Characterization of diseased primary human hepatocytes in an all-human cell-based triculture system.

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-ß were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period.

An All-Human Hepatic Culture System for Drug Development Applications.

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.

Interpretable predictive models of genome-wide aryl hydrocarbon receptor-DNA binding reveal tissue-specific binding determinants.

The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand-activated AhR binds to DNA at dioxin response elements (DREs) containing the core motif 5'-GCGTG-3'. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor (TF)-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible TFs.

Development of a microphysiological skin-liver-thyroid Chip3 model and its application to evaluate the effects on thyroid hormones of topically applied cosmetic ingredients under consumer-relevant conditions.

All cosmetic ingredients registered in Europe must be evaluated for their safety using non-animal methods. Microphysiological systems (MPS) offer a more complex higher tier model to evaluate chemicals. Having established a skin and liver HUMIMIC Chip2 model demonstrating how dosing scenarios impact the kinetics of chemicals, we investigated whether thyroid follicles could be incorporated to evaluate the potential of topically applied chemicals to cause endocrine disruption. This combination of models in the HUMIMIC Chip3 is new; therefore, we describe here how it was optimized using two chemicals known to inhibit thyroid production, daidzein and genistein. The MPS was comprised of Phenion® Full Thickness skin, liver spheroids and thyroid follicles co-cultured in the TissUse HUMIMIC Chip3. Endocrine disruption effects were determined according to changes in thyroid hormones, thyroxine (T4) and 3,3',5-triiodothyronine (T3). A main part of the Chip3 model optimization was the replacement of freshly isolated thyroid follicles with thyrocyte-derived follicles. These were used in static incubations to demonstrate the inhibition of T4 and T3 production by genistein and daidzein over 4 days. Daidzein exhibited a lower inhibitory activity than genistein and both inhibitory activities were decreased after a 24 h preincubation with liver spheroids, indicating metabolism was via detoxification pathways. The skin-liver-thyroid Chip3 model was used to determine a consumer-relevant exposure to daidzein present in a body lotion based on thyroid effects. A "safe dose" of 0.235 µg/cm2 i.e., 0.047% applied in 0.5 mg/cm2 of body lotion was the highest concentration of daidzein which does not result in changes in T3 and T4 levels. This concentration correlated well with the value considered safe by regulators. In conclusion, the Chip3 model enabled the incorporation of the relevant exposure route (dermal), metabolism in the skin and liver, and the bioactivity endpoint (assessment of hormonal balance i.e., thyroid effects) into a single model. These conditions are closer to those in vivo than 2D cell/tissue assays lacking metabolic function. Importantly, it also allowed the assessment of repeated doses of chemical and a direct comparison of systemic and tissue concentrations with toxicodynamic effects over time, which is more realistic and relevant for safety assessment.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

Hepatocyte size fractionation allows dissection of human liver zonation.

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.

Increasing the availability of quality human tissue for research.

Advances in 3D and other in vitro tissue model platforms have led to fundamental improvements in research on human disease, development of novel therapies, and safety testing. In addition, histological and cellular investigations of human tissues continue to serve as keystones in understanding disease and health processes. In recognition of the importance of human tissues in research, the Physicians Committee for Responsible Medicine held a workshop. Working closely with key stakeholders from the research community, regulatory agencies, and organ procurement organizations, the goal was to explore, understand, and address the barriers to increased use of human organs, tissues, and cells in research. Workshop participants were tasked with identifying the challenges of accessing and qualifying tissues for research purposes and creating a strategy to help meet the needs of the research communities to increase the availability and quality of human tissues in biomedical and translational research. Break-out groups identified significant challenges in the areas of policy, scientific development, and public engagement with respect to the provision and application of tissues and cells for scientific advancement. Following working group recommendations, stakeholders concluded that there is a need to facilitate the availability and quality of human tissues for the research community, as well as provide a framework for education of the public, medical professionals, and researchers to foster donation and utilization for research in place of animal models. The success of these new initiatives will facilitate greater access to high-quality human tissues for biomedical and translational research and help ensure the transition away from the dependence on animal models.

ERRATUM.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening.

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

Identifying qualitative differences in PPARα signaling networks in human and rat hepatocytes and their significance for next generation chemical risk assessment methods.

In this paper, we evaluate the PPARα signaling network in rats, examining transcriptional responses in primary hepatocytes exposed to a PPARα specific ligand, GW7647. These transcriptomic studies were complemented with ChIP-seq studies of PPARα binding and transcription binding motif identification for PPARα responsive genes. We also conducted a limited study of GW7647 dosing the in intact rat to examine differences in transcriptional responses for primary hepatocytes in vitro and in the intact liver. The rat network has a much larger number of down-regulated genes and pathways than we had found in the human and the PPARα binding motifs in rat differed for upregulated and down regulated genes. Based on these results and comparison with our previous work with the human PPARα signaling network, we identified qualitative differences in the transcriptional networks controlled by PPARα activation in the two species that provide an explanation of the interspecies differences in the responses of humans and rodents to GW7647 and likely to other PPARα agonists. These studies also allow some observations on the manner in which in vitro, fit-for-purpose assays in human hepatocytes could form the basis for risk assessment without recourse to in-life studies in rodents or other test species.

Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development.

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Hepatocyte-Derived Exosomes Promote Liver Immune Tolerance: Possible Implications for Idiosyncratic Drug-Induced Liver Injury.

