Acoustically driven rapid spheroid formation at a tunable gas-liquid interface

Growing societal attention to bioethics has increased the demand for alternatives to animal testing, prompting the development of new in vitro models. Spheroids are cell aggregates that provide microenvironments closely resembling in vivo tissue conditions, making them promising candidates for such alternatives. However, conventional methods such as sedimentation and hanging drop techniques typically require over 24 h for spheroid formation and are labor-intensive due to manual handling. Other formation strategies often rely on microfluidic platforms, but these complicate downstream handling and limit compatibility with standard bioanalytical workflows. Here, we introduce an acoustofluidic method that enables rapid spheroid formation via bubble oscillation-induced microstreaming at a pneumatically controlled gas-liquid interface. The interface, formed at the tip of a microcapillary, oscillates under acoustic excitation to produce localized vortical flows that drive efficient cell aggregation. We investigated how interface curvature affects vortex dynamics and cell aggregation behavior. Incorporation of ECM-based bioadhesives during the process further stabilizes spheroids. The technique is fully compatible with standard 96-well plate formats, making it readily applicable to existing workflows. This method offers a rapid, scalable platform for functional spheroid generation, suited for tissue engineering, organoid models, and drug screening.