Geometric interface-guided self-formation of hollow multicellular spheroids in engineered composite structured hyaluronic acid (HA) hydrogels: Experiment and modeling.

The extracellular matrix (ECM) is a noncellular structure component that provides mechanical support and mediates chemical and physical guidance for cellular behaviors. Hydrogels are typical ECM-mimicking materials, which are intricately associated with cellular self-organization. This study pioneers a composite hyaluronic acid (HA) hydrogel system with micropatterned interfaces that directs mesenchymal cells to self-organize into 3D hollow spheroids-unachievable in conventional homogeneous hydrogels. By designing rectangular groove interfaces between stiffness-differentiated hydrogel domains (cell-laden soft gel within grooves of cell-free stiff gel), we establish a spatiotemporally regulated niche for multicellular morphogenesis. The experimental findings reveal that 3D hollow multicellular spheroids with large internal cavities (100-250 μm in diameter) preferentially form adjacent to the engineered groove interfaces. Computational simulations reveal that the self-formation of hollow spheroids is mediated by the designed geometry of the hydrogel interface separating the sub-domains with differing stiffness. Hydrogel sub-domains with distinct mechanical properties and tailored sizes exhibit differential biomolecular diffusion and cell migration kinetics, thereby establishing a complex spatiotemporal regulation of the reaction and diffusion processes of biomolecules and cells. Experimental data align with our activator-inhibitor-substrate (AIS) model, demonstrating that interfacial geometry controls reaction-diffusion dynamics to induce cavity formation. This platform enables the on-demand fabrication of 3D tissue analogues with luminal structures, laying a foundation for advancing organoid engineering and pathological modeling.