Figure 3: Real-time impedance monitoring of HepaRG-based liver-on-chip device following 24 hour APAP challenge.

(a) Protocol time line: Following 24 hours rifampicin induction in confluent HepaRG cells, cells were treated for 24 hours with APAP. (inset key: untreated cell control; 5 mM; 10 mM; and 20 mM). (b) Post-challenge resistance kinetics: APAP caused a dose-dependent decline in normalized resistance - a global indicator of cellular status. (c) Rb (cell-cell tight junctions): APAP disrupted TJs in a concentration- and time-dependent manner; compared with control values, Rb decreased significantly (by 6 hours) at 10 and 20 mM APAP (P < 0.01; Supplementary Table 1). (d) z-alpha: Cell-substrate adhesion disruption was detected at <10 hours, suggesting loosening of cells from the electrode surface. At 5 mM APAP, cell adhesiveness decreased 10% at 6 hours, to 25% by 12 hours, and below 50% at 24 hours (P < 0.01; Supplementary Table 1). No change in cell behaviour was observed with standard biochemical toxicity assays at this dose (Supplementary Fig. 2). (e) Quantitative Cm values (membrane capacitance), reflecting cell membrane integrity, were significantly compromised both at high APAP doses (10–20 mM), and earlier (12 hours), than those of cell viability measured in corresponding 24 hour biochemical toxicity assays. See Supplementary Table 1 and Supplementary Fig. 3 for corresponding non-normalized data.