Cellular Behavior of RAW264.7 Cells in 3D Poly(ethylene glycol) Hydrogel Niches

Abstract

Although macrophages undergo dynamic cellular responses in diverse extracellular environments, macrophage research has mostly relied on conventional culture methodologies such as two-dimensional and suspension cultures. In contrast, recent efforts have revealed evidence of the characteristic cellular behaviors of macrophages in actual tissues using a three-dimensional (3D) culture matrix. In this work, we exploited a poly(ethylene glycol)-based hydrogel as a macrophage culture matrix and observed cellular behaviors in 3D by manipulating the matrix properties. In the 3D microenvironment, macrophage-like RAW264.7 cells proliferated and formed spherical clusters by degrading the surrounding hydrogel network. Interestingly, we observed the significant upregulation of matrix metalloproteinases (MMPs) (i.e., MMP9 and MMP14) as well as M1 polarization markers (i.e., iNOS, COX2, TNF-α) in 3D, whereas M2 polarization markers (i.e., CD206, Arg1, TGF-β) were downregulated. Specifically, the expressions of both M1 and M2 markers were simultaneously increased in a stiff matrix compared to those of a soft matrix. In addition, matrix degradability significantly influenced the TNF-α secretion of encapsulated RAW264.7 cells. The MMP sensitivity of the hydrogel decreased TNF-α expression in a soft matrix, whereas it upregulated TNF-α in a stiff matrix compared to those of MMP-insensitive hydrogel. These findings suggest that the highly tunable poly(ethylene glycol) hydrogels can dictate macrophage behavior by altering the surrounding 3D microenvironment.