Microfluidic-assisted engineering of hydrogels with microscale complexity.

Hydrogels have emerged as a promising 3D cell culture scaffold owing to their structural similarity to the extracellular matrix (ECM) and their tunable physicochemical properties. Recent advances in microfluidic technology have enabled the fabrication of hydrogels into precisely controlled microspheres and microfibers, which serve as modular units for scalable 3D tissue assembly. Furthermore, advances in 3D bioprinting have allowed facile and precise spatial engineering of these hydrogel-based structures into complex architectures. When integrated with microfluidics, these systems facilitate microscale heterogeneity, dynamic shear flow, and gradient generation-critical features for advancing organoids and organ-on-a-chip systems. In this review, we will discuss (1) microfluidic strategies for the preparation of hydrogel microspheres and microfibers, (2) the integration of microfluidics with 3D bioprinting technologies, and (3) their transformative applications in organoids and organ-on-a-chip systems. STATEMENT OF SIGNIFICANCE: Microfluidic-assisted preparation and assembly of hydrogel microspheres and microfibers have enabled unprecedented precision in size, morphology and compositional control. The diverse configurations of these hydrogel modules offer the opportunities to generate 3D constructs with microscale complexity-recapitulating critical features of native tissues such as compartmentalized microenvironments, cellular gradients, and vascular networks. In this review, we discuss the fundamental microfluidic principles governing the generation of hydrogel microspheres (0D) and microfibers (1D), their hierarchical assembly into 3D constructs, and their integration with 3D bioprinting platforms to generate and culture organoids and organ-on-a-chip systems. The synergistic integration of microfluidics and bioprinting overcomes longstanding limitations of conventional 3D culture, such as static microenvironments and poor spatial resolution. Advances in microfluidic design offer tunable hydrogel biophysical and biochemical properties that regulate cell behaviors dynamically. Looking forward, the growing mastery of these principles paves the way for next-generation organoids and organ-on-a-chip systems with improved cellular heterogeneity, integrated vasculature, and multicellular crosstalk, closing the gap between in vitro models and human pathophysiology.