JPWO2020045488A1 - 多孔質三次元細胞培養用足場材料及びその製造方法 - Google Patents
多孔質三次元細胞培養用足場材料及びその製造方法 Download PDFInfo
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Abstract
Description
項1. 架橋された疎水化多糖ナノゲル粒子から構成される多孔質三次元細胞培養用足場材料。
項2. 架橋された疎水化多糖ナノゲル粒子が、フィブロネクチンでコーティングされたものである、項1に記載の多孔質三次元細胞培養用足場材料。
項3. 架橋性基を有する疎水化多糖ナノゲルが、多糖部分、疎水性部分及び重合性部分を含む、項2に記載の多孔質三次元細胞培養用足場材料。
項4. 多糖部分が、プルラン、アミロペクチン、アミロース、デキストラン、ヒドロキシエチルデキストラン、マンナン、レバン、イヌリン、キチン、キトサン、キシログルカンまたは水溶性セルロースである、項3に記載の多孔質三次元細胞培養用足場材料。
項5. 疎水性部分が炭素数8〜50の炭化水素基またはステリル基を含む、項3に記載の多孔質三次元細胞培養用足場材料。
項6. 疎水性部分がコレステリル基を含む、項5に記載の多孔質三次元細胞培養用足場材料。
項7. 重合性部分がアクリロイル、メタアクリロイル、ビニルまたはアリルを含む、項3に記載の多孔質三次元細胞培養用足場材料。
項8. 疎水化多糖ナノゲル粒子の架橋に用いられる架橋剤がメルカプトエチルポリエチレングリコール誘導体である、項2に記載の多孔質三次元細胞培養用足場材料。
項9. 多孔質三次元細胞培養用足場材料の断面における平均細孔径が5〜250μmである連続した細孔を含む、項1〜8のいずれか1項に記載の多孔質三次元細胞培養用足場材料。
項10. 架橋した疎水化多糖ナノゲル粒子を凍結融解し、その後に凍結乾燥することを特徴とする、項1〜9のいずれかに記載の多孔質三次元細胞培養用足場材料の製造方法。
*NanoClik gel: Nanogel-crosslinked gel (凍結融解前の架橋ナノゲルであって、この時点では多孔質ではない)
*NanoCliP gel: Nanogel-crosslinked Porous gel (凍結融解により多孔質になった架橋ナノゲル)
*NanoCliP-FD matrix: Nanogel-crosslinked Porous freeze-dried matrix (凍結融解して得られたNanoCliP gelをさらに凍結乾燥したもの。乾燥しているので長期保存に適している。)
*NanoCliP-FD gel: Nanogel-crosslinked Porous freeze-dried gel (NanoCliP-FD matrixに溶液または細胞懸濁液等を加えてhydrationした架橋ナノゲル。NanoCliP-FD gelはNanoCliP gelよりも、凍結乾燥を行ったことで細孔数と細孔径が増大している)
*CHP:コレステリルプルラン
*CHPOA:アクリロイル基(エステル、OA)で修飾されたコレステリルプルラン
*CHPOA-Rh:アクリロイル基で修飾され、ローダミン(Rh)で標識されたコレステリルプルラン
図1のように、CHPOA を自己組織化させることでCHPOA nanogelを調製することができ、CHPOA naogelをPEGSHで架橋してNanoClik gelを調製することができる。NanoClik gelは多孔質ではない。NanoClik gelを凍結融解し、多孔質のNanoCliP gelを調製することができる。さらにNanoCliP gelを凍結乾燥してNanoCliP-FD matrixを調製することができ、NanoCliP-FD matrixを溶液または細胞懸濁液等でhydrationすることによってNanoCliP-FD gelを調製することができる。
Fibronectin-coated NanoCliP-FD gelの調製法。
図1のように、1×1×10 mmの大きさのNanoCliP gelをFreeze-dryし、NanoCliP-FD matrix を調製した。これを50μg/mLのhuman Fibronectin solution中に 6hr 浸漬した後、70%エタノールにて2回洗浄し、真空乾燥した。これがFibronectin-coated NanoCliP-FD matrixである。これをhydrationして、Fibronectin-coated NanoCliP-FD gelとした。
図1のように、1×1×10 mmの大きさのNanoCliP gelを調製した。純水にて洗浄後、 50μg/mLのhuman Fibronectin solution で 6hr 浸漬した。その後70%エタノールにて2回洗浄し、Fibronectin-coated NanoCliP gelとし、PBS中で保存した。
KUSA-A1 : Japanese Collection of Research Bio-resources Cell Bank (JCRB, Osaka, Japan).
