Cultured corneal epithelial grafts generated from organ–cultured limbal tissue: phenotypic analysis and factors influencing epithelial cell growth

Abstract

Abstract: : Purpose:Whereas cultured human limbal epithelial cells are currently used in patients with limbal stem cell deficiency, limbal stem cells have not yet been formally identified and limbal epithelial cell culture technique is not yet standardized. The aim of this study was to explore mitotic potential of human limbal epithelial cells after 31°C organ–culture preservation and to investigate putative factors influencing limbal epithelial cell growth in culture. Methods: 185 cultures of limbal explants obtained from 42 human organ–cultured corneas were carried–out. Cell suspension cultures were compared to explant cultures. Light and transmission electron microscopy as well as immunohistology were used to identify cell phenotype in culture. Univariate and multivariate analysis were performed to determine factors influencing limbal epithelial growth in culture. Results:Epithelial cell growth > 100mm² was observed in 52% of cultures, with an average growth area of 440 + 265 mm². A short death to retrieval time (p<0,03) and female donors (p<0,001) were found to be associated with higher epithelial cell growth. Enzymatic treatment of explants by trypsin, but not by dispase, decreased cell proliferation at two weeks (p<0,03), but not at three weeks. Corneal storage time and media (Inosol or Corneamax) and donor age did not significantly influence cell growth. Cultured cells expressed cytokeratin 19 and vimentin during the first week. During the second and third weeks, they were positive for cytokeratin 3, but they still expressed vimentin and, to a lesser extent, cytokeratin 19. Conclusions:Organ–culture preserved limbal tissue shows high but heterogeneous mitotic potential. Culture success rate could be improved by controlling factors influencing epithelial cell growth. Limbal tissue should be excised before circulatory arrest. Donor sex influence on epithelial growth in culture needs further investigations.

Keywords: cornea: epithelium • cornea: storage