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

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 explants of C57Bl/6 lpr/lpr mice and from spleen explants 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.

The Generation of Macrophages from Precursor Cells Incubated with Brain Endothelial Cells. A Release of CSF-l Like Factor from Endothelial Cells.

“…MHC Class II positive monocyte-like cells are observed in the experimental metastatic tumors of the brain that preserve intact blood-brain barrier (Schackert et al 1988). These studies suggest that brain phagocytes can be derived from circulating monocytes (Lombard et al 1988). As was observed with our EC-binding cells, direct contact with the layer was necessary (Lombard et al 1988).…”

“…These studies suggest that brain phagocytes can be derived from circulating monocytes (Lombard et al 1988). As was observed with our EC-binding cells, direct contact with the layer was necessary (Lombard et al 1988). However, unlike the nonadherent spleen cells described here, the macrophage-like cell lines from peritoneum, spleen, or lung described by Lombard et al seemed to be derived from mature macrophages.…”

Flow cytometry distinction between adherent and phagocytized yeast particles

“…The culture medium contained <1 ng/ml endotoxin as assayed by the Limulus test. The cells, which has been shown to bear mannose receptors on their surface (15), were regularly passaged as described by Lombard (13).…”

Macrophage cell lines derived from major histocompatibility complex II-negative mice

“…Cell lines overcome the disadvantages of using fresh cells including difficulty in isolating sufficient numbers of cells and the heterogeneity of freshly isolated macrophage. Cell lines retaining the phenotypic and functional properties of mature macrophages provide a useful tool for molecular studies on functional and secretot 7 properties of this cell population (1, 23,24,28,31,32).…”

“…Because experiments often require large numbers of homogenous macrophages, several laboratories have developed macrophage cell lines to study problems in macrophage biology (28,31,32).…”