Application of collagenase perfusion and digestion techniques in the primary culture of endothelial cells from human umbilical vein in vitro

Objective To investigate the primary culture method for the human umbilical vein endothelial cells (HUVECs). Methods Endothelial cells in human umbilical vein were got by the method of 0.1 % Ⅰtype collagenase perfusion and digestion techniques for about 10 min,and then cultured with M199 mixed culture medium in Incubator with 5% CO2 at 37 ℃,0.25% trypsin were used for cell transfer of culture. The cultured endothelial cells were identified by cell morphology,cell micro-structure,Ⅷ factor antigen immunohistochemitry. Results The primary cultured HUVECs could attached to flask completely after 24 hours,which arrayed like pitching stone under microscopy.By transmission electron microscopy,HUVECs were found to have distinctive Weibel-Palade bodies in the cytoplasm and a lot of microvilli in the cell surface. Immunohistochemitry and immmunofluorescent staining by Ⅷ factor were all positive reaction. All of these were the typical features of primary cultured HUVECs. Within five generations,the morphology of cells was not obviously changed. Conclusions High purity HUVECs can be obtained by the technique of collagenase perfusion and digestion,which further can be passaged successfully in vitro,and cells within five generations can be used in research.