In vitro purification and culture of dendritic cells obtained from peripheral blood

Objective To establish a practical method to generate dendritic-cells(DCs) from peripheral blood and to study their morphology and in vitro function.Methods Peripheral blood mononuclear cells(PBMNCs) were isolated from peripheral blood of healthy donors by Ficoll-hypaque gradient centrifugation.Monocytes were purified by either using the MACS CD14 isolation kit or collecting the plastic adherent fraction of PBMNCs.To generate standard monocyte-derived DCs(moDCs),monocytes were cultured in the medium supplemented with rhGM-CSF and rhIL-4 for 5 days and subsequently incubated with a combination of proinflammatory mediators(rhGM-CSF+ rhIL-4 + rhTNF-α)for 12 days.Morphology of DCs was observed under inverted microscope and electronic microscope,respectively.Phenotypes on the cells were identified by flow cytometer.Cell populations were phenotyped with the following FITC-or PE-conjugated MoAbs: CD80,CD83,CD86,CD1α,HLA-DR.The proliferation of allogenetic T cells stimulated by DCs was determined by MTT assay.Results Mature DCs with typical morphology(elongated dendritic processes were observed under inverted microscope and electronic microscope) were induced from monocytes.DCs showed high phenotypic expressions of DC markers of CD80,CD83,CD86,CD1α,HLA-DR.DCs could stimulate the proliferation of allogenetic T cells.Conclusion Large amount of mature DCs could be induced from human peripheral blood.