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

Abstract

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.