An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.
As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.
Department of Dermatology, University of Erlangen, Germany. manfred.lutz@derma.med.uni-erlangen.de
M B
MB
Lutz
N
N
Kukutsch
A L
AL
Ogilvie
S
S
Rössner
F
F
Koch
N
N
Romani
G
G
Schuler
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology*; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor