| Project/Area Number |
13558119
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Tokyo Women's Medical University |
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Principal Investigator |
YAMATO Masayuki Tokyo Women's Medical University, Department of Medicine, Associate Professor, 医学部, 助教授 (40267117)
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| Co-Investigator(Kenkyū-buntansha) |
SHINOZAKI Kazumi Tokyo Women's Medical University, Department of Medicine, Research Assistant Professor, 医学部, 助手 (10256521)
HORI Sadao Tokyo Women's Medical University, Department of Medicine, Professor, 医学部, 教授 (20143498)
SHIMIZU Tatsuya Tokyo Women's Medical University, Department of Medicine, Assistant Professor, 医学部, 講師 (40318100)
青柳 隆夫 東京女子医科大学, 医学部, 助教授 (40277132)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2001: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | thermoresponsive polymers / poly(N-isopropylacrylamide) / cornea / stem cell / tissue engineering / regenerative medicine / cell sheet engineering / 温度応答性表面 / 細胞シートのマニピュレーション / 角膜上皮細胞 / ディスパーゼ / 移植 / 細胞間接着 / 基底膜 |
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| Research Abstract |
Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37℃. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20℃. Histologic analyzes, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.
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