An improved method for stem cell derivation from human great saphenous vein / 生物工程学报

ABSTRACT

The objectiue was to explore how to improve stem cell derivation from human great saphenous vein. After the saphenous vein was cut into small pieces, the cells of the vessel wall were obtained by tissue adherent method and digestion with type Ⅱ collagenase. The morphological changes of blood vessel wall were observed under inverted microscope. The survival of vascular wall cells was assessed by trypan blue staining. Stem cells doubly positive for CD34 and CD117 were sorted out by immunofluorescent staining and flow cytometry. The cells obtained by tissue adherence method exhibited signs of fibrotic changes and aging at the third passage (P3), while the cells extracted by enzymatic digestion still showed colony-like growth. Survival rates of these two groups of cells were (91.7±1.2)% and (97.2±0.7)%, (P=0.005). The results of flow cytometry showed that the positive rates of CD34 and CD117 double positive cells in these two groups were (0.16± 0.05)% and (0.44±0.07)%, respectively, with statistical significance (P=0.005). Immunofluorescent staining showed that the positive rates of double positive stem cells in the two groups were (89.41±2.06)% and (94.03±1.83)%, P<0.05 one week after the sorted stem cells were cultured. The positive rates of CD31, VEGF2 and SMA in the stem cells determined by flow cytometry were (0.12±0.01)%, (0.19±0.02)% and (0.45±0.01)%, respectively, which were not statistically different from those of the control groups. This could rule out substantial inclusion of mature endothelial cells and smooth muscle cells. Tube forming experiment confirmed that these vascular stem cells had developmental plasticity. More viable and morphologically healthy vascular stem cells can be derived by enzymatic digestion. These cells can be widely used in clinical and basic research.