ISOLATION AND CHARACTERIZATION OF VASOFORMATIVE ENDOTHELIAL-CELLS FROM THE RAT AORTA

We describe here a modified nonenzymatic method for the isolation of r at aortic endothelial cells with vasoformative properties. Aortic ring s placed on plastic or gelatin-coated surfaces generated outgrowths pr imarily composed of endothelial cells. Prompt removal of aortic explan ts after endothelial migration minimized fibroblast contamination. How ever, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers. Thi s potential pitfall was avoided by weeding out fibroblasts with the ro unded tip of a bent glass pipette. Primary endothelial colonies free o f fibroblasts were segregated in cloning rings, trypsin-treated, and t ransferred to gelatin-coated dishes. Endothelial cells were cultured i n MCDB 131 growth medium containing 10% fetal bovine serum, endothelia l cell growth supplement, and heparin. Using this technique, pure endo thelial cell strains were obtained from single aortic rings. Confluent endothelial cells formed a contact-inhibited monolayer with typical c obblestone pattern. The endothelial cells were positive for Factor VII I-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplic ifolia-isolectin-B4. Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-P alade bodies and caveolae, and were connected by tight junctions. In a ddition, they reorganized into a network of microvascular cords and tu bes when overlaid with a second layer of collagen and formed microvasc ular sprouts in response to fibroblast-conditioned medium. This isolat ion procedure yields stable strains of vasoformative endothelial cells , which can be used to study aortic endothelium-related angiogenesis a nd its mechanisms.