Culture of human retinal pigment epithelial cells from peripheral scleral flap biopsies

PURPOSE: We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture. METHODS: Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged. RESULTS: There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged. CONCLUSIONS: These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.