Different Enzyme Digestion in Isolation and Culture of Human Umbilical Vein Endothelial Cells

Objective To compare the efficacy of three enzyme digestion to isolate the primary endothelial cells from human umbilical vein in vitro. Methods Trypsin (0.125%, 0.25%) or 0.1% type Ⅰ collagenase was respectively infused into 20-centimeter human umbilical vein and remained inside for 6, 8, 10, 12, 15, 20 min, then the digestion solution and PBS washes were collected to obtain endothelial cells. After the collected endothial cells were cultured with routine method, the cells were observed under inverted phase contrast microscope, their factor Ⅷ related antigen was detected by immunofluorescence method and their ability to uptake DiI-acLDL was examined. Results The optimal digestion time by 0.125% trypsin, 0.25% trypsin or 0.1% type Ⅰ collagenase for primary culture of human umbilical vein endothelial cell was 15 min, 12 min and 15 min respectively. The passaged cells displayed the typical morph of endothelial cells, factor Ⅷ related antigen-positive and the ability to uptake DiI-acLDL. Conclusion Enzyme digestion is simple and reliable to isolate primary endothelial cells from human umbilical vein. The digestion time should be controlled strictly to minimize the cell injury. 0.1% type Ⅰ collagenase may be the best choice to isolate the endothelial cells.