Limbal Epithelial Cell Cultures Obtained from Organ-cultured Human Corneas

Abstract

Abstract: : Purpose. To characterize limbal epithelial cell cultures obtained from organ-cultured human corneas. Methods. Limbal explants obtained from 77 organ-cultured human corneas were cultured on human amniotic membranes for 3 to 5 weeks. The influence of culture conditions on epithelial cell growth was studied in logistic regression. Transmission electron microscopy and immunochemistry were used to evaluate the epithelial cells at the end of culture. Results. Epithelial cell growth was observed in 62% of the cultures obtained from corneal explants which had not been treated with Dispase II, as against 39% of cases when they had been (p=0.04). It was observed in 75% of cases when fibroblast growth occurred and in only 25% of cases only when no fibroblast growth was noted (p=0.01). In successful cultures, a mutilayered epithelium was found in semi thin sections and TEM showed the presence of desmosomes and cytokeratin filaments. In immunochemistry, the cultured epithelial cells expressed CK3 and showed no expression of vimentine and bcl-2. Some cells showed strong staining with the anti-CK3 antibody whereas others showed low or no staining. Both CK3 staining and staining with a broad-spectrum anti-cytokeratin antibody decreased with culture time. Conclusions. Corneal fibroblasts increase limbal epithelial cell growth in culture. A 3-week period of culture seems to be the best time for grafting such cultures. Assessing cultured cells for the expression of CK3 and the lack of expression of vimentine before grafting would be useful to make sure that transplants contain limbal stem cells and are devoid of fibroblasts.

Keywords: cornea: epithelium • immunohistochemistry • microscopy: electron microscopy