Cell Count Reagent SF (Nacalai) Lot:V9F0261
Hoechst 33342 (Dojindo) Lot:KR057
Alexa FluorTM 488 phalloidin (Life Technologies Corporation, Eugene, Oregon ) Lot : 1834338
Alizarin Red S (Sigma Aldrich)
約16時間培養後、各スキャフォールドを新しい24ウェルプレートに移し替え、PBSにてWash後、基礎培地を添加し、Cell Count Reagent SF (Nacalai) Lot:V9F0261を用いて 細胞のviabilityを検討した。各ウェルに培地の10%になるように上記試薬を添加し、2時間呈色反応を行い、吸光度を比較した。結果を図3に示す。
約16時間培養後、各スキャフォールドをPBSにて2回洗浄し、4%PFAにて30分固定した。その後PBSにて2回洗浄し、下記で染色した。
Hoechst 33342 (Dojindo) Lot:KR057
Alexa FluorTM 488 phalloidin (Life Technologies Corporation, Eugene, Oregon ) Lot : 1834338
染色はそれぞれの製品のプロトコルに従った。
播種翌日、各ウェルの培地を基礎培地から骨分化培地(osteogenic medium:DMEM medium supplemented with 50 μg/mL ascorbic acid, 10 mM β-glycerol phosphate, 100 nM dexamethasone and 10% FBS)に交換した。3日に1度、同じ培地にて培地交換を行い、Day7にて各スキャフォールドを回収した。実施例2と同様に4%PFAにて固定後、Alizarin red S solution (Sigma Aldrich)を用いてアリザリンレッドS染色を行った。
Rhodamine-labelled NanoCliP gelおよびRhodamine-labelled NanoCliP-FD gelを調製し、共焦点レーザー顕微鏡によって3次元的に撮影を行なった。画像はxy方向として667×667 μm、z方向として31.4 μm間隔で3枚の断面画像を取得した。この撮影をxy(およびz)の位置が異なる3箇所について行った(n = 3)。得られた画像についてImageJ(NIH無償提供ソフト)のAnalyze Particles機能によって、各断面に存在する細孔の数と面積を算出した。この面積を円の面積として換算した時の各細孔の直径(細孔径)を計算した。この中で直径が5 μm以下の細孔を除外し、最大径を400 μmとして各細孔を20 μm間隔に20の集団に分割したヒストグラムを作成した。各間隔にNx個の細孔が含まれており、各間隔の平均直径Dxを用いたとき、以下の式により面積平均の細孔径を算出した。
Claims (10)
- 架橋された疎水化多糖ナノゲル粒子から構成される多孔質三次元細胞培養用足場材料。
- 架橋された疎水化多糖ナノゲル粒子が、フィブロネクチンでコーティングされたものである、請求項1に記載の多孔質三次元細胞培養用足場材料。
- 架橋性基を有する疎水化多糖ナノゲルが、多糖部分、疎水性部分及び重合性部分を含む、請求項2に記載の多孔質三次元細胞培養用足場材料。
- 多糖部分が、プルラン、アミロペクチン、アミロース、デキストラン、ヒドロキシエチルデキストラン、マンナン、レバン、イヌリン、キチン、キトサン、キシログルカンまたは水溶性セルロースである、請求項3に記載の多孔質三次元細胞培養用足場材料。
- 疎水性部分が炭素数8〜50の炭化水素基またはステリル基を含む、請求項3に記載の多孔質三次元細胞培養用足場材料。
- 疎水性部分がコレステリル基を含む、請求項5に記載の多孔質三次元細胞培養用足場材料。
- 重合性部分がアクリロイル、メタアクリロイル、ビニルまたはアリルを含む、請求項3に記載の多孔質三次元細胞培養用足場材料。
- 疎水化多糖ナノゲル粒子の架橋に用いられる架橋剤がメルカプトエチルポリエチレングリコール誘導体である、請求項2に記載の多孔質三次元細胞培養用足場材料。
- 多孔質三次元細胞培養用足場材料の断面における平均細孔径が5〜250μmである連続した細孔を含む、請求項1〜8のいずれか1項に記載の多孔質三次元細胞培養用足場材料。
- 架橋した疎水化多糖ナノゲル粒子を凍結融解し、その後に凍結乾燥することを特徴とする、請求項1〜9のいずれかに記載の多孔質三次元細胞培養用足場材料の製造方法。
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