The long term objective of this study is to determine whether corneal endothelium, harvested from a normal cornea and subsequently amplified in quantity using the technique of cell culture, can be used as a reliable replacement endothelium on donor corneas subsequently employed to restore vision by transplantation. Previous and ongoing studies in our laboratory indicate that cultured allogeneic corneal endothelium offers promise as a reliable replacement. An endothelial pump mechanism functioning in concert with an endothelial permeability barrier act to maintain cornea stromal deturgescence and corneal transparency. Conditions required to insure effective pump and barrier function for cultured endothelial cell relined cornea transplants have not been established and, therefore, form the central focus of the proposed studies. We expect that cell density, regulated by growth factors as well as cell-substratum interactions in culture and during the relining process, are critical to successful, long-term maintenance of differentiated endothelial cell function in allogeneic transplants. Differentiated endothelial cell function in cultured cells and in relined corneas, relative to native corneal endothelium, will be assessed by monitoring: 1) expression and localization of active Na pump sites by ligand binding with [3H]ouabain; 2) Na+/H+ antiport and HCO3-Na+ symport activities that, together with the Na pump sites, are essential for HCO3- secretion. Transporter activities will be measured in cells loaded with pH-sensitive fluorescent dyes; 3) endothelial barrier function as revealed by transmural extracellular tracer flux and analyses of junctional cytoarchitecture. Data arising from these studies will contribute to our understanding of basic corneal endothelial cell biology as well as having direct clinical relevance regarding the use of cultured corneal endothelial cells either as replacements or as augmenting cells on corneal transplants.
