JP6314458B2 - 温度応答性を有する細胞培養基材およびその製造方法 - Google Patents
温度応答性を有する細胞培養基材およびその製造方法 Download PDFInfo
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Description
(1)温度応答性を有する細胞培養基材の製造方法であって、
温度応答性ポリマーを基材に配置する工程、および
基材に配置された温度応答性ポリマーに放射線を照射することにより温度応答性ポリマーを基材に固定化する工程
を含み、
温度応答性ポリマーが、(メタ)アクリルアミド系モノマー由来の構成単位と側鎖に放射線反応性官能基を有する構成単位とを含むコポリマーである、
前記方法。
(2)(メタ)アクリルアミド系モノマーがN−イソプロピルアクリルアミドである、(1)に記載の方法。
(3)放射線反応性官能基が(メタ)アクリロイル基、(メタ)アリル基およびビニル基からなる群から選択される、(1)または(2)に記載の方法。
(4)細胞培養基材上に温度応答性層を形成するための、温度応答性ポリマーを含む放射線重合性組成物であって、
温度応答性ポリマーが、(メタ)アクリルアミド系モノマー由来の構成単位と側鎖に放射線反応性官能基を有する構成単位とを含むコポリマーである、
前記組成物。
(5)基材上に温度応答性ポリマーが固定化されてなる細胞培養基材であって、
温度応答性ポリマーが、(メタ)アクリルアミド系モノマー由来の構成単位と側鎖に放射線反応性官能基を有する構成単位とを含むコポリマーである、
前記細胞培養基材。
(温度応答性ポリマーの合成)
300ml三口フラスコにN−イソプロピルアクリルアミド(NIPAAm)20.4gとヒドロキシエチルメタクリレート(HEMA)2.4ml、開始剤N,N’−アゾビスイソブチロニトリル(AIBN)0.0821gを入れ、メタノール100mlを加えて溶解させた。フラスコ内をアルゴン雰囲気下にし、60℃で24時間撹拌した。生成物を透析チューブによって3日間透析し、4日間凍結乾燥させた。得られたポリ(NIPAAm−HEMA)7g、トリエチルアミン3.5mlを三口フラスコに加え、脱水テトラヒドロフラン(脱水THF)75mlを加えて溶解させ、氷浴によって氷点下に冷却した。塩化アクリロイル1mlを20mlの脱水THFに溶解させた塩化アクリロイル溶液を三口フラスコに滴下していき、氷点下を3時間維持した後、室温で12時間撹拌した。その後エーテルによって再沈させ、NIPAAmとHEMA−acrylateの共重合比(モル比)が90:10であるポリ(NIPAAm−co−HEMA−acrylate)を得た。分子量は、Mn32,000であり、Mw167,000であった。
OPSフィルム(旭化成ケミカルズ製、製品コード:GM、厚さ:50μm)をA5サイズにカットし、基材とした。i−プロパノール(IPA)にポリ(NIPAAm−co−HEMA−acrylate)を0.8wt%になるようにそれぞれ溶解させた。溶液をミヤバー(番手2)で塗布後、室温で1時間乾燥させた。各フィルムに加速電圧200kV、照射線量120kGyの電子線を照射し、温度応答性ポリマー層を基材に固定化した。高圧水洗によって固定化されていないポリマーを除去した。
(温度応答性ポリマーの合成)
