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192 ENRICHMENT OF CORNEAL EPITHELIAL PROGENITORS BY ISOLATION METHOD AND CULTURE ENVIRONMENT M. Kawashima, T. Kawakita, S. Shimmura, K. Tsubota Ophthalmology, Keio University, Shinjuku-ku, Japan Purpose: For clinical use of better cultivated corneal epithelial sheet, we compared two isolating methds, collagenase-method and previous dispase-method from the point of the number of progenitor cells and proliferating cells. We also compared the characteristics of epithelial cells cultured with different culture medium, serum free kerationcyte medium or DMEM/F12 medium. Methods: Human limbal tissues from donor cornea were evenly divided. Enzymatic treatment was peformed by dispaseII(37C 1h) or type 1A collagenase(37C 16h). Isolated cell fractions were assessed for viable cell numbers, fibroblast contamination, and colony forming efficiency. Cytospin smears were double immunostaining with p63 (4A4) and other progenitor markers to determine which methods include more number of progenitor cells. Results: After enzymatic treatment, viable epithelial cell numbers were 1.7x10^5 ±0.9 (mean + SD) and 8.3x10^5±0.9 cells in dispase and collagenase methods, respectively. The isolated limbal cells were highly positive for p63 with compact cell morphology in both methods. Cells with vimentin with elongated morphology were not observed after 7 days culture in serum free keratinocyte medium in both methods. Conclusion: The combination of collagenase isolation and cultivation in serum-free medium showed the best result in expansion and enrichment in progenitor cells. For limited cell source in application, this method may be useful for cultivating corneal epithelial cells.
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