Hyaluronic Acid Molecular Weight Modulates Chitosan–Gelatin Scaffold Properties and Cancer Cell Organization in 3D Culture

Reconstructing physiologically relevant in vitro tumor microenvironments (TMEs) that capture coupled biophysical and biochemical complexities remains a significant challenge in cancer modeling. Here, we present a tunable chitosan–gelatin–hyaluronic acid (HA) scaffold platform stabilized by Schiff-base (C=N) crosslinking to investigate how HA molecular weight (MW) affects scaffold architecture, hydration behavior, mechanical properties, degradation stability, and cancer cell organization. We established an isocompositional series of HA-containing scaffolds, with HA MW as the primary variable and polymer composition held constant across all HA-containing groups. Varying HA MW modulated pore morphology, swelling behavior, compressive modulus, and degradation profiles, demonstrating distinct effects on scaffold structural organization. Medium-MW HA yielded a balanced scaffold architecture characterized by high porosity, controlled swelling behavior, and stable mechanical performance under hydrated conditions. A549 cells exhibited a compact, spheroid-like organization, whereas PANC-1 cells displayed more protrusive, spread morphologies that varied with scaffold formulation. Conversely, HA-free scaffolds showed reduced structural stability and less organized three-dimensional (3D) cell morphology. Collectively, these findings substantiate that HA MW is an effective design parameter for tuning scaffold physicochemical properties and influencing scaffold-associated cancer cell organization in 3D culture. This study provides a tunable scaffold platform for future in vitro tumor microenvironment modeling.