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-Mphi). 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-Mphi were characterized phenotypically and functionally. Through this method, 9 x 10(6)/mouse Sp-Mphi were obtained in comparison to 2 x 10(5)/mouse when Mphi 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 Mphi-specific markers. The increased Sp-Mphi 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-Mphi. Moreover, we compared the maturation of generated Sp-Mphi to conventional bone marrow-derived Mphi (BM-Mphi) in vitro. Interestingly, Sp-Mphi 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-Mphi cultured for the same time period. Importantly, Sp-Mphi upregulated the expression of several surface markers such as MOMA-2 and CD68 while downregulating SIGN-R1 after 7 days, indicating that these Sp-Mphi undergo further maturation in vitro due to culturing in M-CSF. Taken together, we describe and validate a method for generating Sp-Mphi in large quantities and high purity. These data should prove valuable in future studies characterizing the functions and maturation of Sp-Mphi.