High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

The translation of 3D multicellular systems into clinical applications has been constrained by the need to balance physiological relevance and scalability. Current biofabrication methods primarily depend on passive cell aggregation or capillary- and viscosity-limited segmentation, resulting in stochastic heterogeneity that limits high-throughput screening (HTS). Here, we present OsciSphere, a chip-free droplet microfluidic platform that utilizes Weber-number-driven inertial forces to enable deterministic bioassembly of uniform 3D multicellular systems. Through programmable oscillatory acceleration, OsciSphere achieves precise, high-frequency droplet generation in standard well plates, eliminating the requirement for complex microfabrication. We demonstrate the versatility of this platform by generating miniaturized multicellular tumor spheroids (µMCTs) for drug screening, tissue-derived organoids (µTDOs) for pharmacological studies, and patient-derived organoids (µPDOs) that support tumor-immune co-cultures. In comparison to conventional Matrigel domes, OsciSphere-assembled 3D multicellular systems display improved uniformity, viability, and chemosensitivity. The platform's scalability enabled the screening of 49 commensal gut bacterial secretomes, leading to the identification of Eubacterium species that modulate cancer apoptotic pathways. Furthermore, µPDOs generated with OsciSphere support efficient infiltration of autologous PBMCs, enabling quantitative assessment of PD-1 blockade. This platform provides a robust, accessible approach to bridging the gap between complex tissue modeling and large-scale functional screening in precision oncology.