Predose (measured at day 4) albumin, urea, and LDH levels stratified across different donors. Hu1601, Hu1604, Hu1624, Hu8150, and Hu8181 are lot numbers corresponding to five different donors. “Pooled” refers to the pool of hepatocytes from the five donors, and “All donors” refers to the data from all five donors merged together. Red lines correspond to the mean of the data, purple boxes extend the mean by ± 1 S.D., and pink boxes correspond to 95% confidence intervals around the mean. LDH levels are expressed in optical density (OD) units at 490 nm.

Comparison between the predose (measured at day 4) and the postdose (measured at day 6 for diclofenac, propranolol, lidocaine, and ibuprofen and days 5 and 7 for phenacetin and prednisolone, respectively) albumin, urea, and LDH levels stratified across different treatments. Data from both the five donors and the pooled hepatocytes are shown. Red lines correspond to the mean of the data, purple boxes extend the mean by ± 1 S.D., and pink boxes correspond to 95% confidence intervals around the mean. Thin black lines connect the pre- and postdose levels in a given donor (or pool of donors) and well. Asterisks inside each subplot indicate significant differences between pre- and postdose levels (*P < 0.05, **P < 0.01, ***P < 0.001). LDH levels are expressed in optical density (OD) units at 490 nm.

Volcano plot that illustrates the average fold-change in gene expression between the liver MPS (day 6) and freshly thawed hepatocytes along with the associated statistical significance. The log₂ of the fold-change is plotted on the x-axis; thus, positive values indicate upregulation in the liver MPS compared with the freshly thawed hepatocytes, whereas negative values indicate downregulation. Genes outside the two black vertical lines are up- or downregulated more than 3-fold. On the y-axis, the –log₁₀ of the P value is plotted; thus, the higher values indicate stronger statistical evidence of a significant difference in gene expression between the liver MPS and freshly thawed hepatocytes. The genes for which significant differences were detected after multiple testing correction are highlighted in red, and the respective gene names are reported.

Drug depletion data available for the pharmacokinetic analysis. Hu1601, Hu1604, Hu1624, Hu8150, and Hu8181 are lot numbers corresponding to five different donors. “Pool” refers to the pool of hepatocytes from the five donors. The small numbers on the right of each concentration point (values of 1, 2, or 3) aim to distinguish different wells across the same donor (or pool of donors).

Visual predictive checks of the developed mixed-effect models with regard to the observed individual-donor drug depletion data. Closed gray circles represent the observed concentrations in medium; highlighted with purple are the areas between the 5th and 95th percentiles of model simulations that take into account the different levels of variability (90% prediction intervals), whereas the red solid line represents their median (median prediction); the horizontal dashed black line represents the limit of quantification.