HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE

TLDR

Human umbilical cord vein endothelial cells were isolated by collagenase digestion and cultured in Medium 199 supplemented with 20 % fetal calf serum. Cultured endothelial cells formed confluent monolayers within a week, displayed characteristic Weibel‑Palade bodies, exhibited region‑dependent DNA synthesis that increased after denudation, and proved useful for studying endothelial regeneration.

Abstract

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [(3)H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [(3)H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [(3)H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.