Electromagnetic validation of a multichannel coplanar four-electrode bioimpedance sensing platform for perfused 3D tumor-on-chip systems.

Three-dimensional tumor-on-chip systems better reproduce the spatial organization of tumor cells within extracellular-matrix-based microenvironments than conventional two-dimensional cultures, yet their integration with quantitative, non-destructive monitoring tools remains limited. Electrical bioimpedance provides a label-free approach for real-time assessment of living 3D tissues, but its implementation in perfused millimeter-scale microfluidic environments requires rigorous electrical and geometrical validation. In this study, we designed and electromagnetically validated an integrated coplanar four-electrode bioimpedance sensor embedded within a perfused 3D tumor-on-chip platform compatible with collagen-based hydrogels. Three-dimensional finite-element simulations were used to compute sensitivity fields and quantify the effective measurement volume. Calibration with conductivity standards showed quantitative agreement between experimental measurements and numerical predictions, with relative deviations below 10%, enabling reliable extraction of the cell constant. Chamber height was identified as the dominant geometrical parameter affecting impedance magnitude, whereas lateral misalignment had limited impact. Temperature-dependent conductivity changes induced impedance drifts of up to 25% during thermal equilibration, highlighting the need for strict temperature control. Acellular collagen I hydrogels exhibited concentration-dependent impedance variations, while increased gel loading before chamber closure modified impedance, likely through matrix compression and reorganization. These results demonstrate that the extracellular matrix significantly contributes to the measured electrical response. In static 3D cultures, embedding pancreatic cancer cells within collagen I produced a reproducible decrease in impedance magnitude at 10 kHz compared with acellular controls, confirming a measurable cell-dependent baseline shift. Under continuous perfusion, gradual cellular evolution did not generate a reproducible impedance trajectory, whereas Triton X-100-induced cell disruption increased impedance toward the acellular collagen I baseline. Overall, this study demonstrates that coplanar impedance sensing can detect cell-dependent baseline differences and strong endpoint perturbations in millimeter-scale 3D tumor models. However, dynamic impedance variations under perfusion remain influenced by matrix, thermal, geometrical, and conductive-pathway contributions, emphasizing the need for careful calibration before biological interpretation.