Liver Spheroids Research Articles

Self-Organized Liver Microtissue on a Bio-Functional Surface: The Role of Human Adipose-Derived Stromal Cells in Hepatic Function.

The maintenance of hepatocyte function is a critical research topic in liver tissue engineering. Although an increasing number of strategies have been developed, liver tissue engineering using hepatocytes as a therapeutic alternative remains challenging owing to its poor efficacy. In this study, we developed a multicellular hepatic microtissue to enhance the function of induced hepatic precursor cells. Mouse induced hepatic precursor cells (miHeps) were self-organized in 3D with human adipose-derived stem cells (hASCs) on a bio-functional matrix. We found that hepatic phenotypes, such as levels of albumin, asialoglycoprotein receptor-1, and cytochrome P450, were enhanced in miHeps-hASC microtissue comprising miHeps and hASCs relative to two-dimensional-cultured miHeps-hASCs. Additionally, the secretome of 3D-cultured hASCs increased the hepatic function of mature miHeps. Furthermore, hepatic gene expression was reduced in mature miHeps treated with conditioned media of hypoxia-inducible factor 1α (HIF1α)-depleted hASCs relative to that with conditioned media of control hASCs. Our results suggested that the hepatic function of 3D-co-cultured miHeps could be enhanced by HIF1α-dependent factors secreted from stromal cells. This study provides an insight into the factors regulating hepatic function and shows that self-organized hepatic microtissue could act as liver spheroids for liver regenerative medicine and liver toxicity tests.

Mechanism of baricitinib supports artificial intelligence‐predicted testing in COVID‐19 patients

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID‐19 infection via proposed anti‐cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID‐19 infection. We validated the AI‐predicted biochemical inhibitory effects of baricitinib on human numb‐associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID‐19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS‐CoV‐2 viral load, inflammatory markers, and IL‐6 levels. Collectively, these data support further evaluation of the anti‐cytokine and anti‐viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID‐19 patients.

3D Microscopy Keeps Getting Faster, Smarter, Leaner

3D Microscopy Keeps Getting Faster, Smarter, Leaner

Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents

A microfluidic multi-organ chip emulates the tissue culture microenvironment, enables interconnection of organ equivalents and overcomes interspecies differences, making this technology a promising and powerful tool for preclinical drug screening. In this study, we established a microfluidic chip-based model that enabled non-contact cocultivation of liver spheroids and renal proximal tubule barriers in a connecting media circuit over 16 days. Meanwhile, a 14-day repeated-dose systemic administration of cyclosporine A (CsA) alone or in combination with rifampicin was performed. Toxicity profiles of the two different doses of CsA on different target organs could be discriminated and that concomitant treatment with rifampicin from day6 onwards decreased the CsA concentration and attenuated the toxicity compared with that after treatment with CsA for 14 consecutive days. The latter is manifested with the changes in cytotoxicity, cell viability and apoptosis, gene expression of metabolic enzymes and transporters, and noninvasive toxicity biomarkers. The on chip coculture of the liver and the proximal tubulus equivalents showed its potential as an effective and translational tool for repeated dose multi-drug toxicity screening in the preclinical stage of drug development.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung–liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.

A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model.

Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip? Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1week and the co-cultures reproduced specific natural and drug-induced liver-testis systemic interactions. Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver-testis axis. However, these are important to study the biotransformation of substances. Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver-testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated. The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively. Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture. N/A. The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured. This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level. This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis.

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which...

Gaining New Biological and Therapeutic Applications into the Liver with 3D In Vitro Liver Models.

Three-dimensional (3D) cell cultures with architectural and biomechanical properties similar to those of natural tissue have been the focus for generating liver tissue. Microarchitectural organization is believed to be crucial to hepatic function, and 3D cell culture technologies have enabled the construction of tissue-like microenvironments, thereby leading to remarkable progress in vitro models of human tissue and organs. Recently, to recapitulate the 3D architecture of tissues, spheroids and organoids have become widely accepted as new practical tools for 3D organ modeling. Moreover, the combination of bioengineering approach offers the promise to more accurately model the tissue microenvironment of human organs. Indeed, the employment of sophisticated bioengineered liver models show long-term viability and functional enhancements in biochemical parameters and disease-orient outcome. Various 3D in vitro liver models have been proposed as a new generation of liver medicine. Likewise, new biomedical engineering approaches and platforms are available to more accurately replicate the in vivo 3D microarchitectures and functions of living organs. This review aims to highlight the recent 3D in vitro liver model systems, including micropatterning, spheroids, and organoids that are either scaffold-based or scaffold-free systems. Finally, we discuss a number of challenges that will need to be addressed moving forward in the field of liver tissue engineering for biomedical applications. The ongoing development of biomedical engineering holds great promise for generating a 3D biomimetic liver model that recapitulates the physiological and pathological properties of the liver and has biomedical applications.

