An animal component-free, serum-free culture method for generation of human dendritic cells

Abstract

Dendritic cells (DCs) are central regulators of adaptive immune responses that potentially have major applications in the immunotherapy of cancer and autoimmune disorders. DCs are rare in peripheral blood (PB) and are therefore often generated in vitro from PB monocytes. We developed an animal component-free (ACF) and serum-free medium, ImmunoCult-ACF DC, that supports the generation of DCs from monocytes in culture. CD14+ monocytes were isolated using EasySep immunomagnetic separation and cultured for 5 days in ImmunoCult-ACF DC medium and cytokines (GM-CSF and IL-4) to promote their differentiation into CD14−CD83− immature DCs. The immature DCs were then stimulated for 2 days with the ImmunoCult-DC Maturation Supplement, a combination of cytokines and pro-inflammatory mediators, to promote their maturation to CD14− CD83+ DCs (93 ± 5% CD83+, n=23). The yield of mature DCs was 42 ± 23% (n=23), similar to the yield in a control serum-free medium (49 ± 24%, n=16). Mature DCs produced high levels of IL-12, on average 371 pg/mL (range: 27–1756, n=9 in cultures initiated with 106 monocytes). Mature DCs loaded with CMV, EBV and Flu virus peptides efficiently stimulated the proliferation of autologous CD8+ T cells in 7 day co-cultures. An 18-fold increase in T cell numbers (range: 5–31, n=4) was observed when compared to control culture conditions containing only T cells without mature DCs. In conclusion, functional DCs can be efficiently generated by differentiation of monocytes in a completely animal (including human) component-free and serum-free medium. This medium and culture method will enable further research into the development and application of DCs for cellular therapy.