Phenotypic and functional comparison between flask adherent and magnetic activated cell sorted monocytes derived dendritic cells.

BACKGROUND

Generation of an effective dendritic cell (DC) based cancer vaccine depends on appropriate differentiation of monocytes in vitro.

OBJECTIVE

To compare the effects of monocyte separation methods, flask adherence (Flask-DC) and magnetic activated cell sorting (MACS-DC), on phenotypic and functional characteristics of resultant DCs.

METHODS

DCs from healthy volunteers were generated from plastic adherence and MACS isolated monocytes in the presence of GM-CSF and IL-4 as well as TNF-α and monocyte conditioned medium (MCM) in 7 day cultures. Mature DCs were then subjected to phenotypic analysis using anti-CD14, anti-CD83 and HLA-DR monoclonal antibodies. Functional and cytokine release assays were carried out using [3H] thymidine uptake test and commercially available ELISA kits for the determination of IL-12, IL-10, IFN-γ and IL-4, respectively.

RESULTS

We found that MACS-DCs were more homogenous and the yield and viability were fairly higher than Flask-DCs. MACS-DCs expressed higher levels of CD83 and HLA-DR as well as CD14 compared to the Flask-DCs. Induction of T cell proliferative responses were higher in Flask-DCs and also they elicited higher levels of IL-12: IL-10 and IFN-γ: IL-4 ratios in cytokine generation assays.

CONCLUSION

MACS method was superior for mass production of viable homogenous and fully mature DCs but their cytokine profile had the potential to polarize the immune system toward Th2 type immune response.