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

Abstract

Purpose: To develop a method for grafting endothelial cells isolated from organ-cultured adult human corneas onto the denuded Descemet's membrane of human recipients.

Methods: Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipient 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 seeding density of transplanted cells required for optimal results also was determined and the final numeric cell density achieved on recipient corneas after culturing for 7-20 days ascertained. The influence of incubating source cells with fibroblast growth factor (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of regrafted endothelium was assessed in 24-h perfusion experiments.

Results: The seeding of between 150,000 and 700,000 cells onto recipient corneas, followed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm2 and 1,850/mm2 in porcine and human lines, respectively. Recipient corneas were most effectively denuded of their own endothelium by freezing-and-thawing. The newly established endothelial monolayer remained stable for up to 20 days in organ culture (longest period monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect on their morphology. Only those recipient corneas that exhibited a well-differentiated monolayer of seeded endothelial cells underwent stromal deswelling near to physiologic levels.

Conclusion: A practical working model has been developed, whereby recipient corneas stripped of their own endothelium can be furnished with a "new," near-normal endothelium by appropriate manipulations of the seeded-cell population. This now paves the way for a realistic tackling of the problem of endothelial cell paucity in donor corneas destined for transplantation.