title: 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-alpha and IL-1beta 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 naïve 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 naïve TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models.
  
  
  BACKGROUND
  DC vaccines/dendritic cell development/fetal calf serum-free culture conditions for DC/in vivo therapeutic DC approaches.
authors.affiliation: Clinical Research Unit, Department of Dermatology, University of Mainz, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Müller
authors.forename: A
authors.initials: A
authors.lastname: Müller
authors.forename: H
authors.initials: H
authors.lastname: Jonuleit
authors.forename: K
authors.initials: K
authors.lastname: Steinbrink
authors.forename: C
authors.initials: C
authors.lastname: Szalma
authors.forename: L
authors.initials: L
authors.lastname: Paragnik
authors.forename: K
authors.initials: K
authors.lastname: Lingnau
authors.forename: E
authors.initials: E
authors.lastname: Schmidt
authors.forename: J
authors.initials: J
authors.lastname: Knop
authors.forename: A H
authors.initials: AH
authors.lastname: Enk
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002417:Cattle / Q000196:embryology; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000502:physiology; D003470:Culture Media / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D003877:Dermatitis, Contact / Q000503:physiopathology; D005312:Fetal Blood / Q000502:physiology; D007375:Interleukin-1 / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic / Q000235:genetics; D010047:Ovalbumin / Q000494:pharmacology; D010641:Phenotype; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D013234:Stem Cells / Q000166:cytology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D003470:Culture Media; D007375:Interleukin-1; D011948:Receptors, Antigen, T-Cell; D014409:Tumor Necrosis Factor-alpha; D010047:Ovalbumin