The Initial Exploration Method Of Vascular Endothelial And Smooth Muscle Cells Co-cultured Vascular Tissue Engineering Construction

Objective: To evaluate the methods of primary co-culture.To Exploration the method of vascular endothelial cells (EC) and smooth muscle cells (SMC)co-cultured and to exploration its culture, amplified conditions groping for construction of TEBVs.Materials and Methods: At frist,we collected fresh umbilical cord in May 2007 to February 2008 from Shanxi Medical University affiliated first hospital department of obstetrics and gynecology and Shanxi Province People's Hospital department of obstetrics and gynecology under sterile conditions.Then peeling umbilical cord arteriovenous carefully by trypsin digestion , collagenase digestion, pancreatin and collagenase digestion method, ROSS method by which endothelial cells and smooth muscle cells co-cultured and amplified cells with 20% fetal calf serum containing DMEM medium culture,.Using inverted phase contrast microscope, immunofluorescence method, immunohistochemistry method endothelial cells and smooth muscle cells were identified. To freeze and resus co-cultured cells which provide guidance for further PGA stent implanted cells and construction of TEBVs .Results: After repeated attempts, using trypsin digestion method,we found that cell growth is scarce in the inverted microscope; by collagenase digestion, pancreatin and collagenase digestion method in the inverted phase microscope we also found that cell growth is scarce . In the inverted microscope cells eruption can be observed in 5-7 days by using ROSS method.Then after the generations a number of vascular endothelial and smooth muscle cells can be cultivating which showed paving stones endothelial cells, smooth muscle spindle-like mixed growth characteristics. Immunofluorescence chemical detection and Immunohistochemistry showedⅧfactor endothelial cells, smooth muscle cellsα-actin positive. The cell proliferation can be reached for 8×106～10×106 in the 3-5 timesConclusion: We can get the conclusions from the exploration(1) Cord blood endothelial cells and smooth muscle cells co-cultured can be succeed in the exploration.(2) Trypsin digestion, collagenase digestion, pancreatin and collagenase digestion cells co-cultured experimental results is not satisfactory. And specific enzymatic digestion and the extent and effectiveness of the mechanism should be further explored.(3) The endothelial cells, smooth muscle cells can be obtain and observed by using ROSS method. The cells were co-cultured in vitro which has sufficient quantities for construction of TEBVs(4) There is good workability in the clinical experiment which provide guidance for further PGA stent implanted cells and construction of TEBVs .