Fetal calf serum-free generation of functionally active murine dendritic cells suitable for in vivo therapeutic approaches

Standard protocols to generate mouse dendritic cells (DC) generally use cul ture medium supplemented with fetal calf serum; however, reinjection in viv o of DC cultured in fetal calf serum results in priming to xenogeneic prote ins that clearly limits the use of such DC. We therefore established a feta l calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI sup plemented with 1.5% syngeneic mouse serum. Although the yield of DC grown u nder fetal calf serum-free conditions was somewhat lower than that of the s tandard culture, large numbers of DC could be generated without the exposur e to xenogeneic proteins. The yield of fetal calf serum-free cultured DC wa s further enhanced by addition of the proinflammatory cytokines TNF-alpha a nd IL-1 beta with the combination resulting in up to 10% more DC. Phenotypi cally, CD11c(+) DC cultured fetal calf serum-free homogenously coexpressed the DC-specific molecule DEC-205 as well as the costimulatory molecules CD4 0, CD80, and CD86. In contrast, only a subpopulation of the CD11c(+) DC cul tured in fetal calf serum-containing medium coexpressed these molecules. Fu nctionally, fetal calf serum-free DC showed strong stimulatory capacity for naive allogeneic CD4(+) and CD8(+) T cells. Importantly, fetal calf serum- free DC showed spontaneous in vivo migratory activity. Moreover, 5 x 10(5) subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneou s injection of a single dose of 5 x 10(5) OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in naive TC R transgenic animals. Thus DC cultured under fetal calf serum-free conditio ns are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models.