Isolation and Culture of Capillary Endothelial Cells from the Eel, Anguilla rostrata

Abstract

There has been little development of endothelial cell cultures from vertebrates other than mammals. In this report the isolation of capillary endothelial cells from the rete mirabile of the eel, Anguilla rostrata, is described. The cells are isolated with collagenase H and dispase II. The cells are plated into fibronectin–hyaluronic acid coated flasks. The culture medium is M199 with Earle's salts supplemented with NaCl, HEPES, NaHCO3, glutamine, pyruvate, heparin, antibiotics, endothelial cell growth supplement, and 20% serum. Cultures are incubated at 25°C in humidified air. The rete mirabile contains pericytes in addition to endothelial cells. Variations in plating time, serum concentrations, and growth matrices were tried to separate the two cell types. The total number of endothelial cells and the ratio of endothelial cells to pericytes are the most important factors in obtaining pure cultures of capillary endothelial cells. Endothelial cells are isolated also from the endocardium, bulbus arteriosus, and large vessels. The initial isolates usually take 3–6 weeks to grow to confluence with subcultures taking about 2 weeks to confluence.