Retinal pigment epithelium wound closure in vitro. Pharmacologic inhibition

Early-passage bovine retinal pigment epithelium (RPE) cells were grown to confluence in 24-well plates, and a central defect was created mechanically in the monolayer within quadruplicate wells, sequentially over 9 days. Closure of the wounded area occurred by single-cell migration of elongated RPE cells from the edge of the wound and subsequent cell proliferation. Ten days after wounding, the cultures were fixed, stained, and photographed, and the residual wound area was quantified by computerized planimetry. Cell counts of unfixed cultures were determined with a Coulter counter. Wound closure was complete after 10 days. Using this technique, we assessed the response of RPE to various concentrations of 5-fluorouracil (5-Fu), colchicine (COL), and cytochalasin-B (CYT-B). 5-Fu (10 micrograms/ml) and COL (0.1 and 1 microgram/ml) inhibited migration and proliferation of RPE cells. CYT-B (5 micrograms/ml) inhibited migration. This model allows in vitro study of the response of RPE cells after loss of contact inhibition. The technique provides a quantitative model for assessing the dynamic capabilities of RPE cells in response to a localized mechanical defect and for assessing the pharmacologic modulation of these responses.