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... Show moreQuarter-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) mu m]. 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 alpha SMA. 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. Show less
Aim Studying cell migration of corneal endothelial cellsin vitrois challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on... Show moreAim Studying cell migration of corneal endothelial cellsin vitrois challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, we report a thermoresponsive culture technique designed to maintain cellular viability, and to reduce tissue handling in order to analyzein vitroendothelial cell migration from corneal grafts. Materials and Methods As a test tissue, fifteen Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts were used that were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2-3 weeks in a humidified atmosphere at 37 degrees C and 5% CO2. Results All grafts could be successfully cultured inside the thermoresponsive polymer solution for periods of up to 21 days. Using this system, cell migration could be assessed by light microscopy at fixed time intervals. At the end of the culture period, the gel could be removed from all grafts and immunohistochemistry analysis showed that endothelial cells were able to maintain confluence, viability, and junctional integrity. Some problems were encountered when using the thermoresponsive cell culture system. These were mostly structural inconsistencies during the sol-to-gel transition phase that resulted in the formation of tiny bubbles in the matrix. Additionally, areas with different viscosity resulted in optical distortions showing up as folds throughout the matrix which can persist even after several cycles of culture medium exchange. These effects had impact on the imaging quality but did not affect the viability of the explant tissue. Conclusion This study proves that temperature-reversible hydrogel is a very useful matrix for studyingin vitrocorneal endothelial cell migration from explant grafts and allows for subsequent biological investigation after gel removal. Show less
