Purpose: To study phenotypic variation in primary cultures of rat retinal pigment epithelium (RPE) and to correlate cell morphology with rates of binding and ingestion of rod outer segments (ROS).
Method: Replicate cultures were prepared using RPE cell sheets isolated with Dispase from Royal College of Surgeons normal (RCS rdy+ p+) and dystrophic (RCS p+) rats. Retinal pigment epithelial morphology was analyzed, and phagocytosis was assessed by fluorescence microscopy in cultures fixed at 2-hour intervals from 3 to 19 hours after continuous incubations with fluorescein isothiocyanate (FITC)-stained ROS.
Results: A wide range of RPE cell size, shape, and pigmentation was present at confluence; however, distinct morphologic subtypes were recognized, defined as types 1 to 3, and studied separately. In both normal and dystrophic cultures, the extent and rate of ROS binding varied with RPE phenotype. In normal cultures, highly spread pigmented binucleate cells (type 3) bound and rapidly ingested multiple ROS per cell starting at 3 hours and reached a peak at 9 hours. Lightly pigmented daughter cells (type 2) bound and ingested far fewer ROS per cell than did type 3 RPE, which had not divided. Patches of hexagonally packed cells with in vivo morphology (type 1) bound large numbers of ROS per cell only after prolonged (9- to 11-hour) incubations and ingested them synchronously. Comparison of normal versus dystrophic RPE subtypes 1 to 3 revealed the known ingestion defect in all three mutant phenotypes but indicated delayed ROS binding in type 2 and type 3 cells as well.
Conclusions: Kinetics of ROS binding and ingestion differ markedly among phenotypic variants of RPE cells typically found in primary cultures at confluence. Thus, accurate quantitation requires comparison of equivalent microscopic fields or like RPE subtypes, and the heterogeneous responses of various RPE subtypes should be considered when interpreting phagocytic data obtained from entire cultures at a particular time.