再生医療用ナノブラシ固定化細胞培養基盤の調整と幹細胞保持並びに培養

2011 年度 研究成果報告書

• 研究課題/領域番号: 21500436
• 研究種目: 基盤研究(C)
• 配分区分: 補助金
• 応募区分: 一般
• 研究分野: 医用生体工学・生体材料学
• 研究機関: 独立行政法人国立成育医療研究センター
• 研究代表者: 樋口 亜紺 独立行政法人国立成育医療研究センター, 生殖・細胞医療研究部, 共同研究員 (30189766)
• 研究期間 (年度): 2009 – 2011
• キーワード: バイオマテリアル / 再生医療 / 造血幹細胞 / ナノブラシ / 生体適合性材料 / 細胞培養基板

研究概要

親水性セグメント-疎水性セグメント-親水性セグメントよりなる三元共重合体は、親水性ドメイン並びに疎水性ドメインを有する温度応答性高分子である。一般に、親疎水性ドメインを有する表面は、単に親水性の表面より生体適合性が優れていることが報告されている。しかしながら、表面基板の縦方向に親疎水性ドメインを有する生体適合性材料は、これまで研究されてこなかった。本申請研究は、様々なセグメント長(分子量)並びに様々な親疎水性量比を有する親疎水三元共重合体(プルロニック)を表面反応により細胞培養基板上に固定化することにより、ナノブラシ状親疎水三元共重合体固定化細胞培養基板(ナノブラシ固定化細胞培養基板)を調製した。いかなる分子設計の親疎水三元共重合体を用いてナノブラシ固定化細胞培養基板を調製するのが、造血幹細胞の保持・培養に適しているかを検討することが本申請研究の第1の目的とした。親疎水三元共重合体の温度応答性を利用して、ナノブラシ固定化細胞培養基板上に培養した細胞の剥離操作に、従来のトリプシン処理ではなく、細胞培養基板温度を低温にさせるのみで細胞剥離を効率良く行うことの実証と細胞剥離のための最適なナノブラシ固定化細胞培養基板の調製を第2の目的とした。親水性部位が70%有し、分子量が1万の三元共重合体を適度な密度で固定化させた基板上において、最も造血幹細胞の保持・培養に優れていた。高密度のナノブラシ固定化細胞培養基板上での造血幹細胞の保持・培養は、適しておらず、最適なナノブラシ固定化密度の存在が本研究で明らかとなった。
様々な細胞外マトリックス(ジェラチン、コラーゲン、ラミニン、ビトロネクチン、フィブロネクチン、マトリゲル)をポリスチレン製基板上に共有結合並びにコーティング法にて固定化させた。いずれの基板上に間葉系幹細胞を培養しても、間葉系幹細胞の表面マーカーの発現率に、顕著な違いは見られなかった。ジェラチン並びにビトロネクチンをコーティングした細胞培養基板上では、骨芽細胞への分化は促進されたが、様々な細胞外マトリックスを共有結合で固定化させた基板上では、骨芽細胞への分化は促進されなかった。一方、様々な細胞外マトリックスを共有結合で固定化させた基板上では、神経細胞への分化が促進されていた。特に、マトリゲル固定化基板並びにラミニン固定化基板で顕著であった。さらに、プルロニックを細胞外マトリックスとともに固定化させた基板では、細胞培養液を4度に低下させることにより、間葉系幹細胞の細胞剥離を引き起こすことが可能であった。

