Optimization of Vascularized Intestinal Organoid Model

Abstract Vasculature is crucial for maintaining organ homeostasis and metabolism. Although 3D organoids can mimic organ structures and patterns, they still lack vascular systems, limiting the recapitulation of physiological complexities. Although vascularization of organoids has been demonstrated by mixing Matrigel in fibrin, how the mixed gel niche affects endothelial cells (ECs) and organoids remains unclear. Existing protocols rely on fibroblasts to promote vascular network formation. This study explores how varying the ratio of Matrigel in fibrin‐Matrigel co‐gel affects vascular network formation and intestinal organoid growth. A fine‐tuned hydrogel is developed by adding aprotinin and 15% Matrigel in fibrin. Medium for co‐culturing ECs and organoids is modified with basic fibroblast growth factor (bFGF) and heparin. In combination with fine‐tuned hydrogel and modified medium, vascular network formation and organoid vascularization are successfully generated in the absence of fibroblast. Furthermore, structural cues and pore architectures are critical for angiogenesis and vascularization. By incorporating engineered thick collagen fiber bundles into the system, vascular network formation is guided by bundle architectures, enhancing interactions between vascular networks and organoids. The results demonstrate an optimized system that advances tissue and organoid vascularization by combining fiber bundles with fine‐tuned hydrogel and modified medium.