3D Bio‐Printing and Dynamic Maturation of Heart Valve Organoids Made of Human Induced Pluripotent Stem Cell‐Derived Valve Interstitial Cells

Abstract Heart valve organoids hold promise for modeling human valve biology, yet existing platforms often lack human‐relevant cell sources, preserved multicellular architecture, and physiologic mechanical cues, limiting their capacity to recapitulate extracellular matrix (ECM) formation and structural maturation. Here, a strategy is established that integrates human induced pluripotent stem cell‐derived valve interstitial cells (hiPS‐VICs), spheroid organization, bio‐printable hydrogels, and controlled mechanical conditioning to engineer advanced valve organoids. 3D bio‐printing of hiPS‐VIC spheroids is conducted within GelMA/ChSMA constructs, systematically compares spheroid‐based bio‐printing (SBB) with single‐cell bio‐printing (SCB), and establishes mechanically‐stimulated spheroid‐based bio‐printing (MSSB) to guide ECM remodeling. Spheroid bioprinting preserved cell–cell organization and improved post‐print structural integrity, resulting in substantially enhanced ECM deposition and assembly over time when compared with SCB. Superimposing physiologic loading further promoted cellular spreading and ECM deposition, accompanied by transcriptional signatures associated with matrix production, matrix stabilization, and mechano‐responsive pathways. Together, this spheroid‐based, mechanically matured, human iPSC valve organoid platform provides a robust and physiologically relevant system that supports ECM development and structural organization beyond dissociated‐cell approaches, offering a scalable foundation for human valve biology research, disease modeling, and translational applications.