In vitro induction and amplification of marrow-derived dendritic cells in mice and exploring difference under different growth conditions

Abstract:

Objective：To obtain a large number of high-purity mouse marrow-derived dendritic cells（DC） through in vitro induction and amplification，and to explore its biological characteristics with different cultivating time and under different growth conditions. Methods：Marrow-derived cells of mouse were induced to differentiate into DCs in vitro by using recombinant-murine granulocyte-macrophage colony-stimulating factor（rmGM-CSF）. The cell count，survival rate and morphology changes of DCs were observed under the light inverted Microscope at 0 h，1 d，4 d，7 d，9 d and 11 d. On day 9，suspended cells and adherent cells without stimulation of mouse interleukin-4（rmIL-4） or with stimulation of rmIL-4 were collected，respectively；expression levels of CD11c，CD80，CD86 and MHC-Ⅱ on the surface of suspended cells and adherent cells by Flow Cytometry. On day 9 and 11，the DC was collected and the expression level of CD11c of BMDC was analyzed by FACS. SPSS 17.0 software was used for statistical analysis，and t-test was used for comparison between two independent groups. Results：On days 0 h，1 d，4 d，7 d，9 d and 11 d，the survival rate of BMDCs ranged from 90% to 95%. On day 4，a large number of colony cells were formed and gradually increased. On day 9，BMDCs became significantly larger and atypical protrusions and pseudopodia were able to be obviously found on the surface. On day 9，flow cytometry showed that expression ratio of cell-surface markers including CD11c+ CD80+，CD11c+CD86 and CD11c+MHC-Ⅱin suspended cell population were significantly higher than those in adherent cell population [（88.10±2.41）% vs. （79.17±2.32）%，（84.60±3.26）% vs. （75.40±4.41）%，（92.90±3.93）% vs. （82.77±4.80）%]，with significant differences（all P<0.05）. The expression of CD11c+ in BMDCs had no significant dif－ference in rmIL-4 groups and non-rmIL-4 groups（P>0.05）. After cultivating for 11 days，both the suspended CD11c+ cells population and adherent CD11c+ cells population were significantly higher than those after cultivating for 9 days（P<0.05）. Conclusion：A simple induced cultivation in vitro can obtain highly purified marrow-derived DC with different characteristics. There is no difference of the CD11c+ cells between the differentiation of DCs with rmGM-CSF alone and combination of rmGM-CSF and rmIL-4. The maturity of suspended cells population is higher than that of adherent cells population and prolonged cultivating time can increase the production and purity of CD11c+ DC.