VitroOrganoSphere (VOS): an automated droplet-engineered organoid sorting platform.

Industrialization and clinical translation applications based on organoids are often limited by low automation, high matrix consumption, and poor reproducibility. Here we present the VitroOrganoSphere (VOS) system, an automated droplet-engineered organoid sorting platform, which integrates droplet generation, photoelectric detection, fluorescence-based content evaluation, sorting, and automated dispensing into 96-well plates. Monodisperse droplets (CV <3%) are deposited as single droplets per well with Matrigel consumption as low as ∼0.3 μL. Real-time quality control enables rejection of empty or overloaded droplets and selective collection of droplets with appropriate encapsulation of viable organoids. Under the rigorous screening of two droplet selection criteria (size and sample content), the VOS system ultimately achieved a recovery rate of over 90% and accuracy of over 95%. Encapsulated organoids maintain viability and exhibit enhanced growth compared with manual methods. Drug sensitivity testing using patient-derived tumor organoids reliably distinguishes drug-sensitive and drug-resistant samples and reveals interpatient heterogeneity across multiple therapeutic agents. The VOS system establishes a standardized workflow for automated organoid-based drug screening and supports applications in precision medicine.