The separation, long-term cultivation, and maturation of the human monocyte

Monocytes and macrophages, collectively termed the mononuclear phagocyte system, are intimately involved in a variety of physiological and pathological events (1-5). They participate in the inflammatory process, secrete numerous biologically active products, and are recognized as major effectors of immunity against neoplastic disease and infectious agents. Our present understanding of the physiology and biochemistry of this cell system is based in large part on studies of rodent cells. Although the circulating human monocyte is readily available and is recognized as the precursor of tissue macrophages, its study has been impeded by inefficient isolation and cultivation methods. The yield of monocytes from blood has ranged from 30 to 75% with different isolation procedures (6-11), with the subsequent loss of up to 90% of monocytes during the first 3 days of in vitro cultivation (8). In the present report, we have evaluated the yield of monocytes obtained with Ficoll-Hypaque and albumin gradients and defined optimal conditions for the cultivation of monocytes in vitro. The physiologic changes which occur during the in vitro differentiation of monocytes into macrophages were also examined with respect to the secretory enzyme lysozyme, the lysosomal enzyme myeloperoxidase and plasma membrane markers, such as the Fc and complement receptors and 5'-nucleotidase. A marked increase in 5'-nucleotidase activity was observed during cultivation which was further studied in terms of the effects of phagocytosis, serum deprivation, and inhibition of protein synthesis.