300ml三口フラスコにN−イソプロピルアクリルアミド(NIPAAm)21.5gとN−ヒドロキシメチルアクリルアミド(HMAAm)1.0g、開始剤N,N’−アゾビスイソブチロニトリル(AIBN)0.0821gを入れ、メタノール100mlを加えて溶解させた。フラスコ内をアルゴン雰囲気下にし、60℃で24時間撹拌した。生成物を透析チューブによって3日間透析し、4日間凍結乾燥させた。得られたポリ(NIPAAm−HMAAm)10g、トリエチルアミン2.2mlを三口フラスコに加え、脱水テトラヒドロフラン(脱水THF)90mlを加えて溶解させ、氷浴によって氷点下に冷却した。塩化アクリロイル7.8mlを22mlの脱水THFに溶解させた塩化アクリロイル溶液を三口フラスコに滴下していき、氷点下を3時間維持した後、室温で12時間撹拌した。その後エーテルによって再沈させ、NIPAAmとHMAAm−acrylateの共重合比(モル比)が95:5であるポリ(NIPAAm−co−HMAAm−acrylate)を得た。分子量は、Mn91,000であり、Mw264,000であった。
OPSフィルム(旭化成ケミカルズ製、製品コード:GM、厚さ:50μm)をA5サイズにカットし、基材とした。i−プロパノール(IPA)にポリ(NIPAAm−co−HMAAm−acrylate)を2.0wt%になるようにそれぞれ溶解させた。この溶液をミヤバー(番手2)で塗布後、室温で1時間乾燥させた。このフィルムに加速電圧200kV、照射線量120kGyの電子線を照射し、温度応答性ポリマー層を基材に固定化した。高圧水洗によって固定化されていないポリマーを除去した。
(温度応答性ポリマーの合成)
300ml三口フラスコにN−イソプロピルアクリルアミド(NIPAAm)22.6gと開始剤N,N’−アゾビスイソブチロニトリル(AIBN)0.0821gを入れ、メタノール100mlを加えて溶解させた。フラスコ内をアルゴン雰囲気下にし、60℃で24時間撹拌した。生成物を透析チューブによって3日間透析し、4日間凍結乾燥させ、ポリNIPAAm(PNIPAAm)を得た。
OPSフィルム(旭化成ケミカルズ製、厚さ:50μm)をA5サイズにカットし、基材とした。i−プロパノール(IPA)にPNIPAAmを2.0wt%になるように溶解させた。この溶液をミヤバー(番手2)で塗布後、室温で1時間乾燥させた。このフィルムに加速電圧200kV、照射線量120kGyの電子線を照射した。高圧水洗によって固定化されていないポリマーを除去した。
(細胞培養評価)
実施例1、実施例2および比較例で得られた細胞培養基材をNunc製細胞培養ディッシュ底面に固定化した。得られた細胞培養基材上にて、定法によりウシ血管内皮細胞を37℃で培養した(使用培地:10%FBS含有DMEM(シグマ製))。培養4日後に培養した細胞がコンフルエントの状態となったことを確認し、20℃で30分インキュベートし冷却することで、細胞シートを剥離、回収可能か判断した。
Claims (4)
- 温度応答性を有する細胞培養基材の製造方法であって、
温度応答性ポリマーを基材に配置する工程、および
基材に配置された温度応答性ポリマーに放射線を照射することにより温度応答性ポリマーを基材に固定化する工程
を含み、
温度応答性ポリマーが、(メタ)アクリルアミド系モノマー由来の構成単位と、(メタ)アクリロイル基、(メタ)アリル基およびビニル基からなる群から選択される、放射線照射により基材との間に共有結合を形成する放射線反応性官能基を側鎖に有する構成単位とを含むコポリマーである、
前記方法。 - (メタ)アクリルアミド系モノマーがN−イソプロピルアクリルアミドである、請求項1に記載の方法。
- 細胞培養基材上に温度応答性層を形成するための、温度応答性ポリマーを含む放射線重合性組成物であって、
温度応答性ポリマーが、(メタ)アクリルアミド系モノマー由来の構成単位と、(メタ)アクリロイル基、(メタ)アリル基およびビニル基からなる群から選択される、放射線照射により基材との間に共有結合を形成する放射線反応性官能基を側鎖に有する構成単位とを含むコポリマーである、
前記組成物。 - 架橋剤を含まない、請求項3に記載の放射線重合性組成物。
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