Three-Dimensional Spheroids With Primary Human Liver Cells and Differential Roles of Kupffer Cells in Drug-Induced Liver Injury

Drug-induced liver injury (DILI) remains a challenge and a leading risk for drug discovery. Three-dimensional liver spheroids made from primary human hepatocytes (PHHs) with, or without, other liver cell types can provide more physiological relevance. In comparison to conventional 2-dimensional monolayer culture, our tests with 100 drugs of known DILI status indicate that PHH spheroids are significantly more sensitive in detecting drug-induced hepatotoxicity. To evaluate the role of Kupffer cells (KCs) in drug-induced liver toxicity, we have established conditions for generating co-culture spheroids with PHH and KCs. Inflammatory responses as shown by interleukin 6 secretion can be recapitulated in co-culture spheroids when treated with endotoxin lipopolysaccharides. KCs potentiated the cytotoxicity induced by trovafloxacin in co-culture spheroids at 48 h, but the differences between PHH spheroids and co-culture spheroids became less obvious after a 5-day treatment. Interestingly, a protective role of KCs was shown in co-culture spheroids treated with both acetaminophen and lipopolysaccharides. Additional tests with 14 DILI compounds comparing PHH spheroids and co-culture spheroids showed differential roles of KCs that were compound dependent. In summary, these 3-dimensional liver spheroid models are useful tools to understand the complex mechanisms underlying DILI.

Multiscale modelling of drug transport and metabolism in liver spheroids

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo. While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

Regorafenib Alteration of the BCL-xL/MCL-1 Ratio Provides a Therapeutic Opportunity for BH3-Mimetics in Hepatocellular Carcinoma Models.

Background: The multikinase inhibitor regorafenib, approved as second-line treatment for hepatocellular carcinoma (HCC) after sorafenib failure, may induce mitochondrial damage. BH3-mimetics, inhibitors of specific BCL-2 proteins, are valuable drugs in cancer therapy to amplify mitochondrial-dependent cell death. Methods: In in vitro and in vivo HCC models, we tested regorafenib’s effect on the BCL-2 network and the efficacy of BH3-mimetics on HCC treatment. Results: In hepatoma cell lines and Hep3B liver spheroids, regorafenib cytotoxicity was potentiated by BCL-xL siRNA transfection or pharmacological inhibition (A-1331852), while BCL-2 antagonism had no effect. Mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-3 activation mediated A-1331852/regorafenib-induced cell death. In a patient-derived xenograft (PDX) HCC model, BCL-xL inhibition stimulated regorafenib activity, drastically decreasing tumor growth. Moreover, regorafenib-resistant HepG2 cells displayed increased BCL-xL and reduced MCL-1 expression, while A-1331852 reinstated regorafenib efficacy in vitro and in a xenograft mouse model. Interestingly, BCL-xL levels, associated with poor prognosis in liver and colorectal cancer, and the BCL-xL/MCL-1 ratio were detected as being increased in HCC patients. Conclusion: Regorafenib primes tumor cells to BH3-mimetic-induced cell death, allowing BCL-xL inhibition with A-1331852 or other strategies based on BCL-xL degradation to enhance regorafenib efficacy, offering a novel approach for HCC treatment, particularly for tumors with an elevated BCL-xL/MCL-1 ratio.

P161 - Quantitative metabolic profiling of slowly cleared chemicals in in vitro models including 3D liver spheroid and hepatopac and hepatic clearance predictions from in vitro data using gastroplus PBPK modelling following topical application

P161 - Quantitative metabolic profiling of slowly cleared chemicals in in vitro models including 3D liver spheroid and hepatopac and hepatic clearance predictions from in vitro data using gastroplus PBPK modelling following topical application

P136 - Optimization of 3D liver spheroid culture conditions of individual hepatocyte donors and pooled hepatocyte donors

P136 - Optimization of 3D liver spheroid culture conditions of individual hepatocyte donors and pooled hepatocyte donors

