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.