Standardized Isolation and Cytokine Priming of Murine Bone Marrow-Derived Mesenchymal Stromal Cells for Enhanced Immunomodulation

Mesenchymal stromal cells (MSCs) possess strong regenerative and immunomodulatory functionality. These properties make MSCs potent cellular pharmaceuticals for treating multiple immune disorders. Bone marrow is the primary and most frequently utilized source of MSCs. Preclinical mechanistic studies using murine models are crucial for gaining knowledge of the optimal drug delivery strategies for BMSCs in human clinical trials. Hence, standardization of the murine MSCs' manufacturing process is also critical for obtaining better therapeutic outcomes. A central goal of this protocol is to demonstrate how controlled interferon-gamma (IFN-γ) priming enhances the intrinsic immunosuppressive properties of BMSCs. We describe the generation of conditioned media (CM) from untreated or IFN-γ-prelicensed BMSCs and evaluate their biological activity using primary peritoneal macrophages. Through ELISA and flow-cytometric profiling, the protocol details how BMSC-derived secretome -- particularly when IFN-γ-primed -- drives anti-inflammatory macrophage polarization characterized by elevated IL-10 production and M2-associated surface markers. Overall, this protocol establishes a unified framework for producing functionally consistent murine BMSCs and for assessing their immunomodulatory potency. By reducing technical variability and providing visual and procedural clarity, it supports improved reproducibility in preclinical MSC research and facilitates optimized MSC deployment strategies for translational studies.