Noninvasive Viability Profiling of 3D Spheroids via Flow-through Electrical Impedance Spectroscopy

Three-dimensional (3D) cell models, such as spheroids and organoids, are powerful tools to enhance the predictive power of preclinical in vitro studies and support clinical translation of anti-cancer therapies. However, their integration into drug discovery pipelines remains limited, particularly for high-throughput screening, where rapid and reliable viability assessment is essential. Conventional viability assays, often dye-based or destructive, remain labor-intensive and poorly suited for high-throughput applications and downstream re-use of the models. In this study, we demonstrate, for the first time, that electrical impedance spectroscopy (EIS) enables label-free viability profiling of individual spheroids. For this purpose, a differential EIS sensor was integrated into a plastic microfluidic chip in flow-through configuration with self-aligned facing electrodes. Using six probing frequencies, we measured the EIS responses of liver cancer spheroids exposed to varying concentrations of dimethyl sulfoxide and doxorubicin, a chemotherapeutic compound. We identified an optimized frequency pair that enabled us to monitor spheroid viability, independent of spheroid size. Comparison with conventional viability assays, namely fluorescence imaging and ATP quantification, confirmed that the EIS method reliably reflected viability changes and revealed inter- and intra-treatment group differences owing to the method's single-spheroid resolution. The same frequency pair could be used to detect viability changes for both compounds, suggesting transferability of the EIS method across treatments. Furthermore, we showed that utilizing additional EIS features improved classification of spheroids with subtle viability differences. Together, these results demonstrate that EIS is a promising approach for noninvasive, single-spheroid viability profiling and a valuable tool for high-throughput drug screening with spheroid models.