Challenges and solutions in measuring commonly used biomarkers for drug-induced liver injury in a liver-on-a-chip platform.

Liver-on-a-chip (liver-chip) is designed to better maintain in vitro-cultured hepatic cells and improve the prediction of drug-induced liver injury (DILI). Albumin, urea, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) are proposed translational biomarkers for the prediction of DILI using liver microphysiological systems (MPS), including liver-chip. However, the performance of commonly-used assays for these biomarkers in liver MPS may vary. While using the Emulate® liver-chip, we observed that the activity of ALT, but not AST, measured using the extensively validated International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Reference Procedure, spontaneously and remarkably decreased in the perfusing medium after 24 h. Furthermore, ALT and AST activity remained largely below the detection limit (5 U/L) after acetaminophen treatment when their protein concentrations were increased by approximately 20-fold, as determined by an enzyme-linked immunosorbent assay (ELISA). The protein concentrations of ALT and AST, but not activity, showed good correlation with LDH activity, which was nearly eliminated after a freeze-thaw cycle. For viability of non-parenchymal cells, an unusually high background signal, largely attributable to CultureBoost and fetal bovine serum in the perfusion medium, prevented the use of LDH activity assays; however, LDH ELISA appeared to be a useful alternative. Of two widely used urea assays, the enzymatic approach was profoundly affected by medium supplements, wherein sample dilution increased the limit of detection, making it impossible to observe drug-induced urea inhibition. By contrast, the chemical assay offered adequate and improved specificity and sensitivity. For albumin, an ELISA had to be adopted, since routinely used dye-binding methods were not sensitive enough. These findings provide a plausible explanation for some controversies in the literature and highlight the significance of establishing reliable and reproducible assays for translational DILI biomarkers in liver MPS.