Effects of Substrate Stiffness on Macrophage Differentiation and Mechanobiology

Background: Monocytes are immune cells that circulate in the blood and can differentiate into macrophages with specific functions; those include fighting infection and reconstructing tissue to maintain homeostasis. The different Macrophage phenotypes and functions are co-regulated by the biochemical and the biophysical features of their microenvironment. Many diseases, such as cancer, are accompanied by changes in stiffness of the surrounding tissue and microenvironment. However, little is known about the influence of physical factors on the polarization, gene expression, and specified functionality of macrophages.

Methods: We evaluate the effects of substrate stiffness on the differentiation of monocytes into macrophages, by using THP-1 human cell lines. Those are seeded on a two-dimensional (2D), synthetic, polyacrylamide (PAM) gel-substrates with controlled stiffness; stiffness is chosen in soft-tissue range and verified by rheometry. Monocyte differentiation into macrophages is chemically initiated and flow cytometry is used to detect changes in cell size, granularity, cytokine secretion, and specific cell markers indicative of differentiation. Concurrently, we run a particle-phagocytosis assay to identify increase in internalization, typical for macrophages.

Results: Cells that are differentiated on 2D PAM substrate with stiffness of 2.4-9.1 kPa, form three-D clusters of varying sizes. At different differentiation stages - 0,1,2,5 and 7 days post initiation the uptake of 2 µm beads increases.

Conclusion: The physical characteristics of the macrophages’ microenvironment can affect their differentiation and activation, drive or inhibiting progression of disease.