Separation of bovine pigmented ciliary epithelial cells by density gradient and further characterization in culture

Abstract

Dissociated pigmented epithelial (PE) cells from ciliary processes (CP) of bovine eyes were separated by isopycnic centrifugation in metrizamide gradients and further cultivated. The level of cellular contamination during separation of PE by non-pigmented epithelial (NPE) cells was estimated to be 5–10% and by non-epithelial cells (i.e. endothelial, fibroblasts and blood cells) less than 1%. Bovine PE cells were found to have different serum requirements than NPE to grow in vitro. PE cells were actively stimulated to proliferate in the presence of fetal calf serum for a few generations. Short-term culture of NPE was only obtained in media containing a low concentration of l-tyrosine and no serum. Two morphological markers, namely desmosomes and intermediate-sized filaments (IF) of the vimentin type, were found by immunolabeling to be coexpressed in cultured PE and were used as criteria to define the epithelial identity of these cells. The present study was unable to detect any tyrosinase activity in cultured PE cells.