Systematic multi-parameter optimization of an injectable thermosensitive CS/β-GP/HEC hydrogel for 3D cell culture applications.

Injectable thermosensitive hydrogels have been widely investigated as minimally invasive scaffolds for three-dimensional (3D) cell culture. In this study, we systematically screened a chitosan (CS)/β-glycerophosphate (β-GP)/hydroxyethyl cellulose (HEC) ternary hydrogel system using a 2 × 2 × 2 factorial design. By evaluating gelation kinetics, injectability, and structural stability through a weighted scoring model, two superior formulations (G1 and G3) were selected for their ability to balance rapid gelation at 37 °C with reliable room-temperature injectability. Subsequent biological evaluations demonstrated that, compared to conventional 2D cultures, the 3D hydrogels supported robust cellular viability and spatial proliferation through Day 10. The stable biomimetic matrix provided essential physical anchorage, enabling Huh7 cells to self-assemble into dense tumor spheroids and BMSCs to establish spatial networks. This study presents a structured formulation-screening strategy and demonstrates the effectiveness of the CS/β-GP/HEC hydrogel as a reliable platform for in vitro 3D cell culture applications.