Optimization of the in vitro culture system for bone marrow-derived dendritic cells in rats

Objective To investigate the optimization of the in vitro culture system for bone marrow-derived dendritic cells(DC)in rats.Methods Immature DC from bone marrow cells of Lewis rats were induced and differentiated in vitro by 20 μg/L recombinant rat granulocyte macrophage colony stimulating factor(rrGM-CSF)and 10 μg/L recombinant rat interleukin(rrIL)-4.On day 6,100 μg/L of lipopolysaccharide was added and the cells were cultured for another 48 h to generate mature DC.The morphological features were observed by invert optical microscope.The immune phenotypes of DC were detected by flow cytometry(FACS).The contents of interleukin(IL)-12 and IL-10 in the DC supernatant were detected by direct sandwich enzyme-linked immune absorbent assay(ELISA).Mixed lymphocyte reaction(MLR)culture was performed to observe the proliferation of T lymphocyte cells by the DC stimulation,while Brown Norway rat splenetic T lymphocyte cells were used as responders.Results The cultured cells were identified as DC by cytomorphology,FACS and MLR.After culture for six days,the cultured cells displayed the typical morphology of dendritic cells,with high expression of OX62(the marker of rat DC).After LPS stimulation,most cells in culture had more dendrite-like characters on the surface and expressed major histocompatibility complex(MHC)class Ⅱ,CD40 and CD86 abundantly.They stimulated allogeneic T cell responses effectively in MLR.Compared with immature DC,mature DC produced significantly more IL-12 and IL-10 to stimulate the proliferation of T lymphocyte cells.Conclusion The improved culture system from bone marrow cells of rats can induce plenty of bone marrow-derived DC in rats through adjusting the doses of rrGM-CSF and rrIL-4.