The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas

Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.