The influence of preparation and storage time on endothelial cells in Quarter–Descemet membrane endothelial keratoplasty (Quarter–DMEK) grafts in vitro

Quarter–Descemet membrane endothelial keratoplasty (Quarter-DMEK) has been introduced as a modification of the standard DMEK technique to increase the pool of endothelial grafts. In this study, we evaluated in vitro changes in endothelial cell distribution, viability and morphology of Quarter–DMEK grafts when stored in organ-culture medium. Quarter–DMEK grafts were prepared from 5 corneas and stored in organ-culture medium for 4, 7 and 11 days. Endothelial cell re-distribution was investigated by light microscopy, cell viability by a Calcein-AM assay, and expression of endothelial and non-endothelial markers by immunohistochemistry. Three standard DMEK-grafts were used as controls. After preparation, all Quarter–DMEK grafts showed a band with no viable endothelial cells along the radial cut graft edges [average width 190 (± 20) µm]. Endothelial cell density in the central graft area decreased by 12%, 23% and 26% after 4, 7, and 11 days of storage, respectively. At the same time, empty bands along the cut edges were re-populated and some cells migrated to the stromal side of the Descemet membrane (DM). These cells showed an altered phenotype, as indicated by expression of migration marker CD73 and fibroblast marker αSMA. Majority of migration occurred within the first 4 days of storage. Our data suggest that endothelial cells on Quarter–DMEK grafts re-distribute during organ-culture storage to re-populate preparation-induced empty bands and after re-distribution, cells may show further migration to the stromal DM side during storage.