3D bioprinting of liver spheroids derived from human induced pluripotent stem cells sustain liver function and viability in vitro

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver’s epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Generation of liver organoids by 3D co-culturing of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells

Background and Hypothesis: Recent progress with combination of chemicals in cell culture media prolongs hepatocyte (HC) function in vitro. However, without other cells of hepatic lineage that comprise natural liver, HC alone are not suitable for the study of complex liver diseases. Hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) play vital roles in physiological and pathological liver function in situ. We hypothesized that coculturing primary HC with HSC and LSEC as 3D liver organoids in the same chemical conditioned media would uphold HC function over time, creating a better physiological 3D liver microenvironment for liver disease research.
 Experimental Design: Freshly thawed primary human HCs were combined with immortalized human HSC alone, or together with immortalized human LSECs, to generate 3D liver spheroids. Spheroids were formed in HC maintenance media (HMM, Lonza) using low adhesion 96-well plates for 6 days before switching to the media with different combination of chemicals (SB31542, Forskolin, IWP2, DAPT, and LDN193189) for culturing another 14 days. Spheroids characterization, albumin secretion, mRNA transcription (CYP3A4), and histological analysis were performed for HC differentiation, maturation, and function. 
 Results: Both co-cultures of HC:HSC (2.5:1 ratio), and HC:HSC:LSEC (2.5:1:1) formed spheroids in HMM within 4 to 6 days (Fig.1A). The introduction of the different chemical-based media affected the roundness and diameter of spheroids differently (Fig.1B). In the presence of all 5 chemicals (5C), HC function was better maintained up to 21 days in HC:HSC:LSEC spheroids, measured by albumin, CYP3A4, and CK-19 secretion (Fig.1C). Surprisingly, 5C-based media significantly upregulated the expression of CK-19, which is one of the markers for cholangiocytes and liver precursor cells. 
 Conclusion and Potential Impact: 3D liver organoids composed of HC, HSC, and LSEC would create a niche environment mimicking the in vivo condition. Optimization of complex hepatic spheroids, as well as optimization of complex hepatic spheroid culturing media, would allow for the generation of a unique model for studying complex liver diseases.

Application of New Cellular and Microphysiological Systems to Drug Metabolism Optimization and Their Positioning Respective to In Silico Tools

New cellular model systems for drug metabolism applications, such as advanced 2D liver co-cultures, spheroids, and microphysiological systems (MPSs), offer exciting opportunities to reproduce human biology more closely in vitro with the aim of improving predictions of pharmacokinetics, drug–drug interactions, and efficacy. These advanced cellular systems have quickly become established for human intrinsic clearance determination and have been validated for several other absorption, distribution, metabolism, and excretion (ADME) applications. Adoption will be driven through the demonstration of clear added value, for instance, by more accurate and precise clearance predictions and by more reliable extrapolation of drug interaction potential leading to faster progression to pivotal proof-of-concept studies. New experimental systems are attractive when they can (1) increase experimental capacity, removing optimization bottlenecks; (2) improve measurement quality of ADME properties that impact pharmacokinetics; and (3) enable measurements to be made that were not previously possible, reducing risk in ADME prediction and candidate selection. As new systems become established, they will find their place in the repository of tools used at different stages of the research and development process, depending on the balance of value, throughput, and cost. In this article, we give a perspective on the integration of these new methodologies into ADME optimization during drug discovery, and the likely applications and impacts on drug development.

The comet assay applied to HepG2 liver spheroids

In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of human toxicity of industrial chemicals and environmental pollutants. In this study we compared cytotoxic and genotoxic responses induced by chemicals in 2D and 3D spheroidal cultures of the human liver cancer cell line HepG2.HepG2 spheroids were prepared by hanging drop technology. Both 3D spheroids and 2D monolayer cultures were exposed to different chemicals (colchicine, chlorpromazine hydrochloride or methyl methanesulfonate) for geno- and cytotoxicity studies. Cytotoxicity was investigated by alamarBlue assay, flow cytometry and confocal imaging. DNA damage was investigated by the comet assay with and without Fpg enzyme for detection of DNA strand breaks and oxidized or alkylated base lesions.The results from the cyto- and genotoxicity tests showed differences in sensitivity comparing the 2D and 3D HepG2 models. This study shows that human 3D spheroidal hepatocellular cultures can be successfully applied for genotoxicity testing by the comet assay and represent a promising advanced in vitro model for toxicity testing.

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

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 (EC50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC50 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.