Characteristics of long-term cultures of proliferating, mononuclear phagocytes from bone marrow.

Abstract

The characteristics of murine bone marrow mononuclear phagocytes in long-term cultures with embryonic fibroblast-conditioned medium were studied to determine the stage of development and state of activation of these cells. Two liquid culture systems were used: for studies on the morphology, cytochemistry, and functional characteristics at the cellular level, the cells were cultured adherent to a glass surface; and for experiments where the cells were needed in suspension (replating experiments, and studies on locomotion, intracellular killing, and cytotoxicity) use was made of Teflon culture systems. Three developmental stages of mononuclear phagocytes could be recognized easily in these cultures: monoblasts, promonocytes, and macrophages. In cultures on a glass surface, these cells grow in colonies separate from granulocytic colonies. When incubation is prolonged beyond 7-9 days, the granulocytes die, leaving pure mononuclear phagocyte cultures. Primary cultures, in which monoblasts, promonocytes, and some macrophages proliferate, can be maintained for 3-4 weeks. Calculation showed that one monoblast present on day 0 gives rise to a progeny of more than 7 X 10(3) mononuclear phagocytes by day 14; after that, the rate of proliferation declines despite the addition of fresh media. Regular replating of the cells cultured on Teflon made it possible to maintain proliferation over a period of almost 200 days. The cells in culture have the typical characteristics of mononuclear phagocytes, as judged by light microscopy, alpha-naphthyl butyrate esterase activity, lysozyme activity, presence of receptors for Fc and C3, and endocytic, microbicidal, and cytotoxic activity. The 5'nucleotidase activity, ingestion of erythrocytes via C3-receptor, locomotion, and antibody-dependent cytotoxicity indicate that the cultured bone marrow mononuclear phagocytes are more active than resident macrophages, and as active as or even more active than thioglycollate-induced macrophages. In conclusion, the population of mononuclear phagocytes in the liquid cultures of bone marrow is heterogenous with respect to developmental stage and state of activation.