Transplantation of adult human or porcine corneal endothelial cells onto human recipients in vitro. Part I: Cell culturing and transplantation procedure

Purpose. To develop a method for grafting endothelial cells isolated from o rgan-cultured adult human corneas onto the denuded Descemet's membrane of h uman recipients. Methods. Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipien t corneas were stripped of their own endothelium by one of three different methods (mechanical, chemical, or physical) and the completeness of removal assessed after vital staining. The utility of each method was evaluated by monitoring the quality of attachment of the seeded-cell population. The se eding density of transplanted cells required for optimal results also was d etermined and the final numeric cell density achieved on recipient corneas after culturing for 7-20 days ascertained. The influence of incubating sour ce cells with fibroblast growth factor (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of re grafted endothelium was assessed in 24-h perfusion experiments. Results. Th e seeding of between 150,000 and 700,000 cells onto recipient corneas, foll owed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm(2) and 1,850/mm(2) in porcine and human lines, respectively. Recipient corneas were most effectively denuded of the ir own endothelium by freezing-and-thawing. The newly established endotheli al monolayer remained stable for up to 20 days in organ culture (longest pe riod monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect o n their morphology. Only those recipient corneas that exhibited a well-diff erentiated monolayer of seeded endothelial cells underwent stromal deswelli ng near to physiologic levels. Conclusion, A practical working model has be en developed, whereby recipient corneas stripped of their own endothelium c an be furnished with a "new," near-normal endothelium by appropriate manipu lations of the seeded-cell population. This now paves the way for a realist ic tackling of the problem of endothelial cell paucity in donor corneas des tined for transplantation.