Culture conditions required for primary isolation and study of mouse blood monocytes

Blood monocytes, the precursors to macrophages found in inflammatory exudates, have several important immunologic functions, and may also contribute to populations of mature macrophages found in tissues and serous cavities. Mouse blood monocytes have not been studied much because they have been difficult to culture. If plated alone, ie, in the absence of blood lymphocytes, they die in media usually used to culture mouse macrophages. Here we describe conditions for plating them alone and maintaining them for several weeks in a standard culture medium (Neuman-Tytell) supplemented only with a normal serum. Plating them at a high concentration (8 x 10(7) cells/ml) and using horse serum instead of fetal calf serum were important, and their survival was improved by adding reduced glutathione or sodium thioglycollate but not 2-mercaptoethanol. Using high concentrations of horse serum (20%) and high plating concentrations of cells both encouraged monocyte replication. We studied the morphologic and functional characteristics of these cells at succeeding intervals during several-day cultures. At the beginning, during, and shortly after replication they had the characteristics of monocytes by several criteria. Later, and when not replicating, they developed into macrophages and varying numbers of epithelioid cells. By moderate manipulation of culturing conditions we have been able, then, to observe various aspects of mouse monocyte-macrophage differentiation and replication in relatively simple medium and using blood-adherent monocytes alone.