JP2009240766A - 骨組織再生シート及びその作製方法 - Google Patents
骨組織再生シート及びその作製方法 Download PDFInfo
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- JP2009240766A JP2009240766A JP2009048890A JP2009048890A JP2009240766A JP 2009240766 A JP2009240766 A JP 2009240766A JP 2009048890 A JP2009048890 A JP 2009048890A JP 2009048890 A JP2009048890 A JP 2009048890A JP 2009240766 A JP2009240766 A JP 2009240766A
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Abstract
【解決手段】生体吸収性合成高分子から成るシート状の多孔質体の片側に軟骨細胞又は軟骨細胞に分化する幹細胞を播種し、播種後の多孔質体を培養液に入れ100〜1000Gの加速度を所定時間加えて播種した軟骨細胞又は軟骨細胞に分化する幹細胞を凝集させた後、凝集させた細胞に加速度を加えずに血清を含まずアスコルビン酸,アスコルビン酸誘導体,デキサメタゾンから選ばれた1種又は2種以上が含まれている培養液で培養して厚さが200μm以上の軟骨細胞層を形成し、この軟骨細胞層を血清を含む培養液で培養することで内軟骨性骨化させることによって厚さが200μm以上の皮質骨組織層を形成させて骨組織再生シート材を作製する。
【選択図】なし
Description
本発明の他の一つは、生体吸収性合成高分子から成るシート状の多孔質体の片側に厚さが200μm以上の軟骨細胞層を形成し、該軟骨細胞を内軟骨性骨化させることによって厚さが200μm以上の皮質骨組織層を形成させることを特徴とする骨組織再生シート材の作製方法である。
この骨組織再生シート材の作製方法において、生体吸収性合成高分子から成るシート状の多孔質体の片側に厚さが200μm以上の軟骨細胞層を形成する方法としては、生体吸収性合成高分子から成るシート状の多孔質体の片側に、軟骨細胞又は軟骨細胞に分化する幹細胞を播種し、播種後の多孔質体を培養液に入れ100〜1000Gの加速度を所定時間加えて軟骨細胞又は軟骨細胞に分化する幹細胞を凝集させた後、凝集させた細胞に加速度を加えずに血清を含まずアスコルビン酸,アスコルビン酸誘導体,デキサメタゾンから選ばれた1種又は2種以上が含まれている培養液で培養して厚さが200μm以上の軟骨細胞層を形成する方法があり、軟骨細胞を内軟骨性骨化させることによって厚さが200μm以上の皮質骨組織層を形成させる方法としては、200μm以上の軟骨細胞層を血清を含む培養液で培養することで軟骨細胞層を厚さが200μm以上の皮質骨組織層に内軟骨性骨化させる方法が好ましいのである。
4週齢のウサギの大腿骨,脛骨から筋肉及び靭帯等を除いてこれを切除し、その両端を切断し、10%ウシ胎児血清,αMEM培地で骨髄内を洗浄し、その洗浄液中でよく懸濁して骨髄液をほぐした後、300Gで5分間遠心分離し、細胞を分離して約7×107個の有核細胞を得た。
骨髄から採取した7×107の有核細胞を前記と同様の10%ウシ胎児血清,αMEM培地の入った75cm2培養フラスコへ播種し、37℃にて5%炭酸ガス存在下で培養した。6日目で培地を交換することによって非接着細胞を除去した。以後3日に1回の割合で培地を交換した。また、5日目からはbFGFを3ng/mlの割合で培地に添加した。その結果、10日前後でほぼ集密的にまで増殖した。この培養フラスコを(0.05%トリプシン+0.2mM MEDTA)で5分間インキュベートして細胞を剥離,回収した。細胞数をCoulterカウンター(Z1シングル,コールター社製)で計測し、そして5×103細胞個/cm2の密度で細胞を二代目の継代培養皿へ播種した。前記の37℃にて5%炭酸ガス存在下で培養するところから、5分間インキュベートして細胞を回収するところまでの操作を繰り返して、この二代目の継代培養皿から得た三代目の細胞を用いた。
分子量230,000のポリ−L−乳酸と分子量250,000のDL−乳酸/グリコール酸共重合体とを所定の割合で混合した高分子ブレンドをジオキサンに溶解させた後、凍結乾燥によって、孔のサイズが平均20μm,有孔率52%,厚さ250μmの生体吸収性合成高分子から成るシート状の多孔質体を作製した。
