[Response surface methodology for optimizing the technology of induction and isolation of dendritic cells by multiple cytokines].

Objective To promote the induction and separation efficiency of bone marrow-derived dendritic cells (BMDCs) in vitro through optimizing the inducing and isolating process by multiple cytokines. Methods The factors to be optimized in single factor tests included recombinant mouse granulocyte macrophage-colony stimulating factor (rmGM-CSF), recombinant mouse interleukine 4 (rmIL-4), lipopolysaccharide (LPS), recombinant mouse tumor necrosis factor α (rmTNF-α) and inducing time. The numbers of immature dendritic cells (imDCs) and mature dendritic cells (mDCs) were investigated as the indicators. Box-Behnken experimental design-response surface methodology was used to analyze and verify the data. Morphological changes were observed using the inverted microscopy and the transmission electron microscopy. Surface molecules including CD11c and CD86 were detected using the flow cytometry. Results The optimum inducing conditions for imDCs were obtained as follows: rmGM-CSF was 46 ng/mL, rmIL-4 was 24 ng/mL, inducing time was 6 days, and the number of imDCs was (4.58±0.28)×106 cells, and the relative deviation was 4.00%. The optimum inducing conditions for mDCs were as follows: LPS was 1.4 μg/mL, rmTNF-α was 30 ng/mL, inducing time was 1 day, and the number of mDCs was (4.21±0.15)×106 cells, and the relative deviation was 3.80%. Sufficient typical imDCs and mDCs were obtained within 5-7 days of induction in vitro. Also, flow cytometry showed that the amplified imDCs had a high expression of CD11c (68.62%±2.3%) and a low expression of CD86 (37.95%±1.8%), and the mDCs had high expressions of both CD86 (90.34%±1.4%) and CD11c (82.05%±1.6%). Conclusion The combination of single factor tests and Box-Behnken design -response surface methodology could optimize the inducing and isolating method for DCs in vitro by multiple cytokines rapidly and efficiently, which provided basic experiment materials for further studies.