Establishment and Characterization of Long‐Term Cultured Cell Lines of Murine Resident Macrophages

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung expiants of C57BI/6 Ipr/lpr mice and from spleen expiants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3‐4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec +, H‐2K+, THY1 ‐, LYT2 ‐, L3T4 ‐). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony‐stimulating factor or gamma‐interferon were released in the medium. Long‐term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long‐term cultured cell lines of macrophages of defined and reproducible characteristics.