A Fully Defined GelMA‐Based Matrix Allows Fine Tuning of Tissue‐Relevant Biomechanical and Biochemical Cues for Organoid Culture

ABSTRACT Organoids are powerful models for developmental biology, disease modelling, and personalized medicine. However, their broader application is limited by the inability of extracellular matrix (ECM) hydrogels to mimic the biomechanical and biochemical properties of native tissues. Dependence on poorly‐defined basement membrane matrices such as Matrigel—a xenogenic, tumor‐derived, nontunable material—introduces organoid growth variability and constrains reproducibility across organoid systems. In this study, we report a fully defined ECM platform that integrates key biomechanical and biochemical features of native tissue to support organoid growth. Using gelatin methacryloyl (GelMA) as a tunable hydrogel backbone, supplemented with laminin–entactin complex, we established a matrix with physiologically relevant stiffness and biochemical composition for mouse small intestinal organoids (mSIOs). The optimized matrix (2% w/v DS95 GelMA with 2 mg mL − 1 laminin–entactin) supported robust mSIO proliferation, morphology, and differentiation comparable to or exceeding that achieved with Matrigel. Importantly, continuous passaging was achieved without affecting organoid quality, suggesting feasibility of long‐term mSIO expansion. These findings demonstrate that coordinated control of matrix mechanics and biochemical cues is critical for organoid development. The simplicity, stability, and tunability of this GelMA‐based system provides a scalable and reproducible alternative to Matrigel, with broad potential for organoid culture, mechanistic studies, and translational applications.