A parallelized rotary-perfusion bioreactor for enhancing hydrodynamic conditions in dECM-based organoid culture

• A parallelized 8-chamber rotary-perfusion bioreactor was successfully developed. • CFD identified 0.03 m/s and 3-6 rpm as optimal for low-shear dECM suspension. • The system maintains stable scaffold suspension with shear stress below 0.2 Pa. • Dynamic culture significantly improved MSC viability and 3D network organization. • The platform enables high-throughput, low-cost fabrication of complex organoids. The scalable and physiological fabrication of organoids remains a pivotal challenge in tissue engineering. Decellularized extracellular matrix (dECM) offers an ideal, organ-specific biomaterial scaffold, yet its full potential is often unrealized in static culture due to limited mass transport and poorly controlled mechanical environments. Here, we present a parallelized rotary-perfusion bioreactor designed to address these limitations by uniquely integrating simulated microgravity with low-shear perfusion-a combination that enables true three-dimensional suspension while avoiding the detrimental hydrodynamic forces associated with conventional dynamic systems. The system integrates eight independent culture chambers with precise control over perfusion and rotation, allowing for high-throughput, low-volume, and co-culture-compatible organoid production. Using computational fluid dynamics (CFD) coupled with experimental validation, we systematically optimized the operating parameters to achieve a stable, low-shear microenvironment ( τ e q , m a x < 0.3 P a , perfusion velocity 0.03 m/s, rotation speed 3-6 rpm). Biological validation using human umbilical cord mesenchymal stem cells cultured on murine dECM demonstrated that, compared to static controls, dynamic culture significantly enhanced cell viability (10 % higher on average), proliferation, and cytoskeletal organization over five days. This work establishes a robust, scalable platform for low-loading dynamic culture of cell-seeded dECM constructs, providing a foundation for subsequent organoid maturation under controlled rotation-perfusion conditions. By decoupling shear control from perfusion and integrating simulated microgravity, our approach addresses a key technological gap and offers a generally applicable strategy for engineering physiologically relevant 3D tissue models. The system holds promising potential for advancing tissue engineering, drug screening, and regenerative medicine applications.