Micropatterned Coculture With 3T3-J2 Fibroblasts Enhances Hepatic Functions and Drug Screening Utility of HepaRG Cells.

Human liver models are useful for assessing compound metabolism/toxicity; however, primary human hepatocyte (PHH) lots are limited and highly variable in quality/viability. In contrast, cell lines, such as HepaRG, are cheaper and more reproducible surrogates for initial compound screening; however, hepatic functions and sensitivity for drug outcomes need improvement. Here, we show that HepaRGs cocultured with murine embryonic 3T3-J2 fibroblasts, previously shown to induce PHH functions, could address such limitations. We either micropatterned HepaRGs or seeded them "randomly" onto collagen-coated plates before 3T3-J2 coculture. Micropatterned cocultures (HepaRG-MPCCs) secreted 2- to 4-fold more albumin and displayed more stable cytochrome P450 activities than HepaRG conventional confluent monocultures (HepaRG-CCs) and HepaRG micropatterned hepatocytes (HepaRG-MPHs) for 4 weeks, even when excluding dimethyl sulfoxide from the medium. Furthermore, HepaRG-MPCCs had the most albumin-only positive cells (hepatic), lowest cytokeratin 19 (CK19)-only positive cells (cholangiocytic), and highest mean albumin intensity per cell than HepaRG random cocultures and monocultures; however, 80%-84% of HepaRGs remained bipotential (albumin+/CK19+) across all models. The 3T3-J2s also induced higher albumin in HepaRG spheroids than HepaRG-only spheroids. Additionally, although rifampin induced CYP3A4 in HepaRG-MPCCs and HepaRG-CCs, only HepaRG-MPCCs showed the dual omeprazole-mediated CYP1A2/3A4 induction as with PHHs. Lastly, when treated for 6 days with 47 drugs and evaluated for albumin and ATP to make binary hepatotoxicity calls, HepaRG-MPCCs displayed a sensitivity of 54% and specificity of 100% (70%/100% in PHH-MPCCs), whereas HepaRG-CCs misclassified several hepatotoxins. Ultimately, HepaRG-MPCCs could be a more cost-effective and reproducible model than PHHs for executing a tier 1 compound screen.

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.

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.

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

BACKGROUND AND AIMS: Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. METHODS: Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. RESULTS: LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. CONCLUSIONS: PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

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.

Exploring Chronic Drug Effects on Microengineered Human Liver Cultures Using Global Gene Expression Profiling.

Global gene expression profiling is useful for elucidating a drug's mechanism of action on the liver; however, such profiling in rats is not very sensitive for predicting human drug-induced liver injury, while dedifferentiated monolayers of primary human hepatocytes (PHHs) do not permit chronic drug treatment. In contrast, micropatterned cocultures (MPCCs) containing PHH colonies and 3T3-J2 fibroblasts maintain a stable liver phenotype for 4-6 weeks. Here, we used MPCCs to test the hypothesis that global gene expression patterns in stable PHHs can be used to distinguish clinical hepatotoxic drugs from their non-liver-toxic analogs and understand the mechanism of action prior to the onset of overt hepatotoxicity. We found that MPCCs treated with the clinical hepatotoxic/non-liver-toxic pair, troglitazone/rosiglitazone, at each drug's reported and non-toxic Cmax (maximum concentration in human plasma) for 1, 7, and 14 days displayed a total of 12, 269, and 628 differentially expressed genes, respectively, relative to the vehicle-treated control. Troglitazone modulated >75% of transcripts across pathways such as fatty acid and drug metabolism, oxidative stress, inflammatory response, and complement/coagulation cascades. Escalating rosiglitazone's dose to that of troglitazone's Cmax increased modulated transcripts relative to the lower dose; however, over half the identified transcripts were still exclusively modulated by troglitazone. Last, other hepatotoxins (nefazodone, ibufenac, and tolcapone) also induced a greater number of differentially expressed genes in MPCCs than their non-liver-toxic analogs (buspirone, ibuprofen, and entacapone) following 7 days of treatment. In conclusion, MPCCs allow evaluation of time- and dose-dependent gene expression patterns in PHHs treated chronically with analog drugs.

Engineered Liver Platforms for Different Phases of Drug Development.

Drug-induced liver injury (DILI) remains a leading cause of drug withdrawal from human clinical trials or the marketplace. Owing to species-specific differences in liver pathways, predicting human-relevant DILI using in vitro human liver models is crucial. Microfabrication tools allow precise control over the cellular microenvironment towards stabilizing liver functions for weeks. These tools are used to engineer human liver models with different complexities and throughput using cell lines, primary cells, and stem cell-derived hepatocytes. Including multiple human liver cell types can mimic cell-cell interactions in specific types of DILI. Finally, organ-on-a-chip models demonstrate how drug metabolism in the liver affects multi-organ toxicities. In this review we survey engineered human liver platforms within the needs of different phases of drug development.

Water-Stable Metal-Organic Framework/Polymer Composites Compatible with Human Hepatocytes.

