Heterogeneous Spheroid Engineering: Spatial Organization, Fabrication, and Tissue-Specific Design.

Abstract

Three-dimensional (3D) spheroid models have emerged as advanced in vitro systems that overcome the limitations of conventional two-dimensional (2D) culture by better recapitulating native tissue architecture, cell-cell and cell-extracellular matrix interactions, and physiologically relevant gradients of oxygen, nutrients, and signaling factors, making them valuable platforms for tissue engineering and regenerative medicine. Realizing their full potential, however, requires rational control over cellular composition and spatial organization. In this review, we classify spheroids by cellular composition and spatial organization into single-cell-type and heterogeneous architectures-including mixed, core-shell, assembloid, and embedded co-culture configurations. We then summarize principal fabrication strategies encompassing static methods, dynamic suspension systems, and scaffold-based approaches. Spatially organized heterogeneous spheroids consistently enhance cell survival, paracrine signaling, vascularization, and tissue-specific differentiation relative to single-cell-type counterparts. We discuss applications across musculoskeletal, cardiovascular, nervous, epithelial, hepatic, pancreatic, renal, and urogenital tissues, highlighting how spatial design governs functional outcomes in each context. Spatial organization and fabrication strategy are decisive determinants of spheroid function for regenerative applications. Persistent challenges in vascularization, standardization, and scalable manufacturing must be addressed to enable reliable clinical translation, while emerging engineering approaches offer promising routes toward functional tissue constructs.