Nanofibrillar Cellulose Hydrogels with Anionic Surface Modifications for Modulating Macrophage Phenotype in 3D Culture.

Anti-inflammatory M2 macrophages are highly relevant in various physiological processes ranging from tissue regeneration to cancer progression. However, conventional two-dimensional (2D) in vitro cell cultures limit our understanding of macrophage phenotypes and how they can be modulated for immunotherapeutic approaches. Moreover, there is a growing demand for scalable, animal-free hydrogels to replace animal-derived materials in three-dimensional (3D) in vitro models. In this study, we explore hydrogels based on plant-derived nanofibrillar cellulose (NFC), also known as cellulose nanofibrils (CNFs) or microfibrillated cellulose (MFC), for generating 3D in vitro models of M2-like macrophages from human blood monocytes. Notably, flow cytometry analysis shows that cells cultured in 3D phosphorylated NFC hydrogels show enhanced expression of the M2 macrophage marker CD206 compared to cells cultured in other negatively charged hydrogels prepared from native NFC or NFCs with carboxylate or sulfate modifications. Furthermore, the upregulation of CD206 expression in 3D phosphorylated NFC is comparable to the induction of CD206 in interleukin 4 (IL-4)-differentiated M2a macrophages. In addition, the cells in the phosphorylated NFC hydrogel show a differential cytokine profile compared to 2D cultured cells, secreting similar levels of tumor necrosis factor α (TNF-α), but 2.6-fold higher amounts of IL-1β and 1.2-fold higher amounts of IL-10. The results suggest that the conversion of monocytes to M2-like macrophages can be controlled by the phosphorylation of NFC, a strategy which does not require the addition of polarization factors like growth factors and cytokines conventionally used to generate macrophages in vitro. The findings highlight the importance of surface chemistry in matrix-guided macrophage polarization, paving the way for xeno-free yet bioactive 3D macrophage culture scaffolds for immunological research.