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

Abstract

Human monocytes were isolated from peripheral blood using albumin or Ficoll-Hypaque gradients and subsequent adherence to plastic and glass surfaces. Cell cultures contained 90-95 monocytes by multiple criteria including morphology, peroxidase cytochemistry, ingestion of latex beads and presence of Fc and complement receptors. The monocyte yield from albumin gradients was 74% of the cells contained in whole blood and greater than that obtained with Ficoll-Hypaque. Determinations of cell number and protein indicated that approximately 50% of the initially adherent monocytes were present after 1 wk of cultivation in plastic wells containing Neuman-Tytell medium supplemented with fresh autologous serum. Monocytes were cultured for up to 3 mo. under these conditions. The morphology of monocytes changed dramatically during cultivation. The initially well spread cells retracted their plasma membranes during the 1st day in culture, returned to original diameter after 2-3 days, and doubled their size after 1 wk. The use of heated autologous serum resulted in severe cell clumping and poor viability. Lysozyme secretion was constant after the 1st day of monocyte cultivation but myeloperoxidase activity was virtually undetectable after 4 days. 5''-Nucleotidase activity increased 11-fold during this period. The increased 5''-nucleotidase activity was the result of endogenous synthesis and not related to either activation of latent enzyme nor to loss of enzyme inhibitors. Enzyme activity did not increase in cells cultured in serum-free medium and was reversibly depressed in monocytes phagocytizing latex particles.