方法としては連続のコート法又は印刷法を使用することもできる。連続のコート法又は印刷法としては、具体的にはホットメルトコート、ホットラッカーコート、グラビアダイレクトコート、グラビアリバースコート、ダイコート、マイクログラビアコート、スライドコート、スリットリバースコート、カーテンコート、ナイフコート、エアコート、ロールコート等の塗布方法が使用できるが、これらは例示に過ぎず、当業者であれば暫時適用可能なものを使用することができる。 In the solid formation, a known coating method such as a gravure coating method, a roll coating method, a slot coating method, a kiss coating method, a spray coating method, or a fountain coating method can be used. In the pattern formation of the pattern layer, a known printing method such as a gravure printing method, a screen printing method, or an offset printing method can be used. As a method of applying the coating composition to the substrate, a continuous coating method or printing method can also be used. Specifically, the continuous coating method or printing method includes hot melt coating, hot lacquer coating, gravure direct coating, gravure reverse coating, die coating, micro gravure coating, slide coating, slit reverse coating, curtain coating, knife coating, and air coating. Application methods such as roll coating can be used, but these are merely examples, and those skilled in the art can use those that can be applied for a while.
（放射線照射工程）
本発明の方法は、前記塗膜に放射線を照射して、基材表面上における前記刺激応答性高分子の形成反応（すなわちグラフト化）を進行させる放射線照射工程を含む。ここでいう形成反応（グラフト化）は、遊離のモノマーが基材表面に結合した後に当該モノマーを基点としてポリマー鎖が伸張する現象だけでなく、放射線照射による重合によってモノマーからin situで形成された遊離のポリマーが基材表面に結合する現象などを包含する。
(Radiation irradiation process)
The method of the present invention includes a radiation irradiation step of irradiating the coating film with radiation to advance a formation reaction (that is, grafting) of the stimulus-responsive polymer on the surface of the substrate. The formation reaction (grafting) referred to here was not only a phenomenon in which the polymer chain stretches after the free monomer was bonded to the substrate surface, but also was formed in situ from the monomer by polymerization by radiation irradiation. It includes a phenomenon in which a free polymer binds to the substrate surface.
使用する放射線としては、α線、β線、γ線、電子線、紫外線等がある。所望のグラフトポリマーを作製するための合成にはγ線と電子線がエネルギー効率が良く、特に生産性の面からも電子線が好ましい。紫外線に関しては適当な重合開始剤や基材とのアンカー剤を組合せることで使用できる。
放射線の線量の範囲は、電子線であれば５Ｍｒａｄ〜５０Ｍｒａｄが好ましく、γ線であれば０．５Ｍｒａｄ〜５Ｍｒａｄが好ましい。
Examples of the radiation used include α rays, β rays, γ rays, electron beams, ultraviolet rays, and the like. In the synthesis for producing a desired graft polymer, γ rays and electron beams are energy efficient, and electron beams are particularly preferable from the viewpoint of productivity. With respect to ultraviolet rays, it can be used by combining an appropriate polymerization initiator and an anchor agent with a substrate.
The range of radiation dose is preferably 5 Mrad to 50 Mrad for electron beams, and preferably 0.5 Mrad to 5 Mrad for γ rays.
（乾燥工程）
本発明の方法は、前記塗膜を乾燥させて塗布用組成物に由来する有機溶媒を除去する乾燥工程を含む。
前記塗布工程で形成される塗膜は残留溶剤量の影響により結晶が形成されることがないため、乾燥前の塗膜に放射線を照射した後、乾燥を行ってもよいし、塗膜を乾燥した後に放射線を照射してもよい。ただし、乾燥前のウェットな状態の塗膜に放射線照射を行うと、環境変化や異物、塗膜厚変動等の影響を受ける可能性があることから、塗膜を乾燥した後に放射線を照射することが好ましい。
乾燥方法としては特に限定されないが、典型的にはドライエア乾燥法、熱風（温風）乾燥法、（遠）赤外乾燥法などが挙げられる。
(Drying process)
The method of the present invention includes a drying step of drying the coating film to remove an organic solvent derived from the coating composition.
