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 generatio n 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% p urity are obtained per mouse after 8 days of culture with GM-CSF. We have i mproved 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 ma jor modifications were: (i) the avoidance of any active depletion of bone m arrow cell subpopulations to circumvent loss of precursors, (ii) a lower pl ating 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 g eneration 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 mole cular level. (C) 1999 Elsevier Science B.V. All rights reserved.