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

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.