A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells

J Immunol Methods. 2002 Apr 1;262(1-2):145-57. doi: 10.1016/s0022-1759(02)00013-3.

Abstract

We established a novel culture method for generating dendritic cells (DC) from mouse bone marrow (BM) cells. Unfractionated bulk BM cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5-7 days and a DC population was isolated by gradient centrifugation with 14.5% (w/v) metrizamide. Through this method, 30-40 x 10(6)/mouse DC with 85-95% purity was obtained on day 7; this yield was higher than those of conventional DC generated by Inaba's method either with GM-CSF alone (conventional-GM DC) or GM-CSF and IL-4 (conventional-GM/4 DC). Bulk-cultured DC have a more matured phenotype than both conventional-GM and -GM/4 DC as shown by higher expression of CD86, MHC class II and CD40. Functional analyses reveal that (1) bulk-DC show less ability in endocytosis than conventional-GM DC and are comparable in IL-12 p70 production with conventional-GM and -GM/4 DC. (2) Bulk-DC exhibit stronger stimulatory capacity in allogeneic T-cell proliferation than conventional DC. (3) By using ovalbumin (OVA) and OVA-specific T-cell receptor (TCR) transgenic mice (DO11.10) system, OVA protein-loaded bulk-DC stimulated CD4 T cells of DO11.10 mice more than conventional-GM DC and comparable with conventional-GM/4 DC. (4) Furthermore, OVA peptide-pulsed bulk-DC stimulated CD4 T cells more than conventional-GM and -GM/4 DC. These data indicate that bulk-DC are functionally more mature than conventional DC. Taken together, bulk-culture method is a simple technique for generating functionally mature BM-DC in large quantities and high purity.

MeSH terms

  • Animals
  • Antigen Presentation
  • Bone Marrow Cells / cytology*
  • Bone Marrow Cells / immunology
  • CD4-Positive T-Lymphocytes / immunology
  • Cell Culture Techniques / methods*
  • Cell Differentiation / drug effects
  • Cell Division
  • Dendritic Cells / cytology*
  • Flow Cytometry
  • Granulocyte-Macrophage Colony-Stimulating Factor / immunology
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
  • Interleukin-4 / immunology
  • Interleukin-4 / pharmacology
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL

Substances

  • Interleukin-4
  • Granulocyte-Macrophage Colony-Stimulating Factor