Since the coating film formed in the coating process does not form crystals due to the effect of the residual solvent amount, the coating film before drying may be irradiated with radiation and then dried. After that, radiation may be applied. However, if radiation is applied to a wet coating before drying, it may be affected by environmental changes, foreign matter, coating thickness fluctuations, etc., so radiation should be applied after the coating is dried. Is preferred.
Although it does not specifically limit as a drying method, Typically, a dry air drying method, a hot air (warm air) drying method, a (far) infrared drying method etc. are mentioned.
（洗浄工程）
上述の各工程を経て形成された細胞培養支持体のポリマー層には、基材表面上に共有結合により固定化されたポリマー分子だけでなく、固定化されていない遊離のポリマー分子や、モノマー又はオリゴマー分子等が存在している。そこでこれらの遊離ポリマー或いはモノマー又はオリゴマー分子を除去するために洗浄を行う洗浄工程を更に含むことが好ましい。
洗浄方法としては特に限定されないが、典型的には浸漬洗浄、遥動洗浄、シャワー洗浄、スプレー洗浄、超音波洗浄等が挙げられる。また洗浄液としては典型的には各種水系、アルコール系、炭化水素系、塩素系、酸・アルカリ洗浄液が挙げられる。洗浄方法と洗浄液の組み合わせは洗浄される細胞培養支持体に応じて適宜選択すればよい。
(Washing process)
The polymer layer of the cell culture support formed through the above-described steps includes not only polymer molecules immobilized by covalent bonding on the substrate surface but also free polymer molecules, monomers or Oligomer molecules exist. Therefore, it is preferable to further include a washing step for washing in order to remove these free polymers or monomer or oligomer molecules.
Although it does not specifically limit as a washing | cleaning method, Typically, immersion washing | cleaning, swing washing | cleaning, shower washing | cleaning, spray washing | cleaning, ultrasonic washing | cleaning, etc. are mentioned. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, acid / alkali cleaning liquids. The combination of the washing method and the washing solution may be appropriately selected according to the cell culture support to be washed.
（本発明の方法で製造された細胞培養支持体）
本発明はまた、本発明の方法により製造された細胞培養支持体に関する。本発明の細胞培養支持体は、細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つが所望の範囲に最適化されている。また発明の方法により製造された細胞培養支持体は、原料モノマー／溶媒混合物を塗布用組成物として用いる従来法により製造された細胞培養支持体と比較してより均一なグラフトポリマー層を有することを特徴とする。
(Cell culture support produced by the method of the present invention)
The present invention also relates to a cell culture support produced by the method of the present invention. In the cell culture support of the present invention, at least one of cell adhesion, cell sheet detachability, and single cell detachability is optimized in a desired range. In addition, the cell culture support produced by the method of the invention has a more uniform graft polymer layer compared to the cell culture support produced by the conventional method using a raw material monomer / solvent mixture as a coating composition. Features.
（細胞培養シートの作成方法）
本発明の細胞培養支持体を用いて、種々の細胞、例えば生体内の各組織、臓器を構成する上皮細胞や内皮細胞、収縮性を示す骨格筋細胞、平滑筋細胞、心筋細胞、神経系を構成するニューロン、グリア細胞、繊維芽細胞、生体の代謝に関係する肝実質細胞、非肝実質細胞や脂肪細胞、分化能を有する細胞として、種々組織に存在する幹細胞、さらには骨髄細胞、ＥＳ細胞等から細胞シートを作製することができる。こうして作製された細胞シートは表面の接着因子が損なわれていないことに加えて、細胞培養面に接した部分が均一な品質を有することから、再生医療などへの利用に適したものである。また、細胞シートを利用することでバイオセンサー等の検出デバイスへの応用へも展開できる。
(Method for creating cell culture sheet)
Using the cell culture support of the present invention, various cells, such as epithelial cells and endothelial cells constituting each tissue and organ in the living body, skeletal muscle cells exhibiting contractility, smooth muscle cells, cardiomyocytes, nervous system Constituent neurons, glial cells, fibroblasts, liver parenchymal cells related to metabolism in the living body, non-hepatic parenchymal cells and fat cells, stem cells existing in various tissues as cells having differentiation potential, bone marrow cells, ES cells A cell sheet can be produced from the above. The cell sheet thus prepared is suitable for use in regenerative medicine and the like because the adhesion factor on the surface is not impaired and the portion in contact with the cell culture surface has a uniform quality. In addition, the cell sheet can be applied to detection devices such as biosensors.
細胞培養支持体の作製
（試験1）
実施例１〜3として、イソプロピルアクリルアミドモノマー（興人社製）とポリイソプロピルアクリルアミド（アルドリッチ社製、565311）を、図1a, bに示す重量％となるようにイソプロピルアルコールに溶解して塗工液（塗布用組成物）とした。図1a, bにおいて重量％とは塗工液全量に対する重量％である。以下実施例では塗工液S、A、B、C、D、E、FおよびGと呼ぶ。
Production of cell culture support (Test 1)
As Examples 1 to 3, an isopropyl acrylamide monomer (manufactured by Kojin Co., Ltd.) and polyisopropyl acrylamide (manufactured by Aldrich, 565311) were dissolved in isopropyl alcohol so as to have the weight% shown in FIGS. (Coating composition). In FIGS. 1a and b,% by weight is% by weight relative to the total amount of the coating solution. In the following examples, they are referred to as coating solutions S, A, B, C, D, E, F, and G.
この塗工液を以下の3種類の基材に塗布した。
基材1: 易接着ポリエチレンテレフタレート(PET)、水接触角:７８
基材2:ポリカーボネート(PC)シート（200μm厚、帝人社製）、水接触角:７２(プラズマ処理後)
基材3:サイクロポアメンブレンシート（20μｍ厚、ワットマン社製。材質ポリカーボネート、孔径0.4μｍ、孔密度1.0 x 10E8／cm2）、接触角:６５(プラズマ処理後)
This coating solution was applied to the following three types of substrates.
Base material 1: Easy adhesion polyethylene terephthalate (PET), water contact angle: 78
Base material 2: Polycarbonate (PC) sheet (200μm thickness, manufactured by Teijin Limited), water contact angle: 72 (after plasma treatment)
Base material 3: Cyclopore membrane sheet (20 μm thick, manufactured by Whatman Corp., material polycarbonate, hole diameter 0.4 μm, hole density 1.0 x 10E8 / cm 2 ), contact angle: 65 (after plasma treatment)
塗工液を機材1〜3にワイヤーバー４番手で約5ｇ／ｍ２の塗工厚で塗工し、ドライヤー乾燥で乾燥面質を確認した。この塗工基材の塗工面に低電圧電子線照射装置（岩崎電気社製 EC 250/15/180Ｌ）で電子線を線量24Mrad照射した。
各条件で作製した塗工フィルムを洗浄・乾燥し、ディッシュサイズに切り抜いたものを粘着剤でディッシュに固定してEOG滅菌して細胞培養支持体を得た。
実施例1〜3は基材1の易接着PET上に塗工液Ｓ（モノマー４０％、ポリマー２％）、Ｂ、Ｅの３種類を用いて、細胞培養支持体を作製した。比較例１として市販のポリスチレン細胞培養ディッュ（Tissue Cell Poly Styrene (TCPS) ベクトンデッキントン社製）を用いた。
The coating solution was applied to the equipment 1 to 3 with a 4th wire bar with a coating thickness of about 5 g / m 2 , and the dry surface quality was confirmed by drying with a dryer. The coated surface of the coated substrate was irradiated with an electron beam at a dose of 24 Mrad with a low voltage electron beam irradiation device (EC 250/15/180 L manufactured by Iwasaki Electric Co., Ltd.).
