The mammalian intestine relies upon continuous renewal of its epithelial lining to sustain digestion and the uptake of nutrients. Disruptions in this process contribute to disorders such as inflammatory bowel disease and colorectal cancer. Intestinal organoids, derived from stem cells, provide a powerful system for modeling epithelial biology; however, their culture most often relies on Matrigel®, an animal-derived basement membrane extract. Researchers widely adopt Matrigel because it is cost-effective and highly effective at supporting organoid growth, yet its ill-defined composition and batch-to-batch variability limit translational potential. Growing evidence suggests that extracellular matrix (ECM) mechanics, including stiffness and viscoelasticity, are significant in intestinal stem cell fate and morphogenesis. Synthetic hydrogel systems, such as poly(ethylene glycol) (PEG)-based platforms, provide tunable alternatives that eliminate the undefined biomechanical signals present in Matrigel, while enabling user-defined incorporation of essential adhesive ligands. This project will exploit photopatternable synthetic PEG-based hydrogels using a hydrogel-in-hydrogel approach, in which PEG is crosslinked inside Matrigel droplets. This hybrid strategy introduces controlled spatial mechanical heterogeneity while preserving biochemical support from Matrigel, allowing investigation of how local mechanics regulate intestinal organoid development. To evaluate feasibility, organoids were encapsulated within Matrigel droplets containing fluorescently labeled PEG, enabling simultaneous monitoring of macromer incorporation and organoid development. Diffusion kinetics were assessed across spherical droplets and flat layers to compare how geometry influenced pattern uniformity. Spherical droplets exhibited diffusion limitations, with macromer penetration more pronounced at the periphery than the core. Smaller, flat droplets improved diffusion through the core and were ultimately chosen for later experiments. Organoid growth was visualized, with budding events, curvature, and lumen architecture quantified over time, and morphology monitored throughout culture. Samples were also stained for secretory markers to assess lineage specification. This work addresses major limitations of current organoid systems by using spatiotemporal control of hydrogel mechanics and lays the groundwork for studies of how mechanical cues orchestrate intestinal tissue organization.
