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

Young Ik Son, Shin Ichi Egawa, Tomohide Tatsumi, Richard E. Redlinger, Pawel Kalinski, Tatsuya Kanto

Research output: Contribution to journalArticle

110 Citations (Scopus)

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, 30-40×106/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.

Original languageEnglish
Pages (from-to)145-157
Number of pages13
JournalJournal of Immunological Methods
Volume262
Issue number1-2
DOIs
Publication statusPublished - 2002 Apr 1
Externally publishedYes

Keywords

  • Bone marrow
  • Bulk-culture
  • Dendritic cell
  • Maturation
  • Mouse

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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