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T. Sumide, K. Nishida, M. Yamato, T. Ide, N. Maeda, H. Watanabe, A. Kikuchi, T. Okano, Y. Tano; Harvest of bioengineered human corneal endothelial cell sheet cultivated on temperature–responsive culture dish . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4787.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously, we reported our temperature–responsive culture system that allowed us to fabricate and harvest, in various cell lines, a single, intact cell sheet retaining junctional proteins as well as extracellular matrix proteins. In this study, we have investigated a method to culture and harvest the human corneal endothelial cell (HCEC) sheets from the temperature–responsive dish for clinical use and studied some physiologic characteristics. Methods: HCECs from the remainder of USA donor corneas after penetrating keratoplasty were seeded on temperature–responsive culture dishes, and cultivated at 37°C in a 10% CO2 atmosphere. Several weeks later, fabricated cell sheets were harvested by low temperature treatment. The Na/K ATPase pump sites in the harvested sheet were examined by immunohistochemistry and quantified by a 3H–ouabain binding assay. Results: The cultivated HCECs could be harvested intact as a monolayer. In these sheets, the Na/K ATPase pump sites were located at the lateral borders of the cells. The 3H–ouabain binding assay demonstrated that the number of the pump sites in the sheet is similar to that in the human corneal endothelium in vivo. Conclusions: We have developed a novel method to fabricate and harvest cultivated HCEC sheets using our temperature–responsive culture system. Location and number of Na/K ATPase pump sites in the sheet are similar to in vivo. This bioengineered tissue construct has potential applications for ocular reconstructive surgery.
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