An improved method of culturing human umbilical vein endothelial cells

    LI Qinª²Shan, FENG Zanª²Jie, LIU Yang, XU Haiª²Yan, QIAN Minª²Zhang

    Department of Biochemistry, Zunyi Medical College, Zunyi 563003, China

    ¡¾Abstract¡¿ AIM: To establish a stable method to isolate and culture human umbilical vein endothelial cells (hUVEC) in vitro. METHODS: Endothelial cells of vein from newborn umbilical cord were successfully isolated using a combination of collagenase and trypsin (1¡Ã1). Cells were not digested with trypsin until the cells reached confluence over 80%. The composition of culture medium was simplified by not adding vascular endothelial cell growth factor and heparin. The cultured cells were verified as hUVEC by morphology and immunofluorescence staining. RESULTS: The endothelial cells initially spread on the bottom of the dishes in 4 h, then coalesced and grew to form confluent monolayers of polygonal cells within 7-10 d. The cultured cells had a cobblestone appearance with a strict monolayer growth and contact inhibition. The purity of endothelial cells was more than 95% identified by immunocytochemistry staining for ¢ø:Ag and KDR. CONCLUSION: Method of injection with mixed Enzyme solution is an optimized protocol which is reliable and of high achievement ratio. Cells harvested with this protocol can be used as models on research of vascular endothelial cells in vitro. ......