研究成果

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• [学会発表] Differentiation Ability of Adipose-derived Stem Cells Separated from Adipose Tissue by a Membrane Filtration Method (2011)
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• [学会発表] Differentiation ability of amniotic fluid-derived stem cells cultured on extracellular matrix-immobilized surface (2011)
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• [学会発表] Differentiation ability of adipose-derived stem cells separated from adipose tissue by membrane filtration method (2011)
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• [学会発表] Ex vivo expansion of hematopoietic stem cells on the surface having nano-segments and extracellular matrix proteins (2011)
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• [学会発表] Differentiation Ability of Adipose-derived Stem Cells Separated from Adipose Tissue by A Membrane Filtration Method (2011)
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  • 著者名/発表者名: Akon Higuchi
  • 学会等名: 第60回高分子年次学会
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  • 年月日: 20110500
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  • 学会等名: 第60回高分子年次学会
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  • 著者名/発表者名: Akon Higuchi
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• [学会発表] Purification and Characterization of Cancer Stem Cells in Colon Cancer Cells Treated by Anti-Cancer Drugs (2010)
  • 著者名/発表者名: Wan-Chun Yu and Akon Higuchi
  • 学会等名: The Tissue Engineering & Regenerative Medicine International Society(Termis) 2010 Asia pasific meeting
  • 発表場所: Sydnet. Australia
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• [学会発表] Cultivation of hematopoietic stem cells on the surface-mosdified materials having nano-segments and extracellular matrix proteins (2010)
  • 著者名/発表者名: Li-Ying Chen, Pei-Tsz Li & Akon Higuchi
  • 学会等名: The Tissue Engineering & Regenerative Medicine International Society(Termis) 2010 Asia pasific meeting
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  • 著者名/発表者名: 樋口亜紺
  • 学会等名: 高分子討論会(60th Symposium on Macromolecules, Polymer Society of Japan(2019))
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• [学会発表] Separation of Mesenchymal Stem Cells from Adipose tissue by Membrane Filtration Method and Their Differentiation Ability (2010)
  • 著者名/発表者名: 樋口亜紺
  • 学会等名: 高分子討論会(60th Symposium on Macromolecules, Polymer Society of Japan(2019))
  • 年月日: 20100900
• [学会発表] Preservation and cultivation of stem cells on the surface having nano-segments (2010)
  • 著者名/発表者名: Chung-Ming Huang, Akon Higuchi
  • 学会等名: The Asia Pacific Chapter of the Tissue Engineering and Regenerative Medicine International Society
  • 発表場所: Singapore, Singapore
  • 年月日: 20100803-05
• [学会発表] Effect of micro-environment on pluripotent gene expression and differentiation of mesenchymal stem cells from second-trimester amniotic-fluid (2010)
  • 著者名/発表者名: Shiau-Chian Huang, Han-Chow Wang, and Akon Higuchi
  • 学会等名: The International Society for Stem Cell Research(ISSCR) 8th Annual Meeting
  • 発表場所: San Francisco, USA
  • 年月日: 20100616-19
• [学会発表] Purification and cultivation of hematopoietic stem cells through surface-modified membranes having nano-segments and extracellular matrix proteins from umbilical cord blood (2010)
  • 著者名/発表者名: Pei-Tsz Li, Li-Ying Chen, and Akon Higuchi
  • 学会等名: The International Society for Stem Cell Research(ISSCR) 8th Annual Meeting
  • 発表場所: San Francisco, USA
  • 年月日: 20100616-19
• [学会発表] Preservation of hematopoietic stem cells from umbilical cord blood stored in a surface having polymer nano-segments (2010)
  • 著者名/発表者名: Akon Higuchi
  • 学会等名: The International Society for Stem Cell Research(ISSCR) 8th Annual Meeting
  • 発表場所: San Francisco, USA
  • 年月日: 20100616-19
• [学会発表] Differentiation Ability of Adipose-derived Stem Cells Separated from Adipose Tissue by AMembrane Filtration Method (2010)
  • 著者名/発表者名: Akon Higuchi, Chung. Wei Chuang
  • 学会等名: 膜Symposium 2010
  • 年月日: 2010-11-20
