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R. Hayashi, H. Takayanagi, Y. Oie, A. Kubota, M. Yamato, T. Okano, K. Nishida; Method for the Validation of Tissue-Engineered Epithelial Cell Sheet. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5747.
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Regenerative therapy with tissue-engineered epithelial cell sheet has been clinically applied to ocular surface diseases. It will be required to develop the method for validation of these cell sheets for standardizing the regenerative therapy. In the present study, we examined the method for validation of tissue-engineered epithelial cell sheet.
Epithelial cells were isolated from USA eye bank cornea or volunteer oral mucosa. Each epithelial cell was seeded on temperature-responsive 6 well culture inserts and then cultured with feeder cells. After detached from the membrane of culture insert by reducing temperature, the cell sheets were divided into 2 pieces. One was treated with trypsin and applied to measurement of cell numbers and FACS analysis for cell viability and epithelial cell purity. The other was applied to immunostaining for ZO-1, MUC16 as a marker of barrier function, p63 and K3 as markers of immature and differentiated cell, respectively.
Corneal and oral-mucosal epithelial cell sheets showed total cell numbers; 8.0-11.0, 8.0-16.0 x 105 cells/sheet, cell viability; 87.1-93.2, 83.3-88.4%, epithelial cell purity; 93.0-97.9, 94.4-98.7%, respectively (N=3). The results of immunostaining demonstrated that each epithelial cell expressed ZO-1 and MUC16 in the surface cells. Additionally, p63 was expressed in the basal layer, and K3 was expressed in almost all layers of each epithelial cell sheet.
These results suggested the possibility that using these methods validation of tissue-engineered epithelial cell sheet could be performed.
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