Cultivation and in vitro identification of endothelial cells from human umbilical vein

Objective To explore the primary culture method of human vascular endothelial cells(ECs) from umbilical vein,enhance the success rate of separating and culturing ECs in vitro,establish the model of human vascular ECs,and provide an experimental method for research of ECs.Methods The umbilical veins in human umbilical cords,20 cm long,were fixed and filled the digestive fluids,0.2% collagenenaseⅡ in one cord and the complex enzyme of 0.25% trypsin and 0.1% collagenaseⅠin an other,and the digestive results were compared.All the cells were collected and cultured in fluid with 10 mg/mL VEGF,their procreation and generation were observed,the morphologic characteristics of ECs were observed with light microscopy and immunohistochemistry under inverted microscope,and the cell cycle was observed by flow cytometer.Results Enough ECs were obtained from both digestive liquids,and collagenenaseⅡ was better than complex enzyme,the effective digesting time was 13 minutes.the culturing cells grew on the wall appeared after 4-5 hours and the monolayer cells were formed after one week.Ⅷ factor in the ECs showed positive reaction by immunohistochemistry.Cell cycle revealed that 50.6% cells were at stage of G0/G1.Conclusion The perfusion with collagenenaseⅡ and the complex enzyme and collagenaseⅠis an effective method to gain ECs,and collagenenase is the digestive fluid for primary choice,and can successfully establish the model for research of ECs.