Isolation and biochemical characterization of frog retinal pigment epithelium cells.

The structure and function of the photoreceptor cell depends on the renewal of its outer segment. Phagocytosis of the rod outer segments by RPE is an essential part of the renewal process. Several methods have been reported in order to isolate RPE cells; however, the isolated cells are heavily contaminated by other cell types, mainly erythrocytes and rod outer segments. The primary aim of this study was the isolation of pure and viable frog RPE cells. Cells were dissociated in a calcium-free Krebs bicarbonate medium and purified by centrifugation in a ficoll density gradient. Viability of the purified cells assessed by trypan blue dye exclusion was 95%. The metabolic activity of the cells was tested by several parameters: RPE cells consume oxygen at a rate of 11.5 ngatoms/min/mg protein, transport and metabolize 14C-glucose by a sodium dependent mechanism, and are able as well to accumulate 14C-leucine and incorporate it into proteins. Results obtained in this study indicate that our isolation procedure yields a more intact preparation of RPE than those described previously; hence, it may be helpful in elucidating the biochemical and metabolic parameters involved in pigment epithelium physiology.