Phenotypic and functional characterization of THP-1-derived macrophages: impact of serum and serum-free...

Macrophages are essential components of the innate immune system, involved in inflammation and host defense. However, primary human macrophages are limited by availability, donor variability, and ethical constraints. The THP-1 human monocytic leukemia cell line is a practical alternative due to its high expansion capacity, ease of culture, and ability to acquire a macrophage-like phenotype using stimulation agent such as phorbol 12-myristate 13-acetate (PMA). To date, most laboratories generate macrophage-like cells by differentiating THP-1 cells, yet protocols vary widely between laboratories. Currently, the impact of differentiation conditions on both surface marker expression and the functional profile of macrophages remain poorly understood. Factors such as serum supplementation, PMA concentration, exposure duration, resting time, and seeding density can significantly influence macrophage behaviour.

In this study, we characterized the phenotype and functionality of THP-1-derived macrophages under different differentiation conditions, with or without serum supplementation. Phenotype was assessed by flow cytometry, and functionality was evaluated through responses to the reference multi-walled carbon nanotube NM-401 following exposure using the VITROCELL® PowderX system for accurate aerosolization. Phagocytic activity was quantified using fluorescence measurements and further characterized through confocal microscopy. Inflammatory responses were assessed by measuring cytokine secretion following lipopolysaccharide (LPS) stimulation.

Our findings demonstrate that differentiation conditions influence the phenotype and functional responses of THP-1-derived macrophages and highlight the importance of optimizing differentiation protocols to achieve consistent and reproducible outcomes, supporting the development of standardized, animal-free in vitro toxicity testing.