Bioimpedance-Based Measurements of In Vitro Biological Cell Barrier Integrity: A Review and Framework for the...

In vitro cell barrier models have been increasingly integrated into pharmaceutical and academic research pipelines to evaluate drug safety and drug delivery due to a shift towards New Approach Methodologies (NAMs) in research and regulatory safety assessment. Such models require reliable and interpretable functional readouts. Bioimpedance-based monitoring, particularly transepithelial/endothelial electrical resistance (TEER), is a widely adopted readout due to its non-invasive and real-time capabilities. However, substantial variability arises from differences in measurement settings, frequency selection, electrode configuration, impedance measuring techniques, and data analysis strategies. In numerous studies, TEER is approximated from single-frequency impedance magnitude measurements, which do not isolate the resistive component associated with tight junction-mediated paracellular transport but instead reflect the combined response of a coupled electrochemical system. This review clarifies impedance measuring techniques and systematically analyzes impedance-based measurement and analysis strategies for in vitro biological cell barrier integrity. We compare mono-frequency and broadband acquisition approaches, examine the influence of electrode-electrolyte interfaces, electrode geometry, and culture configuration, and evaluate equivalent circuit modeling and phase-resolved electrical impedance spectroscopy (EIS). Based on this comparison, we propose a three-level analytical hierarchy adapted to experimental objectives and instrumentation constraints. We conclude that phase-informed impedance analysis and harmonized reporting are essential to improve measurement reproducibility, inter-platform comparability, and integration of impedance-derived cell barrier assessment within NAMs-oriented research workflows.