Comparison of four culture protocols for differentiating bovine peripheral blood mononuclear cells into macrophages

Introduction Macrophages are essential innate immune cells involved in host defense, tissue homeostasis, and inflammation regulation. In vitro differentiation protocols for macrophages derived from peripheral blood mononuclear cells (PBMCs) are well established in human and murine models. However, in bovine species, standardized protocols for macrophage differentiation from PBMCs are underdeveloped, limiting their application in immunological and infectious disease studies. Methods Four in vitro culture protocols for differentiating macrophages from bovine PBMCs were compared, with the aim of comparing their morphological, phenotypic, and phagocytic characteristics. PBMCs were isolated from clinically healthy cattle and cultured under four protocols: (1) macrophage colony-stimulating factor-supplemented RPMI-1640, (2) nutrient-enriched RPMI-1640 without exogenous cytokines, (3) commercial M1-inducing medium, and (4) commercial M2-inducing medium. Results Differentiated macrophages were evaluated using phase-contrast microscopy, quantitative reverse transcription polymerase chain reaction, and flow cytometry for surface marker expression, and phagocytic assays using heat-killed Mycobacterium avium subsp. paratuberculosis (MAP). Under protocol 2, macrophages exhibited more pronounced morphological differentiation and higher expression of CD14, CD11b, and MHC II compared to other conditions, suggesting a more differentiated phenotype. This phenotype was associated with increased phagocytic activity, as evidenced by the highest phagocytic activity against MAP. Discussion Overall, these findings suggest that nutrient-enriched RPMI-1640 medium without cytokine supplementation (protocol 2), a commonly used culture condition, supports macrophage differentiation with favorable morphological and phagocytic characteristics under our experimental conditions. This culture approach provides a valuable in vitro model for studying bovine immunology and macrophage–pathogen interactions.