Purpose.To evaluate some RPE cell functions, such as wound healing, in a preparation more similar toin situconditions, we developed a method to obtain and culture retinal pigment epithelial (RPE) cells as a sheet. And we assessed the effects of fetal bovine serum (FBS) on the rate of RPE wound healing.Methods.We prepared RPE sheet cultures by incubating rat eyes in 0.1% proteinase K for 13 min, peeling away the neural retina RPE complex, and then incubating the tissue for 1 h to promote spontaneous separation of the RPE sheet from the retina. A Iter several days of incubation, the cultured sheets of RPE cells were examined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diameter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%).Results.The RPE cells cultured in sheets retained theirin situleatures. including microvilli, tight junctions and gap junctions, and the distribution of actin and cytokeratin filaments. A wound was noted to close with restoration of a polygonal configuration. The rate of wound closure depended on serum concentration in the culture medium; when supplemented with 10% fetal bovine serum, wound closure was complete in approximately 40 h.Conclusions.The RPE sheet-culture technique we developed thus provides a suitable model for studying such RPE cell functions as wound healing or phagocytosis. Curr Eye Res. 15: 714-718, 1996