Collagen–Hyaluronic Acid Composite Hydrogel as a Scaffold for 3D Culture: Enhancing UC‐MSCs Viability and Trilineage Differentiation Potential

Traditional two‐dimensional (2D) cell culture in petri dishes poorly mimics the in vivo environment, limiting its relevance for studying normal development and cancer progression. Biomaterials, particularly hydrogels with three‐dimensional (3D) networks resembling the extracellular environment, offer solutions, with natural polymer‐based ones favored for biocompatibility. This study investigated a collagen–hyaluronic acid (Col–HA) composite hydrogel for human umbilical cord mesenchymal stem cells (UC‐MSCs) culture, exploring its impact on microstructure and gene expression during spontaneous differentiation. Fabricated via two‐step cross‐linking—premixing in balanced salt solution, then photopolymerization with lithium phenyl (2,4,6‐trimethylbenzoyl) phosphinate under 420 nm blue–violet light—the hydrogel formed a natural polymer network. Its porous architecture enabled excellent permeability by long‐term culture. Scanning electron microscopy (SEM) showed it effectively encapsulated and supported UC‐MSCs in 3D culture. After 14 days in a neutral medium, multilineage mRNA analysis revealed the 3D environment promoted UC‐MSCs spontaneous differentiation. The Col–HA composite hydrogel exhibited high stability, permeability, biocompatibility, and supported differentiation, highlighting its potential to enhance stem cell differentiation and improve 3D culture performance.