Abstract
Collagen type I, a key component of the extracellular matrix, underpins cellular organization and regeneration but remains challenging to process into scalable, reproducible, and biologically faithful 3D materials. Although several recent platforms have achieved high-throughput collagen microgel fabrication, most rely on chemical modification or rigid process parameters that compromise native collagen structure or limit biological versatility. A scalable yet biologically unmodified system remains lacking. Here, we developed a pressure-tunable microfluidic platform that enables the rapid, high-throughput generation of collagen microbeads with controlled size, fibrillar architecture, and mechanical properties for versatile downstream biofabrication. By adjusting collagen concentrations (0.25-3 mg mL(-1)) and thermal self-assembly, we produced monodisperse microbeads (45-1000 μm) at rates exceeding 10,000 beads min(-1) (~166 Hz) while preserving the native triple-helical structure. The microbeads exhibited tunable stiffness (0.6-2.8 kPa) and pore structures confirmed by scanning electron microscopy, enabling systematic modulation of the 3D microenvironment for cell culture and bioprinting. When incorporated into alginate-gelatin bioinks, the collagen microbeads enhanced rheological stability, shear-thinning, and print fidelity, supporting sustained cell growth that reached a 13-fold increase in proliferation over 21 days; independently, cell-laden collagen microbeads functioned as self-contained microbioreactors enabling multicellular spheroid formation and continuous EV secretion. This modular and naturally derived, unmodified collagen microbead technology unites tunability, throughput, and functional scalability, establishing a versatile platform for tissue modeling, drug testing, and therapeutic EV biomanufacturing.