The isolation and culture of endothelial cells and pericytes from the bovine retinal microvasculature: a comparative study with large vessel vascular cells

Abstract

Endothelial cells (BREC) and pericytes (BRP) were isolated from the bovine retinal microvasculature. These cells were first identified by morphological criteria and by their differential staining for Factor VIII related antigen. BREC and BRP responded differently to a number of experimental parameters in vitro; for example, the plating efficiency of BREC was enhanced by the use of a gelatin substratum and medium conditioned by either endothelial cells or pericytes; oxygen tension had no effect. In contrast, the plating efficiency of BRP was only enhanced by low oxygen tension. Conditioned media also stimulated the proliferation of BREC, but not that of BRP. The saturation density reached by BREC was dependent on the initial plating density while BRP plated at different initial densities reached the same final density. The in vitro behavior of the retinal microvascular cells was also compared to that of large vessel (aorta) endothelial cells (BAEC) and smooth muscle cells (SMC). Aortic and retinal endothelial cells showed similar morphology and behavior. When initially plated as a homogeneous cell suspension within a collagen matrix, both BREC and BAEC self-associated to form three-dimensional meshworks; this morphogenesis was accomplished by cell migration and did not involve cell proliferation. By contrast, BRP and SMC divided and remained homogeneously distributed when plated within a collagen gel matrix. BRP and SMC did, however, behave differently when plated on the surface of a collagen gel; SMC migrated extensively into the gel while BRP remained confined to the gel surface. BRP grown on any substratum began to retract upon themselves shortly after confluence, producing characteristic nodules interconnected by cellular strands. BRP and SMC were able to contract a collagen gel substratum, while retinal and aortic endothelial cells were unable to do so. These results provide new means for the in vitro characterization of endothelial cells, smooth muscle cells and pericytes.