Induction Condition Optimization of the M-CSF-Induced Mouse Bone Marrow-Derived Macrophage and the Mechanism Exploration Based on RNA-Seq Analysis

Bone marrow-derived macrophage (BMDM) is key tool cell for studying biological processes such as immunity and inflammation. It is also known as a cell drug that can be used for cytotherapy of many diseases such as liver fibrosis. Given the high demand for BMDMs in research, this study explored the most cost-effective, high-productivity method and its molecular mechanism for obtaining Macrophage Colony-Stimulating Factor (M-CSF)-induced mouse BMDM. A factorial experimental design was employed to examine different induction conditions on BMDM phenotype proportion and productivity, including bone marrow cell (BMC) seeding density, M-CSF concentration, induction time, and removal of bone marrow resident macrophage. Neutral red phagocytosis assay was used to measure phagocytic capacity of BMDM. The mechanism of BMDM proliferation and differentiation was investigated by RNA-seq technology. The most cost-effective and high-productivity BMDM induction protocol is 2 × 10⁵ cells/cm² BMDM seeding density, 80 ng/ml M-CSF induction for 4 days with medium changing once on day 3. BMDM productivity reaches 75.6%. Furthermore, 4-day BMDM showed the strongest phagocytic capacity. RNA-Seq analysis revealed that Cd4, Smad6, Acta2, and Lrrc32 were key target genes regulating BMDM proliferation. Jun, Smad6, Cd4, Fhl2, Hspb1, Id1, and Inhbb were key target genes regulating BMDM differentiation. MAPK, mTOR, TGF-β, and NF-kappa B signaling pathways were the key proliferation or differentiation-related pathways. This study proposes an efficient and economical protocol for BMDM induction. Besides, it explores the regulatory mechanism of M-CSF on BMDM proliferation and differentiation.