| Subject At present, many methods are used for expansion of stem/progenitor cells in vitro. However these methods are focused on various usages of some kinds of cell factors. Moderate pressure influenced on efficiency of expansion of stem/progenitor cells was evaluated in this research. Method Human umbilical blood mononuclear cells (MNCs) were separated with density gradient centrifugation. MNCs were scattered in low glucose and low blood serum DMEM culture medium supplemented with stem cell factor which final concentration was 50ng/ml. The initial concentration of MNCs was 6.5×105/ml. Three groups were established. The control group (P0 group) was cultured at air pressure under the condition of 37℃,5%CO2 and saturated moisture. The other two groups were cultured at 60mmHg (P60 group) or 120mmHg (P120 group) pressure 12 hours a day respectively. Growing status was observed under microscope. And at 1, 3, 5, 7 day, the total number of MNCs was counted respectively. After collection of 106 MNCs and labeled with PE-CD34 monoclonal antibody, the percentage of CD34 positive cells (CD34+ cells) was obtained by analyzed 1×104 cells on flow cytometer (FCM), and total number and expansion multiples of CD34+ cells were calculated. Results On the 3rd day, the total number of MNCs decreased to approximate half in each group compared with that on the 0th day, and from then on, the total number of MNCs maintained at the same level as on 3rd day, but no differences among 3 groups. Meanwhile, the percentage and the total number of CD34+ cells significantly increased, and kept increasing tendency to the 7th day in every groups. In P120 group, the percentage (7.23±0.88 vs 5.82±0.68, p<0.05) and total number of CD34+ cells (52.88±9.81 vs 38.06±5.62, p<0.05) were higher significantly than those in P0 group on the 3rd day. The same results were found in the same way on the 5th day (24.39±2.89 vs 17.53±5.20, p<0.05 and 152.64±22.74 vs 101.29±25.51, p<0.05) and on the 7th day (29.91±2.92 vs 24.85±3.97, p<0.05 and 182.42±26.05 vs 141.73±19.14, p<0.05). Similar results were found when P120 group was compared with P60 group. The expansion multiples in P120, P60 and P0 groups were 8.51±1.21, 7.08±1.07 and 6.61±0.89 by the 7th day, the expansion multiple of P120 group was higher significantly than those of P60 and P0 groups. By the 5th day, the expansion multiple of P120 group had reached to the same level of P0 group on the 7th day.While percentage, total number and expansion multiple of CD34+ cells were higher in P60 group than in P0 group, but no significant difference between them. Conclusion Culture system associated with the pressure of 120mmHg can accelerate expansion speed of CD34+ cells derived from umbilical cord blood, but the same result cannot be obtained with that of 60mmHg.
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