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

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Abstract
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.