Integrated in vitro models for hepatic safety and metabolism: evaluation of a human Liver-Chip and liver spheroid

Abstract

Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose-and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC 50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC 50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC 50 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in 3-Dimensional Models

“…cell-based therapies). Similar limitations are apparent in those liver-on-a-chip microfluidic devices that culture PHHs in monolayers on synthetic or natural membranes 8,9 . Furthermore, in contrast to static multiwell plates amenable to industrial-scale robotic infrastructure, perfusionbased devices (i.e.…”

Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity

“…Due to the small number of cells present in each spheroid it is perhaps unsurprising that the only miRNA reliably detected in cell culture media samples was miR-122. Previous in vitro studies in both 2D and 3D have indicated that miR-122 is at least as sensitive as ATP and LDH in reporting cytotoxicity (Foster et al 2019;Kia et al 2015a, b;Proctor et al 2017), however currently it does not provide any additional mechanistic information. Where miR-122 may come into further importance is for example in the use of multi-organ in vitro systems where tissue-specific markers will be of significant value (Skardal et al 2017).…”

“…In addition, actual ratios in vivo are likely to fluctuate depending on the current inflammatory status of the tissue. Another study focusing on screening DILI compounds for hepatotoxicity utilised spheroids containing 90% PHH and 10% NPC (Foster et al 2019). In this study, the functionality of the NPCs was not assessed.…”

“…LSECs lining the sinusoids. For situations where a retained tissue architecture is vital, microphysiological systems (Foster et al 2019) or bioprinting techniques (Ma et al 2016;Nguyen et al 2016) may provide ways to incorporate key anatomical features, such as the space of Disse. However, these systems in general have much lower throughput than 3D spheroids, which can be produced in 96-or 384-well plates.…”

Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

“…Therefore, we tested the utility of intermittently starved MPCCs for druginduced hepatotoxicity and drug-mediated CYP induction assays after 4 weeks of culture and compared to non-starved controls of the same culture age. Of the 5 hepatotoxic drugs, both the non-starved and starved MPCCs correctly classified these compounds as toxic based on downregulations of albumin and urea secretions, which correlate with compound-induced hepatotoxicity (as assessed with markers such as ATP) in MPCCs and other platforms (3,28,37). However, for the 5 non-hepatotoxic drugs, non-starved MPCCs falsely identified 3 of 5 drugs as toxic, whereas starved MPCCs correctly identified all 5 compounds as nonhepatotoxic, which is consistent with previously published results with treatment of 1-week old and non-starved MPCCs with the same compound set (3).…”