Automated assessment of tumor spheroids via electrical impedance spectroscopy

Abstract

Three-dimensional tumor spheroids (3D-TS) provide a relevant in vitro model that more closely mimics human tumor biology than traditional cell cultures. However, current assessment techniques, including bright-field microscopy and endpoint assays, are limited by their inability to continuously monitor spheroid dynamics. Here, we present a noninvasive, label-free platform that combines electrical impedance spectroscopy with microelectrode arrays (MEAs) to enable real-time monitoring of 3D-TS. We evaluated several clustering approaches and developed an automated threshold-detection algorithm to distinguish electrodes covered by spheroids from uncovered electrodes based on impedance changes. This approach eliminates the need for manual intervention and optical confirmation, providing a robust solution for automated investigations. Using human colorectal cancer spheroids at different developmental stages, we demonstrated that impedance measurements obtained with two MEA types (rigid, glass-embedded ones and flexible, mesh - MEAs) capture spatial and temporal changes in spheroid behavior. Application of our automated algorithm enabled streamlined, high-throughput recording of 3D-TS under control condition and after application of the chemotherapeutic drug Navitoclax. Across both MEA platforms, adherent spheroids maintained stable impedance profiles under control conditions over several days in culture, whereas treatment with Navitoclax induced a significant gradual decline. Notably, the mesh-MEA configuration facilitated long-term measurements by maintaining stable spheroid positioning. Our findings demonstrate that MEA-based impedance sensing, combined with automated analysis, enables continuous monitoring of spheroid physiology and drug responses. The platform may become a powerful tool for preclinical cancer research and drug screening applications.