ENRICHMENT OF CORNEAL EPITHELIAL PROGENITORS BY ISOLATION METHOD AND
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.