[Human limbal epithelial cell growth kinetics in vitro]

Abstract

Purpose: To assess limbal epithelial cell growth kinetics in vitro using tissue retrieved from organ-cultured donor corneas.

Patients and methods: Twenty-one limbal explants were retrieved from corneoscleral rims of donor corneal grafts preserved for 18-24 days in organ culture. The explants were cultured at 37°C for 10, 13, or 18 days. The epithelial cell sheet area was measured during culture by means of morphometry. At the end of culture, the dissociated cells were counted and analyzed by immunocytochemistry.

Results: The curve of the epithelial cell sheet area (S) in relation to culture time (t) was best fitted by a polynomial model (S=0.024t(3) - 0.038t(2) - 0.044t + 0.092). The average duration of the cell cycle was 24h. Cell growth decreased as donor tissue retrieval postmortem time increased. Cultured cells featured various expressions of cytokeratin-3 ranging from absent (few cells) to strong. More than 50% of cells expressed vimentin.

Conclusion: Limbal tissue retrieved from donor tissue that was organ-cultured for 3 weeks maintains a potential for cell renewal and growth compatible with clinical use for limbal allograft transplantation or cultured stem cell transplantation. However, cell growth potential is impaired by donor postmortem ischemia, which implies that tissue must be retrieved as soon as possible after donor death.