The Role of Methylcellulose in the Development of Physiologically Relevant in vitro Models for Cancer Research

Three-dimensional (3D) spheroid models are essential in cancer research, offering greater physiological relevance than 2D cultures by mimicking tumour microenvironment features. This study presents the first systematic, concentration-dependent optimization of methylcellulose as a semi-solid scaffold for spheroid formation. We identified 2.5 mg/mL methylcellulose and a seeding density of 30 000 cells/mL as optimal for generating uniform, structurally stable spheroids with high viability. Unlike previous studies using methylcellulose mainly for aggregation, we highlight its dual function in supporting both spheroid integrity and invasion in a concentration- and density-dependent manner. Lower concentrations (1.25 mg/mL) supported formation at low cell densities, while higher concentrations (>2.5$ mg/mL) reduced viability, likely due to limited nutrient diffusion. Moreover, spheroids formed under optimized conditions and embedded in collagen I exhibited strong TGF-β -induced invasion, replicating epithelial-to-mesenchymal transition features. Our findings position methylcellulose as a tunable, reproducible scaffold for producing viable, invasive 3D tumour spheroids, establishing a physiologically relevant platform for investigating cancer progression and therapeutic response.