A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells.
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.
Son
Young-Ik
YI
Department of Surgery, Division of Surgical Oncology/Biological Therapeutics, University of Pittsburgh Cancer Institute, W1540, Biomedical Science Tower, 200 Lothrop Street, PA 15261, USA.
Egawa
Shin-ichi
S
Tatsumi
Tomohide
T
Redlinger
Richard E
RE
Kalinski
Pawel
P
Kanto
Tatsuya
T
D000818:Animals; D017951:Antigen Presentation; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D003713:Dendritic Cells / Q000166:cytology*; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000494:pharmacology; D015847:Interleukin-4 / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL
D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor