Abstract:

BACKGROUND: A large number of mononuclear cells adhere to the centrifugation tube in the process of densitygradient centrifugation and isolation for the umbilical cord blood mononuclear cells (UCB-MNCs), the correlation between these adherent cells and the success rate of culturing UCB-MNCs in vitro remains unclear.
OBJECTIVE: To compare the achievement ratio of cultivating UCB-MNCs in vitro by four different methods and to elucidate the influence of the cells adhering to the centrifugation tube on the cultivating rate of UCB-MNCs in vitro.
METHODS: A total of 36 samples were obtained from umbilical cord blood of term deliveries and were divided randomly into four groups, with 9 samples in each. A group: methylcellulose isolation method; B group: density gradient centrifugation isolation method; C group: methylcellulose isolation method plus density gradient centrifugation; D group: methylcellulose isolation method plus modified density gradient centrifugation. Mononuclear cells were separated by the above four methods within 6 hours after collection, seeded in 25 cm2 culture flask at a concentration of 1×10⁹/L - 2×10⁹/L and suspended in low glucose-DMEM cell culture medium supplemented with 0.1 volume fraction of fetal bovine serum. The morphology was observed under contrast phase microscope. UCB-MNCs at passage 3 were collected and counted with cell counting plate. The amplification times were calculated. Flow cytometry was used to examine the surface antigen phenotype of the third passage cells.
RESULTS AND CONCLUSION: There were 33 samples successfully isolated out of 36 UCB-MNCs samples, while three samples failed in B group. A large number of adherent cells were seen in 22 of 33 UCB samples (8 samples in Group A; 2 samples in Group B; 5 samples in Group C; 7 samples in Group D). Fibroblastoid cells from 9 samples (3 samples in Group A; 0 sample in Group B; 1 sample in Group C; 5 samples in Group D) were passaged steadily with the achievement ratio of 27.27%. Fibroblast-like cell and round megakaryocyte were the two basic types of adherent cells at 5-7 days after primary culture. At 3-4 weeks after serial subcultivation, UCB-MSCs could be found in A group and D group, they were morphologically similar to MSCs, showing homogeneous fusiform shape. These cells were cultured steadily for 60-90 days without obvious differences in morphology. Flow cytometry showed that, MSCs-related antigens such as CD29 and CD105 were expressed strongly on the surface of UCB-MSCs, but few hematopoietic lineage markers such as CD34 and CD45 nor endothelial markers such as CD106 could be found. Cells adhering to the centrifugation tubes may elevate significantly the achievement ratio of UCB-MSCs in vitro, methylcellulose isolation method combined with modified density gradient centrifugation could fully recover the adherent cells, thus benefiting for amplifying and passaging UCB-MNCs steadily in vitro.