Tuning physical properties of chitosan-based hydrogels to guide 3D spheroid assembly of C2C12 myoblasts

Biobased hydrogels are promising scaffolds for three-dimensional (3D) cell culture, but achieving suitable mechanical stability while minimizing the use of synthetic polymers remains challenging. In this study, chitosan-based hydrogels containing very low concentrations of poly(vinyl alcohol) (PVA) (< 1 wt. %) were developed to investigate how trace amounts of PVA influence hydrogel structure, physical properties and 3D spheroid formation of C2C12 myoblasts. Hydrogels were crosslinked with citric acid and characterized in terms of microstructure, swelling behavior, gel fraction, mechanical stability and adhesion. Chitosan-only hydrogels exhibited porous microstructures, whereas chitosan/PVA blends formed more homogeneous structures without visible pores, even at low PVA contents. All formulations showed non-Fickian swelling behavior and high gel fractions (>95%). Hydrogels containing ~0.8 wt.-% PVA displayed the highest mechanical stability and adhesion after water immersion, while hydrogels with ~0.2 wt.-% PVA promoted rapid formation of complete 3D C2C12 spheroids within two days. These results demonstrate that trace amounts of PVA can effectively tune the properties of chitosan hydrogels, enabling the design of largely biobased scaffolds for 3D cell culture and soft tissue engineering. • Facile physical crosslinking at mild temperature • PVA content in chitosan/PVA hydrogels ≤ 1 wt.-% • High PVA with ~ 0.8 wt.-% citric acid ensured physical stability after water immersion • Low PVA with ~ 0.8 wt.-% citric acid enabled 3D C2C12 spheroids by day 2