A photo-crosslinkable hyaluronic acid-collagen hydrogel maintains epithelial integrity and supports intestinal organoid stemness

Abstract

Organoids provide physiologically relevant platforms for disease modeling and drug screening, but their performance depends critically on the extracellular matrix (ECM). Matrigel, the current standard, suffers from undefined composition, batch-to-batch variability, and limited tunability. We engineered a photo-crosslinkable hyaluronic acid-collagen (HA-collagen) composite hydrogel using riboflavin-mediated blue-light activation and examined how the collagen-to-HA ratio modulates physicochemical and biological properties. Storage modulus scaled with HA content (Col4HA1: 156 Pa; Col3HA1: 216 Pa; Col2HA1: 824 Pa vs 6 Pa for Matrigel), and organoid formation efficiency was inversely correlated with stiffness: Col2HA1 yielded significantly fewer organoids than Matrigel, whereas Col4HA1 fell within a mechanically permissive range and supported organoid formation comparable to Matrigel. Col4HA1 retained 48.6 ± 2.3% of its mass throughout the 14-day organoid culture period, whereas Matrigel underwent complete degradation by day 10. Organoids in Col4HA1 maintained stemness (Lgr5, Sox9) and differentiation markers (Villin), avoided stress-associated lysozyme elevation, and preserved E-cadherin/β-catenin junctions and epithelial polarity. Critically, Col4HA1 sustained stable Lgr5 expression across passages, whereas Matrigel showed passage-dependent Lgr5 elevation indicative of hyperproliferative drift. The photo-crosslinkable HA-collagen hydrogel offers a chemically defined, tunable, and reproducible Matrigel alternative for intestinal organoid research and regenerative-medicine applications.