HARVEST AND STORAGE OF ADULT HUMAN RETINAL-PIGMENT EPITHELIAL SHEETS

Purpose. To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RFE) cells. Methods. Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 5 0% gelatin containing 300 mM sucrose and stored at 4 degrees C. The vi ability of the cells, as well as their ability to proliferate in vitro , was studied for 96 hours after harvesting. Light microscopy (LM), tr ansmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclud e contamination. Results. LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connect ions. The initial viability of intact RPE sheets was 86%, with a progr essive decline in viability with increased storage time. Cells harvest ed within 24 hours after death maintained greater viability than chose harvested after 24 hours (p < 0.05), Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vit ro. Conclusion. Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplan tation if harvested within 24 hours of death and maintained 82% viabil ity for as long as 48 hours if stored at 4 degrees C.