Endothelial cells from the rat aorta in culture

Kim, Joo Young

Abstract

Endothelial cells were isolated from the intima of Sprague-Dawley rat aorta with 0.2% collagenase solution and cultured in MCDB 131 medium. All endothelial cells regardless of passages and the freezing-thawing were cultured in M-199, alpha-MEM, and MCDB 131 media. Thereafter, the morphological findings of the cells were observed under light and electron microscopes. The activities of nitric oxide synthetase of endothelial cells were investigated with NADPH-diphorase staining. Production of von Willebrand factor, cytoskeletal proteins, and extracellular matrix proteins in endothelial cells was identified with PAP. By treating with 0.2% collagenase solution for 30 minutes, and then incubating the cells in MCDB 131 medium for 3 days, only endothelial cells could be separated. Endothelial cells formed a monolayer with typical cobblestone pattern (polygonal-shaped morphology) 7-10 days after seeding. The growth patterns of endothelial cells were similar regardless of their cultured states (primary cultured cells, subcultured or thawed). The existence of nitric oxide synthetase, von Willebrand factor, beta-actin, fibronectin, and laminin in endothelial cells was confirmed. However, above-mentioned materials were quantitatively less in cells grown with MCDB 131 medium compared to those cultured with M-199 and alpha-MEM. Weibel-Palade bodies were observed by transmission electron microscopy. Cultured cells in MCDB 131 medium compared to those cultured with M-199 and alpha-MEM were relatively fewer in number of rough endoplasmic reticulum, mitochondria, free ribosome, vesicle, and liposome and especially showed the absence of basement membrane. Taken together, the cultivation of endothelial cells from the intima of rat aorta was possible. Endothelial cells in vitro synthesized various materials such as von Willebrand factor etc. and had characteristic organelles (Weibel-Palade body etc.). It suggested that it would be more advantageous to culture the endothelial cells in M-199 with 20% fetal bovine serum and alpha-MEM with 10% bovine calf serum rather than in MCDB 131 endothelial cell culture medium only with 0.7% dialyzed serum to maintain characteristics of endothelial cells in vivo