Long-term stroma-dependent cultures from murine spleen have been previously shown to support dendritic cell (DC) development in vitro. Secondary cultures have now been established using a splenic stromal cell layer overlaid with cells from different lymphoid sites. Cells resembling DCs can be generated in vitro from unfractionated murine lymphoid cells in the absence of added growth factors, Bone marrow (BM) cultures are the most successful but some cultures have been derived from spleen and thymus. Large numbers of mononuclear cells with dendritic morphology can be generated from BM within 2 weeks and cell production in many cultures has been maintained for at least 6 months. A significant proportion of cells binds antibodies specific for DC markers. No lymphoid cells, mast cells or granulocytes are detectable in culture by antibody and histochemical staining and light and electron microscopy. As with cells generated in primary cultures, cells grown in secondary cultures are equally potent stimulators of both syngeneic and allogeneic mixed lymphocyte reactions, confirming their function as antigen-presenting cells. They are also capable of endocytosing and presenting protein antigen to the D10.G4.1 Th2 clone and to unprimed T cells.
This study confirmed the presence of DC precursors in multiple lymphoid sites which can be induced to proliferate in the presence of a spleen stromal cell monolayer. The secondary culture system provides an ideal in vitro model for investigation of the development of DC from different tissue sites. It also provides a stable and continuous resource of cells for further studies on DC development.