New method for the isolation of endothelial cells from large vessels.

Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers. With the use of this method, the lumen of a bovine aorta was filled with wash medium and the outer surface of the sample was washed with alcohol for 30 seconds. Under a laminar flow hood, the inner surface of the sample was covered with filter paper. Collagenase type II was dripped onto the filter paper as a digestion enzyme. The digestion fluid was seeded in T25 flasks and fed with complete medium every 3 days. The isolated cells were characterized by markers such as CD31, von Willebrand factor, 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate acetylated low-density lipoprotein and angiogenesis behavior. The purity of endothelial cells was detected by flow cytometry to be of nearly 90% purity; these results were confirmed by immunostaining. Moreover, endothelial cells formed blood vessel-like tubes in a three-dimensional environment, which is specific dynamic behavior for endothelial cells. The new strategy presented in the current report enables isolation of a highly pure population of endothelial cells that can survive long-term culture without inducing an overgrowth of fibroblast cells.