An efficient culture method for generating large quantities of mature mouse splenic macrophages

In this study, we established an efficient in vitro culture method for generating mature splenic macrophages (Sp-Mϕ). Splenocytes were cultured in the presence of conditioned medium containing macrophage colony-stimulating factor (M-CSF) for 7 days post post-isolation and the generated Sp-Mϕ were characterized phenotypically and functionally. Through this method, 9 × 106/mouse Sp-Mϕ were obtained in comparison to 2 × 105/mouse when Mϕ were cultured in regular medium. In addition, the purity of these cells was as high as 80% by day 5 and > 90% by day 7 of culturing, confirmed with Mϕ-specific markers. The increased Sp-Mϕ yields, in the presence of M-CSF, point towards the existence of a precursor population in the spleen that can be influenced to differentiate into Sp-Mϕ. Moreover, we compared the maturation of generated Sp-Mϕ to conventional bone marrow-derived Mϕ (BM-Mϕ) in vitro. Interestingly, Sp-Mϕ exhibited lower capacity to phagocytose dead cells after 3 days of maturation, but showed similar internalizing capacity after 5 and 7 of maturation to BM-Mϕ cultured for the same time period. Importantly, Sp-Mϕ upregulated the expression of several surface markers such as MOMA-2 and CD68 while downregulating SIGN-R1 after 7 days, indicating that these Sp-Mϕ undergo further maturation in vitro due to culturing in M-CSF. Taken together, we describe and validate a method for generating Sp-Mϕ in large quantities and high purity. These data should prove valuable in future studies characterizing the functions and maturation of Sp-Mϕ.