Rocking-Induced Rapid Formation of Tumor Spheroids for Scalable 3D Tumor-Immune Coculture Assays

Tumor spheroids are widely used to model the tumor microenvironment and evaluate immune-mediated cytotoxicity, but conventional methods such as ultralow-attachment and hanging-drop plates typically require 48-72 h to generate stable spheroids, limiting scalability and rapid testing. Here, we report a simple method for generating uniform tumor spheroids within 7-8 h using continuous rocking in standard multiwell plates. Computational fluid dynamics simulations showed that rocking produced mild oscillatory hydrodynamic conditions, with average shear stress ranging from 0.4 to 0.8 dyn/cm² and X-direction shear stress remaining near ±0.2 dyn/cm², promoting controlled cell aggregation while maintaining spheroid integrity. Using this approach, Hep3B cells reproducibly formed spheroids 80-150 µm in diameter across multiple seeding densities, yielding thousands of spheroids per well. The spheroids were embedded in extracellular matrix and transferred to a 36PillarPlate for scalable 3D tumor-immune coculture assays. To demonstrate utility, Hep3B spheroids were cocultured with NK-92 cells in the presence or absence of the immunomodulator berberine. Berberine treatment enhanced NK-92 localization and spheroid disruption, resulting in reduced tumor viability. This approach reduces spheroid preparation time from days to hours and enables same-day integration into 3D assays, providing a practical platform for studying tumor-immune interactions and screening immunomodulatory therapeutics.