JP2004357694A - 組織プラグの製造方法 - Google Patents
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Abstract
【解決手段】培養液が通過できる微細孔を有するチャンバー内に、被検動物又は患者から採取した組織由来細胞の細胞塊を入れ、前記細胞塊の一部が気相に接する程度の量の培養液が前記チャンバー内に含まれるようにして、前記チャンバー内の培養液よりも過剰量の培養液中で前記細胞塊を培養することを特徴とする組織プラグの製造方法。
【選択図】 図1
Description
2.マトリックスの産生が増加中の時点
3.マトリックスが充分量産生されピークになった時点
次に、あらかじめ任意の形状に成形しておいた撥水性又は非細胞接着性のチャンバーに多数の凝集塊を、最終目的の大きさに対応する数(1個又は複数個)だけ移し、培養する。
本実施例は、直径4mm、厚さ2mmの円柱状のヒト由来軟骨細胞だけからなる、軟骨プラグの作製に関するものである。
ウサギ関節軟骨切片にコラゲナーゼ処理を行い、得られた軟骨細胞の単層培養を行った。最終的に軟骨細胞を15cmディッシュ一枚あたり1.0x106得た。この細胞をトリプシン処理して細胞懸濁液にし、アガロースでコートした96マルチウェルにそれぞれ1.0x105個の細胞が入るように播種した。その後 37℃、5%二酸化炭素の条件下で培養を行い、翌日には直径が平均0.5mmの細胞塊が一プレートあたり96個得られた。
ウサギの骨盤より採取した骨髄由来間葉系幹細胞を単層培養した。最終的に間葉系幹細胞を15cmディッシュ一枚あたり1.0x106得た。この細胞をトリプシン処理して細胞懸濁液にし、住友ベークライト社製スフェロイドプレートにそれぞれ1.0x105個の細胞が入るように播種した。その後 37℃、5%二酸化炭素の条件下で培養を行い、翌日には直径が平均0.5mmの細胞塊が1プレートあたり96個得られた。
ウサギ関節軟骨切片にコラゲナーゼ処理を行い、得られた軟骨細胞の単層培養を行った。最終的に軟骨細胞を15cmディッシュ一枚あたり1.0x106得た。この細胞をトリプシン処理して細胞懸濁液にし、アガロースでコートした96マルチウェルにそれぞれ1.0x105個の細胞が入るように播種した。その後 37℃、5%二酸化炭素の条件下で培養を行い、翌日には直径が平均0.5mmの細胞塊が1プレートあたり96個得られた。
本実施例では、ウサギ大腿骨遠位関節面にプラグを移植する実験を行なった(図6)。すなわち、ウサギの骨盤より採取した骨髄由来間葉系幹細胞を単層培養した。最終的に間葉系幹細胞を15cmディッシュ一枚あたり1.0x106得た。この細胞をトリプシン処理して細胞懸濁液にし、住友ベークライト社製スフェロイドプレートにそれぞれ1.0x105個の細胞が入るように播種した。その後 37℃、5%二酸化炭素の条件下で培養を行い、翌日には直径が平均0.5mmの細胞塊が1プレートあたり96個得られた。
このプラグを、細胞採取元のウサギの大腿骨遠位関節面に移植した。ウサギを麻酔後、大腿骨関節面に直径4mmの孔を作成し(図6A)、前述のプラグを移植した(図6B、C)。
ウサギの骨盤より採取した骨髄由来間葉系幹細胞を単層培養した。
Claims (8)
- 培養液が通過できる微細孔を有するチャンバー内に、被検動物又は患者から採取した組織由来細胞の細胞塊を入れ、前記細胞塊の一部が気相に接する程度の量の培養液が前記チャンバー内に含まれるようにして、前記チャンバー内の培養液よりも過剰量の培養液中で前記細胞塊を培養することを特徴とする細胞塊の培養方法。
- 培養液が通過できる微細孔を有するチャンバー内に、被検動物又は患者から採取した組織由来細胞の細胞塊を入れ、前記細胞塊の一部が気相に接する程度の量の培養液が前記チャンバー内に含まれるようにして、前記チャンバー内の培養液よりも過剰量の培養液中で前記細胞塊を培養することを特徴とする組織プラグの製造方法。
- 組織由来細胞が幹細胞又はその分化細胞である請求項1又は2記載の方法。
- チャンバーが撥水性又は細胞非接着性のものである請求項1又は2記載の方法。
- 組織プラグが軟骨プラグ、骨プラグ又は脂肪プラグである請求項2記載の方法。
- 幹細胞が胚性幹細胞、臍帯血由来細胞又は間葉系幹細胞である請求項3記載の方法。
- 間葉系幹細胞が骨髄、皮膚又は皮下脂肪由来のものである請求項6記載の方法。
- 請求項2〜7のいずれか1項に記載の方法により製造された組織プラグ。
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Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2008178670A (ja) * | 2006-12-13 | 2008-08-07 | Depuy Mitek Inc | 軟組織修復にコラゲナーゼを使った組織癒合法 |
WO2008123614A1 (ja) | 2007-03-30 | 2008-10-16 | Kyushu University, National University Corporation | 細胞の立体構造体の製造方法 |
JP2009106214A (ja) * | 2007-10-31 | 2009-05-21 | Tsuneo Takahashi | 培養軟骨製造方法および培養軟骨 |
WO2009081503A1 (ja) | 2007-12-21 | 2009-07-02 | Yukihide Iwamoto | 細胞移植用器具 |
JP2010213631A (ja) * | 2009-03-17 | 2010-09-30 | Japan Health Science Foundation | 細胞分化装置、細胞分化方法、及び象牙芽細胞 |
WO2012081470A1 (ja) * | 2010-12-13 | 2012-06-21 | 株式会社資生堂 | 細胞凝集塊の形成方法 |
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