Study Of Isolation, Culture And Identification Of Human Umbilical Vein Endothelial Cells In Vitro

【Objetive】To explore the method of isolation, culture and identification of human umbilical vein endothelial cells (HUVEC) in vitro and to provide experimental research model for mechanisms and therapeutics of vein graft restenosis after coronary artery bypass graft (CABG).【Methods】Ten fresh umbilical cords were collected from Tongji hospital affiliated Tongji Medical College of Huazhong University of Science and Technology. The two segments came from one umbilical cord vein were perfused with 0.25% trypsin and remained inside for 15min and 25min respectively. Then the digestion solution and PBS washes were collected to obtain endothelial cells (EC) for cell counting. The collected EC were cultured with M199. HUVEC were purified repeatedly from fibroblasts and blood system cells using differential adhesion method. About after a week, identify EC by the technique of immunohistochemistry staining of factorⅧ-associated antigen vWF. SPSS13.0 was used to do statistical analysis and draw HUVEC growth curve.【Results】The number of 25min group digested by 0.25% trypsin were large significantly compared with 15min group [(20.5±2.87)×105/ml vs (11.7±2.47)×105/ml, P<0.05]. It was effective to purify HUVEC using differential adhesion method. Primary EC attached to the bottom of culture plate about after 2h and adherenced completely after 48h. However, the cell survival rate of 15min group was more than 25min group after 48h. After 3 days in culture, EC grew in confluent monolayers as a typical cobblestone. The factorⅧ-associated antigen vWF were positive according to the immunofluorescencal method.【Conclusion】The digestion of umbilical cord vein using 0.25% trypsin for 15min is simple and successful and can provide EC research model for relative basic and clinical study.