The coated film prepared under each condition was washed and dried, and the one cut into a dish size was fixed to the dish with an adhesive and EOG sterilized to obtain a cell culture support.
In Examples 1 to 3, a cell culture support was prepared using three kinds of coating liquid S (
評価方法は以下の３種類で行った。評価に用いた細胞は牛頚動脈正常血管内皮細胞（ヒューマンサイエンス研究資源バンク JCRB0099 通称HH）。培養液はイーグルダルベッコ改変培地に10%の牛血清を加えたものを用いた。
評価1: 細胞接着力の評価
35ｍｍ細胞培養ディツシユにトリプシン処理によって継代・剥離した6×10４個のHH細胞懸濁液を実施例1〜3、比較例1の支持体（ディッシュ）に播種し、播種６時間後に培地交換とともに上清培養液を別の細胞培養ディッシュで18時間培養し、非接着細胞を観察した。
評価2: シングルセルの剥離性の評価
35ｍｍ細胞培養ディツシユにトリプシン処理によって継代・剥離した10４個のHH細胞懸濁液を実施例1〜3、比較例1の支持体（ディッシュ）に播種し、２４時間後に低温インキュベータ（20℃、５％ＣＯ２）へ移動した、移動前（0分）、移動後10分、30分、60分、120分の接着細胞の剥離を観察した。
評価3: 細胞シートの剥離性の評価
35ｍｍ細胞培養ディツシユにトリプシン処理によって継代・剥離した6×10４個のHH細胞懸濁液を実施例1〜3、比較例1の支持体（ディッシュ）に播種し、培養6日後のコンフレントな増殖・単層HH細胞を低温インキュベータ（20℃、５％ＣＯ２）へ移動し、20分後にディッシュ底、約3〜5ｍｍ内側に単層細胞へ切り込みを入れ、円形の細胞シートが剥離しきるのを観察・時間計測した。測定は2回行い、それぞれについて剥離に要する時間を計測した。
The evaluation method was performed by the following three types. The cells used for the evaluation were bovine carotid artery normal vascular endothelial cells (Human Science Research Resource Bank JCRB0099 commonly known as HH). The culture solution used was Eagle Dulbecco's modified medium supplemented with 10% bovine serum.
Evaluation 1: Evaluation of cell adhesion
6 × 10 4 HH cell suspensions passaged and detached by trypsin treatment in 35 mm cell culture dish were seeded on the support (dish) of Examples 1 to 3 and Comparative Example 1, and the medium was changed 6 hours after seeding. At the same time, the supernatant culture was cultured in another cell culture dish for 18 hours, and non-adherent cells were observed.
Evaluation 2: Evaluation of peelability of single cell
35mm cell culture Ditsushiyu implement 10 4 HH cell suspension were passaged, detached by trypsinization to Examples 1-3, were seeded in support of Comparative Example 1 (dish), low temperature incubator (20 ° C. After 24
Evaluation 3: Evaluation of cell sheet peelability
6 × 10 4 HH cell suspensions passaged and detached by trypsin treatment on 35 mm cell culture dish were seeded on the support (dish) of Examples 1 to 3 and Comparative Example 1, and the cells were cultured 6 days later. Proliferation / monolayer HH cells are moved to a low temperature incubator (20 ° C, 5% CO 2 ), and after 20 minutes, the bottom of the dish is cut into the monolayer cells about 3-5mm, and the round cell sheet is completely detached. Was observed and timed. The measurement was performed twice, and the time required for peeling was measured for each.
