Single-cell quantitative characterization of live THP-1 cells: morphology and mechanics

Abstract

Introduction and Objectives: Macrophages are crucial cells in the foreign body response and subsequent tissue regeneration following biomaterial implantation, yet quantitative data describing their morphological and mechanical characteristics remain limited, particularly for human macrophage models. The absence of standardized reference values complicates the interpretation of macrophage responses in biomaterial research and hinders comparisons between studies. The objective of this study was to establish a reproducible Atomic Force Microscopy (AFM)-based protocol for the characterization of live THP-1-derived macrophages and use it to obtain quantitative baseline data on the morphology, elasticity, and viscoelastic behavior of M0 and M1 macrophages cultured under standard conditions. By generating a reference dataset under controlled experimental conditions, this work can provide a foundation for evaluating how biomaterial properties influence macrophage behaviors.
Methods: Human THP-1 monocytes were differentiated into macrophages and subsequently polarized into the pro-inflammatory M1. M0 and M1 macrophages were seeded in Petri dishes and cultured for three days prior to analysis. Morphological and mechanical characterization was performed on live cells under physiological conditions (37 °C) using Atomic Force Microscopy (AFM). Quantitative Imaging mode was employed to obtain high-resolution topographical images and apparent Young's modulus maps, enabling assessment of cell area, height, and stiffness. Viscoelastic properties were evaluated by AFM microrheology using frequency-dependent oscillatory indentation measurements.
Results: This study established a standardized AFM-based protocol for the quantitative characterization of live THP-1-derived macrophage morphology and mechanics, enabling the measurement of macrophages of different subtypes. Quantitative AFM analysis and complementary optical microscopy demonstrated substantial morphological heterogeneity within both macrophage populations. Irregular morphologies predominantly characterized M0 macrophages, whereas M1 macrophages exhibited a greater proportion of elongated cell shapes. Morphological subgroup analysis revealed that elongated cells consistently occupied smaller projected areas and had lower cell heights than round cells. Three-dimensional AFM reconstructions further revealed membrane roughness, cytoplasmic protrusions, and complex topographical features that were not readily detectable by conventional optical microscopy.
Mechanical characterization showed pronounced heterogeneity in apparent Young’s modulus both within and between individual cells. Elasticity maps consistently showed lower stiffness in nuclear regions than in the surrounding cytoplasm. Mean apparent Young’s modulus values were 1.27 ± 0.62 kPa for M0 macrophages and 1.43 ± 0.70 kPa for M1 macrophages, with no statistically significant difference observed between the two activation states. Frequency-dependent rheological measurements revealed similar viscoelastic behavior for both phenotypes. At lower frequencies, macrophages exhibited predominantly elastic behavior, whereas at higher frequencies, a gradual shift toward viscous dominance occurred. Overall, the results indicate that although morphological distributions differed between M0 and M1 macrophages, substantial heterogeneity existed within each population, and no single mechanical parameter was sufficient to clearly distinguish activation states. The quantitative morphological and mechanical dataset generated in this study provides a reference for future investigations of macrophage–biomaterial interactions and may contribute to the development of immune-informed biomaterial design. Future work should integrate advanced imaging techniques, optimized AFM probes, and physiologically relevant culture systems to further elucidate the relationship between cytoskeletal organization, cell mechanics, and macrophage phenotype.