A critical guideline for controlling Monocyte-derived Macrophages phenotypes

Giorgia Moschetti^1*Doriana Oliveri¹Valeria De Matteis²Marta Zaccaria¹Diego Rondelli¹Anna Griego¹Edoardo Scarpa^1*Loris Rizzello^1*

• ¹University of Milan, Milan, Italy
• ²Universita del Salento, Lecce, Italy

Macrophages (Mϕ) are an extremely heterogeneous and rapidly adapting set of innate immune cells that are scattered throughout all tissues in humans from mid-gestation onwards. Their original definition as key players in phagocytosis and defence against pathogens is too restrictive nowadays, as Mϕ are central to tissue homeostasis, repair, and complex immune regulations involving adaptive immunity. The Mϕ exhibit different ontogenies, originating from either embryonic progenitors or bone marrow, and their fate is shaped by tissue-specific microenvironments, which determine their adaptive phenotypes. This results in functional flexibility, exemplified by their ability to polarize into pro- (M1) or anti- (M2) inflammatory states in response to environmental cues. Such a dynamic process is critical for resolving infections, repairing tissue, and maintaining immune balance. Dysregulated Mϕ polarization is indeed implicated in various pathologies, including chronic inflammation, cancer, and fibrosis. Despite their importance, the study of tissue-resident Mϕ is still limited by technical challenges related to their isolation, maintenance, and donor variability. As an alternative, monocyte-derived macrophages (MDMs) represent an easier in vitro system to model human Mϕ biology under controlled conditions. However, MDMs differ from tissue-resident Mϕ in their developmental origin and functional specialization. This review outlines the key principles and limitations of MDM-based models, discusses commonly used differentiation protocols, and proposes methodological strategies to enhance reproducibility and physiological relevance in macrophage research.