A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries.

Our study indicates that when small pieces of lung or muscles of chest wall are cultured, erythrocytes and leukocytes (PMNs) leave the tissues first, followed by vascular endothelial cells (ECs). Fibroblasts and other mixed cells grow after 72 hr culture. The ECs can then be isolated avoiding mechanical and chemical injuries. The lung tissue is obtained from the peripheral surface and muscles from the chest. It is then cut into pieces and cultured with DMEM containing 20% fetal bovine serum. After 60 hr culture, the tissues are discarded. The flask contains only ECs and blood cells. Blood cells can be cleared out after the cells are subcultured once or twice. The primary cells and the subcultured cells cultured on gelatinized culture dish give the capillary-like structure. Cells cultured on untreated dishes have regular cobblestone morphology and junctional contacts. The isolated cells were not mesothelial cells because the cells did not react to antibody against cytokeratin 18, while mesothelial cells reacted strongly to the antibody. The cells can be isolated from the lung tissue without pleura. The primary microvascular ECs are also cultured on microcarriers (cytodex 3). Because both mechanical and proteolytic injuries are avoided, the cells may be more similar to cells in the in vivo state. There are no significant differences in PMN-endothelium adherence and monolayer responses to second messengers, platelet activating factor, and phospholipase A2 when pulmonary and muscular microvascular endothelial cells are compared.