A High-Throughput Microphysiological Liver Chip System to Model Drug-Induced Liver Injury Using Human Liver Organoids

Abstract

Background and aims: Drug-induced liver injury (DILI) is a major failure mode in pharmaceutical development. This study aims to address the limitations of existing preclinical models by assessing a high-throughput, microfluidic liver-on-a-chip system, termed "Curio Barrier Liver Chips," and its capacity to recapitulate the effects of chronic hepatotoxic drug treatment through metabolic and phenotypic characterization.

Methods: Curio Barrier liver chips (Curiochips), fabricated in an 8 × 2 well configuration, were utilized to establish three dimensional liver organoid cultures. Human-induced pluripotent stem cells were differentiated into human liver organoids, and their viability, liver-specific functions, and pharmacological responses were assessed over 28 days.

Results: The Curiochips successfully maintained liver physiology and function, showing strong albumin secretion and cytochrome (CYP) P450 activities for 28 days. Unlike traditional models requiring millimolar drug concentrations to detect hepatotoxicity, this platform showed increased sensitivity for acetaminophen and fialuridine at micromolar concentrations. In situ differentiation of foregut spheroids to liver organoids was also achieved, further simplifying the establishment of liver chips. Furthermore, the chips demonstrated viability, function, and DILI responsiveness for 28 days, making this an improved model for studying idiosyncratic DILI with prolonged drug exposure and high-throughput capabilities compared to other available systems or primary human hepatocytes.

Conclusion: The Curiochips offer an advanced, miniaturized in vitro model for early-stage drug development and a sensitive, responsive, and cost-effective means to detect direct hepatotoxicity. Induced pluripotent stem cell liver organoids, in conjunction with the Curiochip, deliver a high-throughput platform with robust functionality and pharmacological responsiveness that make it a promising tool for improving the prediction and understanding of DILI risk prediction, especially with prolonged drug exposure. The model also opens new avenues for research in other chronic liver diseases.

Keywords: Curiochips; DILI Model; Drug-Induced Liver Injury; Hepatotoxicity; High-Throughput Screening; Microfluidic Liver Chips; Microphysiologic Liver System; iPSC-Derived Human Liver organoids.

Graphical abstract

Figure 1

Diagram of a 16-well HLO Curiochip with a fluorescent image of a single well and detailed configuration.

Figure 2

Twenty-eight-day Curiochip HLO viability. (A) HLOs were plated on Curiochips, and output media was periodically assessed for albumin secretion (n = 16). (B) CYP450 gene expression was quantified from parallel time points after cell lysis (n = 3). Data were compared to day 14 Curiochips. (C) Day 7 Curiochip cultures were assessed for metabolic function by quantifying molecular turnover of acetaminophen (APAP) and darunavir (DRV) exclusively metabolized by CYP450s using LC/MS/MS; n = 3 and 16 for HLO and Curiochips, respectively. Data are expressed as mean ± standard error of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; n.s., not significant.

Figure 3

Functional and phenotypic assessment of Curiochips as a long-term DILI model. (A) Schematic drawing of chronic DILI assay using HLOs on Curiochips. Curiochips were treated for 7 days with either (B) APAP or (C) FIAU and assessed for kinetic hepatocyte function and injury through biochemical analysis of albumin secretion and ALT activity. Multiple comparisons were made between sequential drug treatment time points. (D) Fluorescent images of vehicle control and drug-treated Curiochips stained for DNA and neutral lipid droplets were (E) quantified for total lipid intensity. Data are expressed as mean ± SEM of n = 16 Curiochips per treatment group. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; n.s., not significant.

Figure 4

In situ differentiation of HLOs on Curiochips. (A) Confocal fluorescence imaging of HLOs differentiated directly on Curiochips with cell-specific staining of hepatocytes (CEBPA) and hepatic stellate cells (vimentin). (B) Kinetic albumin secretion from in situ and ex situ differentiation of HLOs (n = 16). Multiple comparisons were made between sequential days of culture. The yellow dotted line represents the date of complete HLO differentiation (corresponds to day 0 in Figure 2). (C) CYP450 gene expression (n = 3) from in situ differentiated Curiochips compared to ex situ differentiation, both at day 21 of differentiation. DILI model evaluation of in situ Curiochips was quantified by ALT activity after (D) APAP and (E) FIAU treatments, over (n = 16 for each condition). Data are expressed as mean ± SEM; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; n.s., not significant.