Primary culture of microvascular endothelial cells from bovine retina: selective growth using fibronectin coated substrate and plasma derived serum

Abstract

To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type, which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels were plated on tissue culture grade plastic in fetal bovine serum.