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

Abstract

Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal 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 supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins. The yield of fetal calf serum-free cultured DC was further enhanced by addition of the proinflammatory cytokines TNF-α and IL- 1β with the combination resulting in up to 10% more DC. Phenotypically, CD11c+ DC cultured fetal calf serum-free homogenously coexpressed the DC- specific molecule DEC-205 as well as the costimulatory molecules CD40, CD80, and CD86. In contrast, only a subpopulation of the CD11c+ DC cultured in fetal calf serum-containing medium coexpressed these molecules. Functionally, 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 105 subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneous injection of a single dose of 5 x 105 OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in naive TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models.