Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope

Purpose. To evaluate the morphology of endothelial monolayers, which have b een regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM). Methods, Material derived from each of the experimental g roups described in part I of this investigation was evaluated in the curren t study. Recipient corneas, denuded of their native endothelium by mechanic al, chemical, or physical debridement, were examined to assess the effectiv eness of each technique in killing and removing cells. Porcine or human don or corneal endothelial cells maintained in monolayer culture for up to 10 p assages then were seeded onto the denuded Descemet's membranes of recipient s in the absence or presence of fibroblast growth factor (FGF), The monolay ers thereby established were examined in the SEM, and the morphologic statu s of individual cells compared with that manifested in normal human donor c orneas maintained for 4 weeks in organ culture (reference control), Isolate d and cultured human keratocytes regrafted onto the denuded Descemet's memb ranes of recipient corneas served as nonendothelial control specimens. Tiss ue was processed for examination in the SEM according to standard technique s. Results. Each of the three methods used to strip recipient corneas of th eir native endothelium was effective and elicited no gross structural damag e to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers, The morphologic status of regra fted cells corresponded to that manifested in monolayer cultures before see ding, porcine ones always being more differentiated than their human counte rparts. Poorly differentiated human endothelial cells had a slender, elonga ted, fibroblast-like appearance, whereas more highly differentiated ones ma nifested broad, flat, polygonal profiles, Monolayers covered the entire cor neal surface and impinged to a variable degree onto the trabecular meshwork , at which juncture cells always assumed a less well-differentiated morphol ogy. FGF consistently effected an increase in differentiation status, and a s this became augmented, the capacity of monolayers to violate the corneal- trabecular meshwork border was correspondingly repressed. Seeded keratocyte s formed dense, multilayered sheaths, resembling retrocorneal membranes, ac ross the entire corneal surface, trabecular meshwork, and iris root. The su rface characteristics of the constituent cells were quite distinct from tho se manifested by endothelial cells, even the least well-differentiated ones . Conclusion. Regrafting of human corneal endothelial cells onto the denude d Descemet's membranes of recipients resulted in the formation of stable mo nolayers. Because the morphologic status of seeded cells closely mimicked t hat manifested in monolayer cultures before transplantation, it may be anti cipated that efforts to refine and optimize culturing conditions would yiel d improvements in this parameter after regrafting. If these expectations ca n be realized, then the possibility of successfully establishing a "new" an d functional endothelium on recipient corneas destined for clinical graftin g may well be brought to fruition in the not-too-distant future.