Simplified methods for consistent and selective culture of bovine retinal endothelial cells and pericytes

Different matrix components, in combination with various media and serum supplements, were evaluated for their ability to promote selectively the growth of bovine retinal endothelial cells in primary culture. The optimal setting for the selective growth of retinal endothelial cells was a fibronectin/hyaluronic acid matrix, Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% pooled human serum and 100 micrograms/ml heparin. These conditions consistently yielded virtually homogeneous cultures of endothelial cells, assessed using specific endothelial markers. Thus obtained, the retinal endothelial cells could be subcultured and maintained in phenotypically stable long-term serial cultivation. Homogeneous cultures of retinal pericytes were obtained when microvessel isolates were seeded to uncoated or gelatin-coated culture dishes and grown in DMEM supplemented with 20% fetal bovine serum. The retinal pericytes could also be subcultured and cultivated for numerous populations doublings. Additionally, observations from this study suggest that two populations of pericytes may be obtained in culture and distinguished on the basis of their relative size and antigenic properties.