Purchase this article with an account.
H. Kim, D. Li, X. Song, C.S. de Paiva, S.C. Pflugfelder; Ex vivo Expansion and Phenotypes of Human Limbal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1349.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the phenotypes and molecular markers expressed in human corneal epithelial cells expanded ex vivo by two culture systems from human limbal tissues. Methods: Human corneal epithelial cells were expanded by limbal explant culture or single-cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunostaining with antibodies for proposed basal or stem cell markers, integrin ß1, cytokeratin 19 (K19), EGFR and nuclear protein p63, and differentiation markers, cytokeratin 3 (K3) involucrin and connexin 43. 24 hour BrdU labeling with 21 day chasing was performed to identify the label-retaining cells in cultures. Results: Epithelial cell growth was dependent on the tissue freshness, the time of death to preservation (p<0.05) and the time of death to culture (p<0.05) ,but not on the donor age. The cell expansion was confluent in 10-12 days in single-cell suspension cultures and 18-21 days in explant cultures, although the cell growth rates were similar, 96.2% (n=40) v.s. 90.8% (n=181), in the two systems. The cell morphology in single-cell suspension culture was smaller and more compact than that in explant culture. Immunostaining showed that a great percentage of the small cells expressed p63, K19, integrin ß1 and EGFR while the large cells stained for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells (2.3-6.5%) were identified in cultures chased for 17 to 21 days after 24 hour labeling. Conclusions: Cells with both stem cell and differentiated phenotypes were maintained in primary cultured human limbal epithelial cells in both culture systems. Slow-cycling cells identified as BrdU-label retaining cells, which are characteristics of stem cell, were identified in these cultures.
This PDF is available to Subscribers Only