Purpose: To investigate the feasibility of transplanting untransformed human corneal endothelial cells as a treatment strategy and possible alternative for penetrating keratoplasty by growing donor cells in culture and then transplanting them to denuded Descemet's membrane of recipient corneas.
Methods: Corneas from adult donors (50-80 years old) were obtained from eye banks. To grow corneal endothelial cells, Descemet's membrane with associated cells was dissected from the stroma. Endothelial cells were released by ethylenediaminetetraacetic acid treatment, grown in medium containing multiple growth factors, and identified as being of endothelial origin by morphology and by reverse-transcription polymerase chain reaction for keratin 12 and collagen type VIII. In transplantation experiments, cultured cells were seeded onto denuded Descemet's membrane of a second donor cornea at 5 x 10(5) cells/mL. The recipient cornea was incubated in organ culture for as long as 2 weeks. The morphology and ultrastructure of the endothelium were evaluated 7 and 14 days after transplantation by transmission electron microscopy, and by immunolocalization of zonula occludins-1 (ZO-1). Endothelial cell density was calculated in transplants by counting ZO-1-stained cells.
Results: Corneal endothelial cells cultured from adult donors consistently grew well in culture medium. Cells were identified as corneal endothelium by characteristic morphology and messenger RNA expression. Morphologic and ultrastructural studies of corneas containing transplanted endothelial cells demonstrated that with time there was an increase in endothelial cell-Descemet's membrane adhesion, in the extent of cell-cell contacts and lateral interdigitation, and in formation of a single cell layer. ZO-1 staining revealed tight junction formation similar to that of corneas in vivo. Mean endothelial cell density in transplanted corneas was 1,895 cells/mm(2) (range, 1,503-2,159 cells/mm(2) ).
Conclusion: Untransformed adult human corneal endothelial cells can be efficiently and consistently cultured and transplanted onto denuded Descemet's membrane. Transplanted cells in organ culture exhibit morphologic characteristics and cell densities similar to corneal endothelial cells in vivo. These results provide evidence for the feasibility of developing methods for in vivo transplantation of untransformed corneal endothelial cells cultured from adult donor tissue.