Generation of healthy human peripheral blood monocyte derived dendritic cell in vitro

Objective To find a way by which dendritic cells(DC) from healthy human monocyte in peripheral blood might be induced to be matured.Methods Mononuclear cells were isolated from peripheral blood of healthy human by Ficoll-Hypaque density gradient separation,and then monocytes(Mo) were purified through adhensiveness.DCs were obtained after a 12 day incubation of monocytes with the media containing granulocyte/macrophage colony stimulating factor(GM-CSF),interleukin-4(IL-4) and tumor necrosis factor-α(TNF-α) which was added on the 6th day.DC's morphology was observed under inverted microscope,the expressions of CD1a,CD83,CD86 and MHC-DR on DCs were analyzed by flow cytometer on the 6th,9th,12th day,and the abilities to stimulate the proliferation of allogenic T cells by DCs were evaluated by MTT assay.Results After a 6 day incubation of monocytes with GM-CSF and IL4,most of them formed clusters,and the expression levels of CD1a,CD83,CD86 and MHC-DR were 53.2%,13.6%,65.5%,75.4%,respectively.On the 9th day in the presence of TNF-α,the expression levels of CD1a,CD83,CD86 and MHC-DR of DCs were 62.1%,73.5%,92.3%,98.4%,respectively,and the proliferation of native T cells induced by DCs was best enhanced.Conclusion It is suggested that combination of GM-CSF and IL-4 is beneficial to expanding and inducing immature DCs,and the matured DCs best induced by adding TNF-α into the media on the 9th day can be used for clinical immunotherapy.