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 tissu es first, followed by vascular endothelial cells (ECs). Fibroblasts an d other mixed cells grow after 72 hr culture. The ECs can then be isol ated avoiding mechanical and chemical injuries. The lung tissue is obt ained from the peripheral surface and muscles from the chest. It is th en cut into pieces and cultured with DMEM containing 20% fetal bovine serum. After 60 hr culture, the tissues are discarded. The flask conta ins only ECs and blood cells. Blood cells can be cleared out after the cells are subcultured once or twice. The primary cells and the subcul tured cells cultured on gelatinized culture dish give the capillary-li ke structure. Cells cultured on untreated dishes have regular cobblest one morphology and junctional contacts. The isolated cells were not me sothelial cells because the cells did not react to antibody against cy tokeratin 18, while mesothelial cells reacted strongly to the antibody . The cells can be isolated from the lung tissue without pleura. The p rimary microvascular ECs are also cultured on microcarriers (cytodex 3 ). Because both mechanical and proteolytic injuries are avoided, the c ells may be more similar to cells in the in vivo state. There are no s ignificant differences in PMN-endothelium adherence and monolayer resp onses to second messengers, platelet activating factor, and phospholip ase A(2) when pulmonary and muscular microvascular endothelial cells a re compared. (C) 1995 Academic Press, Inc.