Abstract:

BACKGROUND: Currently, there is not a standard method for in vitro culture and large scale amplification of umbilical cord blood mesenchymal stem cells (UCB-MSCs).
OBJECTIVE: To investigate the isolation, purification and culture of UCB-MSCs in vitro, and to detect its surface marker variation.
METHODS: The monocytes were harvested from UCB using 1.077 g/cm3 lymphocytes separating solution and density gradient centrifugation, followed by incubation in an incubator containing 5%CO₂ at 37 ℃. The cell morphological changes were observed at different time points and the expression of surface marker was detected using flow cytometry. 
RESULTS AND CONCLUSION: The monocytes isolated from the UCB grew initially into numerous hematopoietic cell clones, most of which were granulocyte/macrophage colony-forming units and burst forming unit-erithroid, increasing by (37.1±2.3) and (10.4±1.7), respectively. Switzerland staining showed most of them were granulocyte clones (80.1±85.2)%, next was erythroid clones (14.2±1.8)%. At 7 days after culture, some shuttle fibroblast-like cells and flat osteogenic-like cell spread the whole plastic well. At 14 days after culture, flow cytometry showed CD38⁺ cells accounted for 1.64%, and CD34⁺ /CD38⁺ cells accounted for 1.71%, and CD34⁺ /CD38^- were 0.55%. PI⁺ and Annexin-V⁺ cells accounted for 0.05% and 0.18% respectively. At 21 days after culture, CD38  ⁺ , CD34⁺ /CD38⁺ and CD34⁺ /CD38^- cells were 74.32%, 1.61%, and 0.24%. The results reveled that UCB-MSCs can be isolated and cultured in vitro.