Supplemental Material for: Endothelial-Fibroblast Organoids Generated in Hydrogel Microchamber Arrays: A Translational Model of Vascular Dysfunction in Cardio-Kidney-Metabolic Disease

Human umbilical vein endothelial cells (HUVECs) were cultured alone or with NIH3T3 fibroblasts, allowing direct comparison of growth configurations within a defined 3D microenvironment. Morphometric parameters were quantified longitudinally following glucose exposure to model diabetic-like conditions and treatment with a glucagon-like peptide-1 receptor agonist/insulin combination (Xultophy)<b>.</b></p><p dir="ltr"><b>Results:</b> The HMC platform supported reproducible formation of monolayers, scaffold-free spheroids, and matrix-embedded organoids.

Fibroblast co-culture enhanced endothelial organization and structural complexity. Glucose exposure induced time-dependent increases in organoid size and reduced circularity, indicating structural remodeling. Xultophy treatment moderated these effects and preserved higher circularity.

Sequential stimulation revealed order-dependent responses, with Xultophy partially attenuating glucose-induced expansion. Organoids exhibited stronger responses than spheroids, underscoring the role of extracellular matrix context.</p><p dir="ltr"><b>Conclusion</b>: The HMC system provides a reproducible platform for studying endothelial morphogenesis and stromal regulation in physiologically relevant 3D environments, enabling controlled investigation of endothelial responses to metabolic stress and therapeutic modulation</p>