Limbal progenitor/stem cells are responsible for the renewal of the corneal epithelium, and reside in the basal layers of the sclerocorneal limbus. These cells are characterized as an oligopotent progenitor cell population, presenting a high nucleus-to-cytoplasm ratio with a slow cell cycle, high proliferative potential and a notable capacity to self-renew, with asymmetric division.
Human limbal epithelial stem cells were cultured on murine 3T3-J2 fibroblast feeder layers. Co-cultures were fed with epithelial medium on the third day of culture and then every other day thereafter. The primary limbal epithelial cultures were sub-cultured after removal of the 3T3 fibroblasts, by incubation with a 0.02% EDTA solution for 30 seconds at room temperature. The remaining limbal epithelial cells were trypsinized and plated at a density of 0.6x104 cells/cm2, in wells pre-plated with inactivated 3T3 fibroblasts. The sub-cultures (passaged cells) were maintained just as the primary cultures were maintained.