評価結果を表2に示す。易接着ＰＥＴを基材にした温度応答性細胞培養支持体へのHH細胞の6時間の接着は、比較例細胞培養ディッシュに比べると若干の細胞接着残り（全て5％以下）を観察したが、目的の細胞を接着、増殖、継代、細胞シート剥離させる上に問題となるほどの差ではなかった。細胞接着残りを塗工液の組成の違う支持体間で比較するとモノマー比が高いもので若干多い傾向が観察された。 The evaluation results are shown in Table 2. The 6-hour adhesion of HH cells to a temperature-responsive cell culture support based on easy-adhesion PET was observed with a slight cell adhesion residue (all less than 5%) compared to the comparative cell culture dish. The difference was not so great as to cause problems in adhesion, growth, passage, and cell sheet detachment of the target cells. When the cell adhesion residue was compared between supports having different compositions of the coating solution, a tendency toward a slight increase was observed with a high monomer ratio.
評価2の支持体面積あたり少ない細胞数を播種して、細胞間接着の余り無い状態から低温化処理で細胞剥離を行うと、温度応答性ポリマーを基材表面にグラフト化されていない比較例1の細胞培養ディッシュ(TCPS)では2時間後も細胞は接着したままであった。しかしグラフト化された易接着ＰＥＴでは経時的に細胞が支持体から剥離し、培養液中に浮遊して行く状態が観察された。1.5ｍｍ角視野を各観察経過時間で塗工液の組成の違う支持体間で比較するとモノマー比が低いもので視野から完全に細胞接着がなくなる時間が早いことが観察された。 Comparative Example 1 in which a temperature-responsive polymer is not grafted on the substrate surface when seeding a small number of cells per support area of evaluation 2 and performing cell detachment by a low temperature treatment from a state where there is not much cell-cell adhesion In the cell culture dish (TCPS), the cells remained adhered after 2 hours. However, in the grafted easy-adhesion PET, it was observed that the cells detached from the support over time and floated in the culture solution. When the 1.5 mm square field was compared between the supports having different composition of the coating solution at each observation elapsed time, it was observed that the cell ratio disappeared completely from the field of view with a low monomer ratio.
HH細胞を支持体上で播種後6日間接着増殖させてコンフレントな状態を得た。この支持体（ディッシュ）を評価3の方法で低温化・細胞シート剥離する状況を観察した。温度応答性ポリマーを基材表面にグラフト化されていない比較細胞培養ディッシュでは1日経過後も切り込み箇所以外に全く細胞シート剥離がみられなかった。易接着PET基材に温度応答性ポリマーをグラフト化した支持体では全てで細胞シートが得られた。塗工液Bで作製した支持体が塗工液S、Eを用いて作製した支持体よりも剥離時間が短いことが示された。このように、易接着PET基材を細胞培養支持体に加工する上での塗工液のモノマー・ポリマー比に最適条件が存在した。 HH cells were allowed to adhere and proliferate for 6 days after seeding on the support to obtain a confluent state. This support (dish) was observed to be cooled and peeled off by the method of Evaluation 3. In the comparative cell culture dish in which the temperature-responsive polymer was not grafted on the surface of the base material, the cell sheet was not peeled at all other than the cut portion even after 1 day. Cell sheets were obtained in all of the supports in which the temperature-responsive polymer was grafted to the easy-adhesion PET substrate. It was shown that the support prepared with the coating liquid B has a shorter peeling time than the support prepared with the coating liquids S and E. Thus, there existed optimum conditions for the monomer / polymer ratio of the coating solution for processing an easily-adhesive PET substrate into a cell culture support.
実施例4〜8は200μｍ厚ポリカーボネートシート（基材2）に塗工液A〜Eを実施例1〜3同様に塗工・乾燥・電子線照射・洗浄・乾燥・ディッシュへ貼付することで細胞培養支持体を得た。この支持体に評価方法1〜3のHH細胞播種による細胞接着・剥離の試験をおこなった。結果を表3に示す。 In Examples 4 to 8, cells were applied to 200 μm-thick polycarbonate sheet (base material 2) by applying coating solutions A to E to coating, drying, electron beam irradiation, washing, drying, and dish as in Examples 1-3. A culture support was obtained. This support was tested for cell adhesion and detachment by HH cell seeding in Evaluation Methods 1 to 3. The results are shown in Table 3.
