A NEW WOUND-HEALING MODEL OF RETINAL-PIGMENT EPITHELIAL-CELLS IN SHEET CULTURE

Purpose. To evaluate some RPE cell functions, such as wound healing, i n a preparation more similar to in situ conditions, we developed a met hod 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 p romote spontaneous separation of the RPE sheet from the retina. After several days of incubation, the cultured sheets of RPE cells were exam ined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diam eter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%). Results. The R PE cells cultured in sheets retained their in situ features, including microvilli, tight junctions and gap junctions, and the distribution o f actin and cytokeratin filaments. A wound was noted to close with res toration of a polygonal configuration. The rate of wound closure depen ded on serum concentration in the culture medium; when supplemented wi th 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 woun d healing or phagocytosis.