Most idiosyncratic drug-induced liver injury appears to result from an adaptive immune attack on the liver. Recent evidence suggests that the T-cell response may be facilitated by the loss of immune tolerance. In this study, we explored the hypothesis that constitutively released hepatocyte-derived exosomes (HDE) are important for maintaining normal liver immune tolerance. Exosomes were isolated from the conditioned medium of primary human hepatocytes via polymer precipitation. Mock controls were prepared by processing fresh medium that was not hepatocyte exposed with precipitation reagent. THP-1 monocytes were then treated with HDE or an equivalent volume of mock control for 24 h, followed by a 6-h stimulation with LPS. HDE exposure resulted in a significant decrease in the LPS-induced media levels of interleukin-1ß and interleukin-8. Gene expression profiling performed in THP-1 cells just prior to LPS-induced stimulation identified a significant decrease among genes associated with innate immune response. MicroRNA (miRNA) profiling was performed on the HDE to identify exosome contents that may drive immune suppression. Many of the predicted mRNA target genes for the most abundant microRNAs in HDE were among the differentially expressed genes in THP-1 cells. Taken together, our data suggest that HDE play a role in maintaining normal liver immune tolerance. Future experiments will explore the possibility that drugs causing idiosyncratic liver injury promote the loss of homeostatic HDE signaling.

Advancing nonclinical innovation and safety in pharmaceutical testing.

Nonclinical tests are considered crucial for understanding the safety of investigational medicines. However, the effective translation from nonclinical to human application is limited and must be improved. Drug development stakeholders are working to advance human-based in vitro and in silico methods that may be more predictive of human efficacy and safety in vivo because they enable scientists to model the direct interaction of drugs with human cells, tissues, and biological processes. Here, we recommend test-neutral regulations; increased funding for development and integration of human-based approaches; support for existing initiatives that advance human-based approaches; evaluation of new approaches using human data; establishment of guidelines for procuring human cells and tissues for research; and additional training and educational opportunities in human-based approaches.

miR-122 Release in Exosomes Precedes Overt Tolvaptan-Induced Necrosis in a Primary Human Hepatocyte Micropatterned Coculture Model.

Idiosyncratic drug-induced liver injury (IDILI) is thought to often result from an adaptive immune attack on the liver. However, it has been proposed that the cascade of events culminating in an adaptive immune response begins with drug-induced hepatocyte stress, release of exosomal danger signals, and innate immune activation, all of which may occur in the absence of significant hepatocelluar death. A micropatterned coculture model (HepatoPac) was used to explore the possibility that changes in exosome content precede overt necrosis in response to the IDILI drug tolvaptan. Hepatocytes from 3 human donors were exposed to a range of tolvaptan concentrations bracketing plasma Cmax or DMSO control continuously for 4, 24, or 72 h. Although alanine aminotransferase release was not significantly affected at any concentration, tolvaptan exposures at approximately 30-fold median plasma Cmax resulted in increased release of exosomal microRNA-122 (miR-122) into the medium. Cellular imaging and microarray analysis revealed that the most significant increases in exosomal miR-122 were associated with programmed cell death and small increases in membrane permeability. However, early increases in exosome miR-122 were more associated with mitochondrial-induced apoptosis and oxidative stress. Taken together, these data suggest that tolvaptan treatment induces cellular stress and exosome release of miR-122 in primary human hepatocytes in the absence of overt necrosis, providing direct demonstration of this with a drug capable of causing IDILI. In susceptible individuals, these early events may occur at pharmacologic concentrations of tolvaptan and may promote an adaptive immune attack that ultimately results in clinically significant liver injury.

Dose-response in a high density three-dimensional liver device with real-time bioenergetic and metabolic flux quantification.

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C-labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C-glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C-glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Editor's Highlight: Modeling Compound-Induced Fibrogenesis In Vitro Using Three-Dimensional Bioprinted Human Liver Tissues.

Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate- and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1ß) during the early culture time period is followed by concentration- and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-ß1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI.

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity.

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.

The Promise of New Technologies to Reduce, Refine, or Replace Animal Use while Reducing Risks of Drug Induced Liver Injury in Pharmaceutical Development.

Drug induced liver injury (DILI) has contributed more to marketed pharmaceutical withdrawals and clinical development failures than any other human organ toxicity. DILI seen in animal studies also frequently leads to the discontinuation of promising drug candidates very early in the pipeline. This manuscript reviews and critically assesses the current regulatory expectations; the current drug development approaches, strategies, and gaps; and the numerous exciting opportunities becoming available to address these gaps through technological advances. Emerging integrated pharmaceutical development strategies, while far from uniform, have generally evolved to currently inform early DILI risk potential using supplemental assays for reactive metabolite formation, mitochondrial toxicity, inhibition of bile salt transport, and cellular imaging endpoints including cytotoxicity. Despite these approaches and robust animal testing, significant gaps in addressing human DILI remain. Increasingly sophisticated in vitro humanized test systems, new animal models, emerging computational models, and novel translational biomarkers are being introduced to improve our ability to more accurately predict DILI. Expectations are high for a future state with more predictive tools and problem solving strategies that will improve pharmaceutical discovery and development in relation to understanding human DILI risk potential and make it less dependent on animal studies for successfully developing safer drug candidates.