Induction of human peripheral blood monocyte-derived dendritic cells in vitro

Objective To investigate the method of culturing dendritic cells (DCs) derived from human peripheral blood monocytes. Methods The first step of the procedure was to isolate mononuclear cells from human peripheral blood by Ficoll-Hypaque density gradient separation,then monocytes were purified through adhensiveness,which were induced in vitro by human recombinant granulocyte / macrophage colony stimulating factor (rhGM-CSF) and human recombinant interleukin 4 (rhIL-4). The second step was to add 100 ng / mL of tumor necrosis factor-α (TNF-α) at day 5 for cultivation for 2 days to induce maturation. Morphology of DCs was observed by inverted microscope,and the expressions of CD83,CD1a,CD86,CD40,and CD14 on DCs were analyzed by flow-cytometry. The proliferation of allogenetic T cells was detected by MTT method. Results After a 5 day incubation of monocytes with rhGM-CSF and rhIL-4,most of the cells formed clusters,and the expression levels of CD83,CD1a,CD86,CD40,and CD14 were 14.3%,12.8%,20.1%,19.9%,and 16.2%,respectively. At day 7 in the presence of rhTNF-α,the expression levels of CD83,CD1a,CD86,CD40,and CD14 of DCs were 29.8%,18.2%,33.6%,28.1%,and 8.0%,respectively. Matured DCs had a stronger capacity of stimulating T cells to proliferate. Conclusion It is suggested that combination of rhGM-CSF and rhIL-4 is beneficial to expand the proliferation of immature DCs,which can be induced to mature by 2 days incubation with TNF-α,and matured DCs can strongly stimulate T cells to proliferate.