Culture and Isolation of Human Umbilical Vein Endothelial Cells in Vitro

Objective:To establish an effective method to isolate,culture and identify human umbilical vein endothelial cells(HUVEC) in vitro.Methods:Trypsin perfusion method was used to isolate HUVEC from fresh umbilical cord,and then the cells were cultivated in M199 with 20% fetal bovine serum.After the collected endothelial cells were cultured and passaged,the cells were observed under inverted phase contrast microscope.The endothelial specific surface marker was assessed by fluorescence activated cell sorter(FACS) analysis.The cells were also identified by the test of up take of acety-lated low density lipoprotein(Ac-LDL).Results:The obtained HUVECs were small-round and clustering.The cells began adhere to the plate in two hours,and attached cells were cord-like structure.After 7 days culture the cells grow into large number and can be confluences a monolayer,which displayed cobble-stone morphology.These cells express endothelial specific markers,including VE-cadherin,vWF and CD31.The function of the cells was identified by up take of Ac-LDL.Conclusion:A large number of high purified endothelial cells could be acquired by digestion of trypsin.HUVEC can be proliferated and passaged successfully in vitro.