生体吸収性合成高分子から成るシート状の多孔質体を直径9mmに切断し、その片面側に前記の間葉系細胞を集密状態のまま播種した。遠心分離器(商品名:小型卓上遠心機,コクサン社製)により播種した間葉系細胞方向から生体吸収性合成高分子から成るシート状の多孔質体へ向けて略垂直に約352Gの加速度を3分間加えた(半径15cm,1500rpm)。その後、軟骨分化誘導培地(50μg/ml アスコルビン酸二リン酸,100μg/ml ピルビン酸,4.5g/l D-(+)-グルコース,2mM L-グルタミン,10ng/ml TGF-β3,10-7M デキサメタゾン,1% ITS+を含有したαMEM)にて37℃,常圧下で4週間培養して軟骨組織のシート材を作製した。
前記の軟骨組織のシート材を骨分化誘導培地(10% ウシ胎児血清,50μg/ml アスコルビン酸二リン酸,2mM L−グルタミン,10-7M Dデキサメタゾン,10mM βグリセロリン酸を含有したαMEM)にて37℃,常圧下で4週間培養して骨組織再生シート材を作製した。
ヒト腸骨骨髄液から採取した細胞を10%ウシ胎児血清,αMEM培地で懸濁した後、有核細胞数 1×107細胞個/10cm直径培養皿へ播種した。37℃にて5%炭酸ガス存在下で培養した。3日目で培地を交換し、非接着細胞(造血系細胞)を除いた。以後3日に1回培地を交換した。bFGFは5日目から3ng/mlで培地に添加した。10日前後でほぼ集密的にまで増殖した。これらの培養皿をトリプシン(0.05 %)+EDTA(0.2 mM)で5分間インキュベートして、細胞を単離した。細胞数をCoulterカウンター(Z1シングル,コールター社製)で計測し、そして5×103細胞個/cm2の密度で細胞を播種した。前記の37℃にて5%炭酸ガス存在下で培養するところから,5分間インキュベーとして細胞を回収するところまでの操作を再度繰り返して,この二代目の継代培養皿から得た三代目の細胞を用いた。
分子量250,000のDL−乳酸/グリコール酸共重合体をジオキサンに溶解させた後、凍結乾燥によって、孔のサイズが平均20μm,有孔率52%,厚さ250μmの生体吸収性合成高分子から成るシート状の多孔質体を作製した。
生体吸収性合成高分子から成るシート状の多孔質体を直径9mmに切断し、その片面側に前記の間葉系細胞を集密状態のまま播種した。遠心分離器(商品名:小型卓上遠心機,コクサン社製)により播種した間葉系細胞方向から生体吸収性合成高分子から成るシート状の多孔質体へ向けて略垂直に約352Gの加速度を3分間加えた(半径15cm,1500rpm)。その後、軟骨分化誘導培地(50μg/ml アスコルビン酸二リン酸,100μg/ml ピルビン酸,4.5g/l D-(+)-グルコース,2mM L-グルタミン,10ng/ml TGF-β3,10-7M デキサメタゾン,1% ITS+を含有したαMEM)にて37℃,常圧下で4週間培養して軟骨組織のシート材を作製した。
前記の軟骨組織のシート材を骨分化誘導培地(10% ウシ胎児血清,50μg/ml アスコルビン酸二リン酸,2mM L−グルタミン,10-7M デキサメタゾン,10mM βグリセロリン酸を含有したαMEM)にて37℃,常圧下で4週間培養して骨組織再生シート材を作製した。培養終了後にシート材を凍結包埋し。厚さ7μmの薄切片を作製し、ヘマトオキシリンエオシンで染色して顕微鏡で観察した結果、シート材に片面から約40μm侵入した部分に続いて約170μmの計約210μmの皮質骨組織層が形成されていることが確認できた。
1)大腿骨・脛骨からの骨髄液の採取
4週齢のラットの大腿骨,脛骨から筋肉及び靭帯等を除いてこれを切除し、その両端を切断し、10%ウシ胎児血清,αMEM培地で骨髄内を洗浄し、その洗浄液中で良く懸濁して骨髄液をほぐした後、300Gで5分間遠心分離し、細胞を分離して約7×107個の有核細胞を得た。
ラット骨髄から採取した7×107個の有核細胞を前記と同様の10%ウシ胎児血清,αMEM培地の入った75cm2培養フラスコへ播種し、37℃にて5%炭酸ガス存在下で培養した。5日目で培地を交換することによって非接着細胞を除去した。以後3日に1回の割合で培地を交換した。また、5日目からはbFGFを3ng/mlの割合で培地に添加した。その結果、10日前後でほぼ集密的にまで増殖した。この培養フラスコを(0.05%トリプシン+0.2mM MEDTA)で5分間インキュベートして細胞を剥離,回収した。細胞数をCoulterカウンター(Z1シングル,コールター社製)で計測し、そして5×103細胞個/cm2の密度で細胞を二代目の継代培養皿へ播種した。前記の37℃にて5%炭酸ガス存在下で培養するところから、5分間インキュベートして細胞を回収するところまでの操作を繰り返して、この二代目の継代培養皿から得た三代目の細胞を用いた。
分子量230,000のポリ−L−乳酸と分子量250,000のDL−乳酸/グリコール酸共重合体とを所定の割合で混合した高分子ブレンドをジオキサンに溶解させた後、凍結乾燥によって、孔のサイズが平均20μm,有孔率52%,厚さ250μmの生体吸収性合成高分子から成るシート状の多孔質体を作製した。
