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K. Nishida, M. Yamato, Y. Hayashida, K. Watanabe, N. Maeda, H. Watanabe, A. Kikuchi, T. Okano, Y. Tano; A Novel Tissue Engineering Approach for Ocular Surface Reconstruction using Bioengineered Corneal Epithelial Cell Sheet Grafts from Limbal Stem Cells Expanded ex vivo on a Temperature-responsive Cell Culture Surface . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3149.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously we reported a cell sheet manipulation technique that utilizes temperature-responsive cell culture surfaces. Confluent cell cultures on these surfaces are harvested as a single contiguous cell sheet retaining cell-cell junctions as well as a deposited extracellular matrix on the basal sheet surface. In this study, we have developed a novel tissue engineering approach for ocular surface reconstruction using limbal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Methods: Rabbit corneal limbal epithelial cells, including stem cells, were collected from corneal limbal tissue. The epithelial cells (1 x105) were co-cultured with MMC-treated 3T3 feeder-layers on temperature-responsive culture dishes for 2 weeks. Cultivated cells were then harvested from the culture dishes by reducing temperature treatment. Some preparations were observed by light and electron microscopy. Autologous transplantation of the cultivated epithelial cells was undertaken to reconstruct the ocular surface in rabbits with total limbal stem cell deficiencies. Results: After 2 weeks in culture, a multilayered corneal epithelial sheet was formed. This was harvested as a transplantable cell sheet simply by reducing the culture temperature to 20 °C. Electron microscopy revealed numerous small microvilli in the apical cell membranes of superficial cells, and prominent desmosomes along cell-cell junctions of middle cells. By autologous transplantation, the sheet readily adhered to the underlying corneal stroma, self-stabilizing without any suturing. The rabbit corneal surface was successfully reconstructed and its transparency was maintained during observation periods up to 90 days. Conclusions: Our epithelial cell sheet engineering technique utilizing temperature-responsive culture surfaces permits rapid, adherence of corneal epithelial sheet to the corneal stroma without any carriers or sutures. The results suggest promising clinical capabilities for our bioengineered corneal sheet ideal for transplantation.
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