A method for the derivation and continuous propagation of cloned murine bone marrow macrophages.

A method is proposed for the initiation and long term propagation of clonal macrophage cell lines descended from single bone marrow precursor cells. The cells were cloned in soft agar at low cell density, and the resultant colonies were picked from the agar and expanded in liquid culture. The optimal medium for liquid culture contained 75% Dulbecco's Modified Eagle's Medium with high glucose/high bicarbonate, 10% fetal calf serum, 5% horse serum, and 10% L cell conditioned medium. During growth in liquid culture, the cells were plastic adherent but they were easily removed from polymethylpentene or polystyrene culture dishes by means of a jet of cold medium directed through a 25-gauge needle. More than 50 cell lines from 17 mouse strains have been subcultured continuously for many months, some for more than 2.5 years with no evidence of culture senescence or appreciable change in exponential growth rates. Cells from these lines were all adherent, phagocytic, Fc receptor positive, and esterase positive. Cells from each cell line tested expressed MHC-encoded surface antigens and were able to stimulate allogeneic mixed lymphocyte reactions. Using these methods of continuous culture, pure, functionally active normal macrophages could be repeatedly harvested in large numbers from any of the lines.