An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow

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Abstract

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 5×106 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., 1–3×108 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-α 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.

Introduction

Dendritic cells (DC) have been described to develop along the myeloid and lymphoid lineage from pluripotent bone marrow stem cells (reviewed in Steinman, 1991; Cella et al., 1997). Little is known so far about the origins of lymphoid DC and their role within the immune system. In contrast, DC of myeloid origin are extensively described as specialized leukocytes to activate naive T cells. However, also studies on myeloid DC are still difficult, because they are rare in all body tissues and therefore isolation procedures are time consuming and cell yields are low (Williams et al., 1994). In mice only about 6×104 Langerhans cells can be isolated from the epidermis of one ear (Ortner et al., 1996), and about 1–10×105 DC from one spleen or thymus (Vremec et al., 1992; Inaba et al., 1998). From the blood of one mouse 1×106 DC can be generated with granulocyte–macrophage colony-stimulating factor (GM-CSF) (Inaba et al., 1992a), whereas bone marrow (BM) precursors treated with GM-CSF reveal about 5×106 DC after 6–8 days from one mouse (Inaba et al., 1992b; Scheicher et al., 1992; Inaba et al., 1993; Yamaguchi et al., 1997). Here we extend the standard method to propagate 1–3×108 BM-DC per mouse after 10–12 days. The achievement of higher cell yields is based on several modifications of the standard culture conditions.

Section snippets

Bone marrow preparation

Mice were kept under SPF conditions in our own facilities. Femurs and tibiae of female, 4–12 weeks old female C57BL/6, BALB/c and ICR mice (Charles River/Wiga, Sulzfeld, Germany) were removed and purified from the surrounding muscle tissue by rubbing with kleenex tissues. Thereafter intact bones were left in 70% ethanol for 2–5 min for disinfection and washed with PBS. Then both ends were cut with scissors and the marrow flushed with PBS using a Syringe with a 0.45 mm diameter needle. Clusters

Development of immature and mature BM-DC populations at days 6–8 of culture

Our protocol to generate BM-DC is adapted from the current standard method (Inaba et al., 1992b; Yamaguchi et al., 1997; Inaba et al., 1998). Fresh BM cells were used after vigorous pipeting to disintegrate cell aggregates but without depletion of other cell types, such as erythrocytes, T and B cells, granulocytes, MHC class II+ or FcR+ cells. A total of 2×105 BM cells were suspended in 10 ml R10 medium containing 200 U/ml rmGM-CSF in 100 mm bacterial petri dishes. Cultures were fed at days 3,

Discussion

We systematically explored factors that might increase yield and maturation of DC propagated from BM under the aegis of GM-CSF. Previous work on the generation of DC from mouse BM with GM-CSF (Inaba et al., 1992b, Inaba et al., 1993, Inaba et al., 1998) employed several treatments to achieve a maximal purity of BM-DC precursors (Table 1). One approach was the depletion of several non-DC precursor cells in the fresh bone marrow. This may however harm certain DC progenitors and/or remove subsets

Acknowledgements

This work was partially supported by an short term EMBO fellowship for Innsbruck and by the Sonderforschungsbereich 263/C13 for M.B. Lutz. We gratefully acknowledge Luciano Adorini (Milano, Italy), Wim. A. Buurman, (Maastricht, the Netherlands), Siamon Gordon (Oxford, UK), Bruno Kyewski (Heidelberg, Germany), Pieter Leenen (Rotterdam, Netherlands), Philippa Marrack (Denver, CO, USA), and Christoph Scheicher (Würzburg, Germany) for providing antibodies and hybridomas, Armin Bender and Monika

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