• [学会発表] Separation and Differentiation of Adipose-Derived Stem Cells by Membrane Filtration Method (2010)
  • 著者名/発表者名: Akon Higuchi
  • 学会等名: The 6th 2010 Annual Meeting of Korean Society of Stem Cell Research
  • 発表場所: Seoul, Korea
  • 年月日: 2010-10-22
• [学会発表] 高分子ナノセグメント固定化表面上における臍帯血中造血幹細胞の保持 (2009)
  • 著者名/発表者名: 樋口亜紺
  • 学会等名: 高分子討論会
  • 年月日: 20090900
• [学会発表] DNA酵素結合アプタマー分析法からのスロンビンの比色分析 (2009)
  • 著者名/発表者名: 樋口亜紺、福嶋久
  • 学会等名: 高分子討論会(58th Symposium on Macromolecules, Polymer Society of Japan(2009))
  • 年月日: 20090900
• [学会発表] Drugs in a culture medium of Lovo Cells Enhance production of Carcinoembryonic Antigen and Identification of Colon Cancer Stem Cells (2009)
  • 著者名/発表者名: Hsin Ling Hsieh, Wen-Yih Chen, and Akon Higuchi
  • 学会等名: 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium(TERMIS WC2009)
  • 発表場所: Seoul, Korea
  • 年月日: 20090831-0903
• [学会発表] Isolation of Adipose Tissue-derived Stromal Cells from Adipose Tissues by Filtration Method through Porous Membrane having Nano-segments and Their Characteristics (2009)
  • 著者名/発表者名: Chuang Chung Wei, and Akon Higuchi
  • 学会等名: 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium(TERMIS WC2009)
  • 発表場所: Seoul, Korea
  • 年月日: 20090831-0903
• [学会発表] Separation of Hematopoietic Stem cells from Human Peripheral Blood and Umbilical Cord Blood through Surface-modified Polyurethane Forming Membranes (2009)
  • 著者名/発表者名: Pei-Tsz Li, Siou-Ting Yang, and Akon Higuchi
  • 学会等名: 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium(TERMIS WC2009)
  • 発表場所: Seoul, Korea
  • 年月日: 20090831-0903
• [学会発表] Effect of Micro-environment on Pluripotent Gene Expression and Differentiation of Mesenchymal Stem Cells from Second-trimester Amniotic Fluid (2009)
  • 著者名/発表者名: Shiau-Chian Haung, Han-Chow Wang, and Akon Higuchi
  • 学会等名: 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium(TERMIS WC2009)
  • 発表場所: Seoul, Korea
  • 年月日: 20090831-0903
• [学会発表] Preservation of Hematopoietic Stem Cells from Umbilical Cord Blood Stored in a Surface Derivatized with Polymer Nanosegments (2009)
  • 著者名/発表者名: Akon Higuchi, Pei-Tsz Li, and Siou-Ting Yang
  • 学会等名: 2nd TERMIS World Congress in conjunction with 2009 Seoul Stem Cell Symposium(TERMIS WC2009)
  • 発表場所: Seoul, Korea
  • 年月日: 20090831-0903
• [学会発表] Separation and ex vivo expansion of hematopoietic stem cells from human blood by membrane filtration method and magnetic associated sorting method (2009)
  • 著者名/発表者名: Siou-Ting Yang, Pei-Tsz Li, Akon Higuchi
  • 学会等名: The Fifth Conference of Aseanian Membrane Society(AMS5)
  • 発表場所: Kobe, Japan
  • 年月日: 20090700
• [学会発表] Visible light regulates gene expression and neurite outgrowth of nerve cells cultured on collagen-coated membranes (2009)
  • 著者名/発表者名: Ling-Yi Ho, Qing-Dong Ling, Akon Higuchi
  • 学会等名: The Fifth Conference of Aseanian Membrane Society(AMS5)
  • 発表場所: Kobe, Japan
  • 年月日: 20090700
• [学会発表] Stabilization of human epidermal growth factor(hEGF) and its bioassay (2009)
  • 著者名/発表者名: Jen-Hao Lee, Toshiko Abe, Motohisa Matsuzaki, Akon Higuchi
  • 学会等名: The Fifth Conference of Aseanian Membrane Society(AMS5)
  • 発表場所: Kobe, Japan
  • 年月日: 20090700
• [学会発表] Permeation and isolation of hematopoietic stem cells and blood cells from umbilical cord blood through surface-modified polyurethane membranes having nanosegments (2009)
  • 著者名/発表者名: Akon Higuchi
  • 学会等名: The Fifth Conference of Aseanian Membrane Society(AMS5)
  • 発表場所: Kobe, Japan
  • 年月日: 20090700
• [学会発表] PRESERVATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS FROM UMBILICAL CORD BLOOD STORED IN A SURFACE DERIVATIZED WITH POLYMER NANOSEGMENTS (2009)
  • 著者名/発表者名: Akon Higuchi, Pei-Tsz Li, Siou-Ting Yang
  • 学会等名: International Symposium of Stem Cells and Bioengineering
  • 発表場所: Taian, Taiwan
  • 年月日: 20090500
• [図書] 医療分野における材料と機能膜(樋口亜紺監修) (2011)
  • 著者名/発表者名: 樋口亜紺
  • 総ページ数: 1-328
  • 出版者: シーエムシー出版
• [図書] Separation & Purification of Stem and Blood cells by Porous Polymeric Membranes, Comprehnsive Membrane Science and Engineering(Eds. E. Driolli, L. Giorno) (2010)
  • 著者名/発表者名: Akon Higuchi