An improved culture method for human umbilical vein endothelial cells

OBJECTIVE The study aimed to establish an efficient, reliable, and economical method for culturing primary human umbilical vein endothelial cells(HUVECs), so to provide an experimental approach for in vitro studies on vascular endothelial cells. METHODS Umbilical cords of healthy C-sectioned newborn infants were removed and digested with 0.1%collagenase I at room temperature to obtain vascular endothelial cells. The cells were incubated under 5% CO2at 37℃ and primary-cultured in M199 culture medium containing 10% fetal bovine serum, 2 mmol / L of glutamine, 50 μg / ml of heparin sodium, 10 u / mlb of FGF(basic fibro blast growth factor), 100 μ / ml of penicillin, and 50 μ / ml gentamicin. Morphological features of the primary and passaged cells were observed under an inverted microscope, and were identified by immunohistochemical methods. RESULTS A considerable amount of HUVECs were obtained by perfusion with 0.1% collagenase I and by using the improved culture medium. Large amount of cell clusters comprised of 3-4 endothelial cells were observed under an optical microscope. The cells began to adhere to the bottom of the flasks 4 h after seeding and reached confluency on around day 5. The primary cells were small triangular, round, or spindle in shape, distributed unevenly, and grew in non-overlapped clusters. On the other hand, the passaged cells were arranged in a flat and polygonal paving stone-like pattern, and the cells could reach confluency in about 3 days. Immunohistochemical analysis revealed that cytoplasm of the endothelial cells contained human factor VIII related-antigens. CONCLUSION Collagenase I perfusion is a feasible method for obtaining HUVECs; it is high in success rate and reliability. The improved medium is useful in promoting culturing of endothelial cells in vitro and proliferation of passaged cells, and has demonstrated to successfully establish in vitro models for studies on vascular endothelial cells.