加工前の基材2にプラズマ処理を施した。プラズマ処理は減圧真空下約60〜200ｍｍTorrで酸素充填し、400 Wで3分間プラズマ放電して行った。
評価1および3の方法でHH細胞を播種したところ、支持体表面への6時間後接着残りの細胞数は塗工液のモノマー比率の高いものほど多く、塗工液ＡとＢを用いた支持体では6日間で十分な接着・増殖が得られず、コンフレントな状態にならなかった。Ｃ〜Ｅは十分な増殖でコンフレント状態を得た。評価3の低温化処理ではＣ〜Ｅ全てで短時間で細胞シートを形成した。ただし、Ｃで細胞間接着が弱く、シート剥離時に破損するものが観察された。
評価２のシングルセル剥離では全ての支持体で60分以内に視野内の細胞が剥離し、培養液中に浮遊した。
The substrate 2 before processing was subjected to plasma treatment. The plasma treatment was performed by filling with oxygen at about 60 to 200 mm Torr under reduced pressure and plasma discharge at 400 W for 3 minutes.
When HH cells were seeded by the methods of Evaluations 1 and 3, the number of cells remaining after 6 hours of support on the support surface was higher as the monomer ratio of the coating solution was higher, and support using coating solutions A and B was used. The body did not get enough adhesion and proliferation in 6 days and did not become confluent. CE obtained a confluent state with sufficient growth. In the low temperature treatment of evaluation 3, cell sheets were formed in a short time with all of C to E. However, it was observed that the adhesion between cells was weak at C and was damaged when the sheet was peeled off.
In the single cell exfoliation of Evaluation 2, the cells in the visual field were exfoliated within 60 minutes on all the supports and floated in the culture solution.
実施例9〜13は20μｍ厚サイクロポアメンブレンシート（基材3）に塗工液C〜Gを実施例1〜3同様に塗工・乾燥・電子線照射・洗浄・乾燥・ディッシュへ貼付することで細胞培養支持体を得た。この支持体に評価方法1〜3のHH細胞播種による細胞接着・剥離の試験をおこなった。結果を表4に示す。 In Examples 9 to 13, coating liquids C to G are applied to a coating, drying, electron beam irradiation, washing, drying, and dish in the same manner as in Examples 1 to 3 on a 20 μm-thick cyclopore membrane sheet (base material 3). A cell culture support was obtained. This support was tested for cell adhesion and detachment by HH cell seeding in Evaluation Methods 1 to 3. The results are shown in Table 4.
加工前の基材3にプラズマ処理を施した。プラズマ処理は減圧真空下約60〜200ｍｍTorrで酸素充填し、400 Wで3分間プラズマ放電して行った。
評価1および3の方法でHH細胞を播種したところ、支持体表面への6時間後接着残りの細胞数は塗工液のモノマー比率の高いものほど多く、塗工液ＣとＤを用いた支持体では6日間で十分な接着・増殖が得られず、コンフレントな状態にならなかった。Ｅ〜Ｇは十分な増殖でコンフレント状態を得た。評価3の低温化処理ではＣ〜Ｅ全てで短時間で細胞シートを形成した。ただし、Ｃで細胞間接着が弱く、シート剥離時に破損するものが観察された。
評価２のシングルセル剥離では全ての支持体で60分以内に視野内の細胞が剥離し、培養液中に浮遊した。
Plasma treatment was performed on the base material 3 before processing. The plasma treatment was performed by filling with oxygen at about 60 to 200 mm Torr under reduced pressure and plasma discharge at 400 W for 3 minutes.
When HH cells were seeded by the methods of Evaluations 1 and 3, the number of cells remaining after 6 hours of adhesion to the support surface was higher as the monomer ratio of the coating solution was higher, and support using coating solutions C and D was used. The body did not get enough adhesion and proliferation in 6 days and did not become confluent. EG obtained a confluent state with sufficient growth. In the low temperature treatment of evaluation 3, cell sheets were formed in a short time with all of C to E. However, it was observed that the adhesion between cells was weak at C and was damaged when the sheet was peeled off.
