Characterization of macrophage differentiation in hydrogel-based 3D models

Details

Supervisors

Dupont-Gillain, Christine C. ; Pierreux, Christophe

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enBreast cancer remains a major health concern and presents significant challenges for the development of effective treatments. In addition to uncontrollable cancer cells, the tumor microenvironment is composed of a 3D extracellular matrix and immune cells such as macrophages, which can adopt a pro-tumoral phenotype and promote cancer progression and therapy resistance. Traditional 2D models fall short in replicating the complexity of the TME, while 3D systems offer a more physiologically relevant alternative by better mimicking the extracellular matrix. This master's thesis aimed to investigate the differentiation of THP-1 monocytes into macrophages in both 2D and 3D environments, and to optimize hydrogel-based bioinks for the reproducible generation of viable 3D bioprinted scaffolds. First, gene expression of macrophage-associated markers was assessed by RT-qPCR in THP-1 cells treated with specific cytokines in 2D culture. In parallel, we optimized the preparation and bioprinting process of two bioinks, alginate-gelatin and hyaluronic acid-gelatin, by testing various mixing protocols and printing parameters. Finally, we compared the expression of the macrophage-associated markers in THP-1 cells embedded in 3D alginate–gelatin hydrogel, cultured for two weeks, to that of THP-1 cells treated in 2D. Our results confirmed that cytokine treatments promote THP-1 differentiation into macrophages in 2D and showed that the use of different markers allows the identification of M0, M1, and M2 macrophages. In 3D, THP-1 cells embedded in alginate-gelatin hydrogels acquired the expression of macrophage differentiation markers, suggestive of an M1-like polarization. This indicates that culturing THP-1 monocytes in a 3D matrix is sufficient, in the absence of cytokines, to trigger monocyte-to-macrophage differentiation. To obtain these bioinks, composed of viable cells embedded in hydrogel, we selected a Sequential mixing protocol that limits mechanical stress and optimizes hydrogel fluidity through gentle mixing and temperature control. Our experiments also demonstrated that while alginate–gelatin bioink supported cell proliferation, it showed greater sensitivity to everyday environmental conditions. Conversely, hyaluronic acid-gelatin bioinks offered greater printing stability but require further optimization for cell embedding and mixing. Altogether, our results advance the development of 3D printed biocompatible scaffolds that could significantly improve our understanding of the complex interactions between cancer cells, macrophages, and the surrounding extracellular matrix.