Expression of proliferating cell nuclear antigen in corneas kept in long term culture

To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture.

Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope.

Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea.

Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.