前記の間葉系細胞を生体吸収性合成高分子から成るシート状の多孔質体を直径9mmに切断し、その上に2x104cell/cm2の濃度となるように播種した。その後、骨分化誘導培地(10% ウシ胎児血清,50μg/ml アスコルビン酸二リン酸,2mM L-グルタミン,10-7M デキサメタゾン,10mM βグリセロリン酸を含有したαMEM)にて37℃,常圧下で4週間培養して骨芽細胞組織のシートを作製した。培養終了後にシートを凍結包埋し。厚さ7μmの薄切片を作製し、ヘマトオキシリンエオシンで染色して顕微鏡で観察した結果、シート材に片面から約40μm侵入した部分に続いて約10μmの計約50μmの薄い骨芽細胞組織しか形成できなかったことが確認できた。
Claims (4)
- 生体吸収性合成高分子から成るシート状の多孔質体の片側に厚さが200μm以上の皮質骨組織が形成されていることを特徴とする骨組織再生シート材。
- 生体吸収性合成高分子から成るシート状の多孔質体の片側に厚さが200μm以上の軟骨細胞層を形成し、該軟骨細胞を内軟骨性骨化させることによって厚さが200μm以上の皮質骨組織層を形成させることを特徴とする骨組織再生シート材の作製方法。
- 生体吸収性合成高分子から成るシート状の多孔質体の片側に厚さが200μm以上の軟骨細胞層を形成する方法が、生体吸収性合成高分子から成るシート状の多孔質体の片側に、軟骨細胞又は軟骨細胞に分化する幹細胞を播種し、播種後の多孔質体を培養液に入れ100〜1000Gの加速度を所定時間加えて軟骨細胞又は軟骨細胞に分化する幹細胞を凝集させた後、凝集させた細胞に加速度を加えずに血清を含まずアスコルビン酸,アスコルビン酸誘導体,デキサメタゾンから選ばれた1種又は2種以上が含まれている培養液で培養して厚さが200μm以上の軟骨細胞層を形成する方法である請求項2に記載の骨組織再生シート材の作製方法。
- 軟骨細胞を内軟骨性骨化させることによって厚さが200μm以上の皮質骨組織層を形成させる方法が、200μm以上の軟骨細胞層を血清を含む培養液で培養することで軟骨細胞層を厚さが200μm以上の皮質骨組織層に内軟骨性骨化させる方法である請求項2又は3に記載の骨組織再生シート材の作製方法。
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2009
- 2009-03-03 JP JP2009048890A patent/JP5554002B2/ja not_active Expired - Fee Related
- 2009-03-09 EP EP09003409.1A patent/EP2100629B1/en not_active Not-in-force
- 2009-03-10 US US12/401,121 patent/US20090228027A1/en not_active Abandoned
-
2015
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KR20170005168A (ko) | 2010-03-01 | 2017-01-11 | 후지필름 가부시키가이샤 | 생체 친화성을 갖는 고분자 블록과 세포로 이루어지는 세포 구조체 |
US9597432B2 (en) | 2010-03-01 | 2017-03-21 | Fujifilm Corporation | Cell construct comprising polymer blocks having biocompatibility and cells |
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US9211266B2 (en) | 2011-08-31 | 2015-12-15 | Fujifilm Corporation | Cell construct for cell transplantation and cell aggregate for cell transplantation |
JP2017006028A (ja) * | 2015-06-18 | 2017-01-12 | 株式会社ジーシー | 細胞工学用支持体、及び細胞工学用支持体の製造方法 |
WO2024004747A1 (ja) * | 2022-06-30 | 2024-01-04 | 国立大学法人広島大学 | 細胞構造体の製造方法、細胞構造体および骨再生剤 |
Also Published As
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EP2100629B1 (en) | 2013-05-22 |
US20090228027A1 (en) | 2009-09-10 |
EP2100629A1 (en) | 2009-09-16 |
JP5554002B2 (ja) | 2014-07-23 |
US20150240207A1 (en) | 2015-08-27 |
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