Abstract: Objective To establish the convenient and efficient cultivation of HUVECs in vitro，so as to provide good seed cells for tissue engineering vascular grafts and related basic medical study. Methods 0.25%Trypsin perfusion method was used to isolate HUVECs，then cultivated in endothelial cell medium（ECM），identified the bionomics of HUVECs by checking Von Willebrand factor (VWF)、CD144 through immunocytochemical method and RT-PCR. HUVECs were suspended in cryopreserving solution containing with 10% glycerine and cryopreserved in liquid nitrogen for 4 weeks. The expression of CD144 and VWF of post-thawed cells were evaluated by Western blot. Results HUVECs grew to monolayer after cultivation for 2~3 days. They presented fusiform and appeared like Whirlpool under the light microscope, and presented lumens structure by passage，highly expressed VWF and CD144. The post-thawed cells maintained high proliferational ability and highly expressed VWF and CD144.Conclusion A large number of high purified HUVECs could be acquired by digestion of trypsin and cultivation of ECM,which could be good source cells for related study.

Key words: umbilical vein, endothelial cell, cell cryopreserving, tissue engineering