In the single cell exfoliation of Evaluation 2, the cells in the visual field were exfoliated within 60 minutes on all the supports and floated in the culture solution.
Claims (13)
放射線照射により重合して前記刺激応答性高分子を形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物を、放射線照射により前記刺激応答性高分子が共有結合を介して導入され得る材料を含む表面を備えた基材に塗布して、前記基材の表面上に塗膜を形成する塗布工程と、
前記塗膜に放射線を照射して、基材表面上における前記刺激応答性高分子の形成反応を進行させる放射線照射工程と、
前記塗膜を乾燥させる乾燥工程とを含み、
前記塗布用組成物中の前記モノマーの配合量が、製造される細胞培養支持体の細胞接着性、細胞シートの剥離性及び単一細胞の剥離性の少なくとも一つが所定の程度となるように、基材の水接触角に基づき決定された配合量であり、
前記オリゴマー又はプレポリマーの分子量が３，３００より大きく、
前記モノマーがイソプロピルアクリルアミドモノマーであり、前記オリゴマー又はプレポリマーがポリイソプロピルアクリルアミドである、前記方法。 A method for producing a cell culture support in which a stimulus-responsive polymer that is a temperature-responsive polymer is immobilized on a surface by covalent bonding,
A coating composition comprising a monomer that can be polymerized by radiation irradiation to form the stimulus-responsive polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent, An application step in which a polymer is applied to a substrate with a surface containing a material that can be introduced via a covalent bond to form a coating on the surface of the substrate;
Radiation irradiation step of irradiating the coating film with radiation to advance the formation reaction of the stimulus-responsive polymer on the substrate surface;
A drying step of drying the coating film,
The blending amount of the monomer in the coating composition is such that at least one of the cell adhesion of the cell culture support to be produced, the peelability of the cell sheet, and the peelability of a single cell is a predetermined level. It is a blending amount determined based on the water contact angle of the substrate,
The molecular weight of the oligomer or prepolymer rather greater than 3,300,
The method, wherein the monomer is isopropylacrylamide monomer and the oligomer or prepolymer is polyisopropylacrylamide .
前記塗布用組成物中の前記モノマーの配合量を、前記基材の水接触角に応じて調節することを含み、
前記オリゴマー又はプレポリマーの分子量が３，３００より大きく、
前記モノマーがイソプロピルアクリルアミドモノマーであり、前記オリゴマー又はプレポリマーがポリイソプロピルアクリルアミドである、前記方法。 A process for producing a cell culture support in which a stimulus-responsive polymer that is a temperature-responsive polymer is immobilized on a surface by covalent bonding, and a monomer that can be polymerized by irradiation to form the stimulus-responsive polymer And a coating composition containing an oligomer or prepolymer obtained by polymerizing the monomer and an organic solvent, and a surface containing a material into which the stimulus-responsive polymer can be introduced through a covalent bond by irradiation with radiation. Applying to the base material to form a coating film on the surface of the base material, and irradiating the coating film with radiation to advance the formation reaction of the stimulus-responsive polymer on the base material surface In the manufacturing process including a radiation irradiation process and a drying process for drying the coating film, at least one of cell adhesion, cell sheet detachability, and single cell detachability of the produced cell culture support. There is provided a method of modulating,
Adjusting the blending amount of the monomer in the coating composition according to the water contact angle of the substrate;
The molecular weight of the oligomer or prepolymer rather greater than 3,300,
The method, wherein the monomer is isopropylacrylamide monomer and the oligomer or prepolymer is polyisopropylacrylamide .
The method according to claim 11 or 12 , comprising adjusting a ratio of the amount of the monomer and the amount of the oligomer or prepolymer in the coating composition according to a water contact angle of the substrate. .
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