[Culture of retinal capillary endothelial cells]

Abstract

The culture of retinal capillary endothelial cells involves certain problems concerning contamination by pericytes, the maintenance of differentiation and the duration of culture viability. A procedure for the isolation and culture of capillary endothelial cells from bovine retina which overcomes these difficulties, is described. Microvessel fragments isolated by mechanical dispersion and filtration techniques adhere strongly to dishes coated with extracellular matrix produced by bovine corneal endothelial cells. The first migrating cells emerge from the original microvessel fragments two days after plating. This technique and subsequent cloning provides migrating and proliferating cells derived only from the retinal capillaries and uncontaminated by other cell types such as pericytes. Endothelial cells were grown on gelatin coated dishes in a serum supplemented medium (10% calf serum). Cell proliferation was significantly enhanced by the addition of basic fibroblast growth factor (1 ng/ml) to the culture medium. In these culture conditions, retinal capillary endothelial cells can be repeatedly passing without the loss of their principal morphological characteristics and some of the differentiated properties of endothelial cells. Primary cultures and subcultures, at least up to the 8th passage, formed a monolayer of small, elongated, tightly-packed, contact inhibited cells which expressed Factor VIII-related antigen. Ultrastructural examination by transmission electron microscopy of confluent bovine retinal capillary endothelial cells showed many tight junctions and Webel Palade granules. These studies provide new means for the isolation and culture of retinal capillary endothelial cells and presents evidence for growth factor requirements for the ability of cells to be repeatedly passing.