Metal-organic frameworks (MOFs) have demonstrated promise in biomedical applications as vehicles for drug delivery, as well as for the ability of copper-based MOFs to generate nitric oxide (NO) from endogenous S-nitrosothiols (RSNOs). Because NO is a participant in biological processes where it exhibits anti-inflammatory, antibacterial, and antiplatelet activation properties, it has received significant attention for therapeutic purposes. Previous work has shown that the water-stable MOF H3[(Cu4Cl)3-(BTTri)8] (H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), or CuBTTri, produces NO from RSNOs and can be included within a polymeric matrix to form NO-generating materials. While such materials demonstrate potential, the possibility of MOF degradation leading to copper-related toxicity is a concern that must be addressed prior to adapting these materials for biomedical applications. Herein, we present the first cytotoxicity evaluation of an NO-generating CuBTTri/polymer composite material using 3T3-J2 murine embryonic fibroblasts and primary human hepatocytes (PHHs). CuBTTri/polymer films were prepared from plasticized poly(vinyl chloride) (PVC) and characterized via PXRD, ATR-FTIR, and SEM-EDX. Additionally, the ability of the CuBTTri/polymer films to enhance NO generation from S-nitroso-N-acetylpenicillamine (SNAP) was evaluated. Enhanced NO generation in the presence of the CuBTTri/polymer films was observed, with an average NO flux (0.90 ± 0.13 nmol cm(-2) min(-1)) within the range associated with antithrombogenic surfaces. The CuBTTri/polymer films were analyzed for stability in phosphate buffered saline (PBS) and cell culture media under physiological conditions for a 4 week duration. Cumulative copper release in both cell media (0.84 ± 0.21%) and PBS (0.18 ± 0.01%) accounted for less than 1% of theoretical copper present in the films. In vitro cell studies performed with 3T3-J2 fibroblasts and PHHs did not indicate significant toxicity, providing further support for the potential implementation of CuBTTri-based materials in biomedical applications.

Stem cell-derived liver cells for drug testing and disease modeling.

Differences between animals and humans in liver pathways now necessitate the use of in vitro models of the human liver for several applications such as drug screening. However, isolated primary human hepatocytes (PHHs) are a limited resource for building such models given shortages of donor organs. In contrast, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be propagated nearly indefinitely and differentiated into hepatocyte-like cells in vitro using soluble factors inspired from liver development. Additionally, iPSCs can be generated from patients with specific genetic backgrounds to study genotype-phenotype relationships. While current protocols to differentiate hESCs and iPSCs into human hepatocyte-like cells (hESC-HHs and iPSC-HHs) still need improvement to yield cells functionally similar to the adult liver, proof-of-concept studies have already shown utility of these cells in drug development and modeling liver diseases such as α1-antitrypsin deficiency, hepatitis B/C viral infections, and malaria. Here, we present an overview of hESC-HH and iPSC-HH culture platforms that have been utilized for the aforementioned applications. We also discuss the use of semiconductor-driven microfabrication tools to precisely control the microenvironment around these cells to enable higher and longer-term liver functions in vitro. Finally, we discuss areas for improvement in creating next generation stem cell-derived liver models. In the future, stem cell-derived hepatocyte-like cells could provide a sustainable cell source for high-throughput drug screening, enabling better mechanistic understanding of human liver diseases for the development of more efficacious and safer therapeutics, and personalized cell-based therapies in the clinic.

Prediction of Drug-Induced Liver Injury in Micropatterned Co-cultures Containing iPSC-Derived Human Hepatocytes.

Primary human hepatocytes (PHHs) are a limited resource for drug screening, their quality for in vitro use can vary considerably across different lots, and a lack of available donor diversity restricts our understanding of how human genetics affect drug-induced liver injury (DILI). Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) could provide a complementary tool to PHHs for high-throughput drug screening, and ultimately enable personalized medicine. Here, we hypothesized that previously developed iPSC-HH-based micropatterned co-cultures (iMPCCs) with murine embryonic fibroblasts could be amenable to long-term drug toxicity assessment. iMPCCs, created in industry-standard 96-well plates, were treated for 6 days with a set of 47 drugs, and multiple functional endpoints (albumin, urea, ATP) were evaluated in dosed cultures against vehicle-only controls to enable binary toxicity decisions. We found that iMPCCs correctly classified 24 of 37 hepatotoxic drugs (65% sensitivity), while all 10 non-toxic drugs tested were classified as such in iMPCCs (100% specificity). On the other hand, conventional confluent cultures of iPSC-HHs failed to detect several liver toxins that were picked up in iMPCCs. Results for DILI detection in iMPCCs were remarkably similar to published data in PHH-MPCCs (65% versus 70% sensitivity) that were dosed with the same drugs. Furthermore, iMPCCs detected the relative hepatotoxicity of structural drug analogs and recapitulated known mechanisms of acetaminophen toxicity in vitro. In conclusion, iMPCCs could provide a robust tool to screen for DILI potential of large compound libraries in early stages of drug development using an abundant supply of commercially available iPSC-HHs.

Microengineered liver tissues for drug testing.

Drug-induced liver injury (DILI) is a leading cause of drug attrition. Significant and well-documented differences between animals and humans in liver pathways now necessitate the use of human-relevant in vitro liver models for testing new chemical entities during preclinical drug development. Consequently, several human liver models with various levels of in vivo-like complexity have been developed for assessment of drug metabolism, toxicity, and efficacy on liver diseases. Recent trends leverage engineering tools, such as those adapted from the semiconductor industry, to enable precise control over the microenvironment of liver cells and to allow for miniaturization into formats amenable for higher throughput drug screening. Integration of liver models into organs-on-a-chip devices, permitting crosstalk between tissue types, is actively being pursued to obtain a systems-level understanding of drug effects. Here, we review the major trends, challenges, and opportunities associated with development and implementation of engineered liver models created from primary cells, cell lines, and stem cell-derived hepatocyte-like cells. We also present key applications where such models are currently making an impact and highlight areas for improvement. In the future, engineered liver models will prove useful for selecting drugs that are efficacious, safer, and, in some cases, personalized for specific patient populations.

Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro.

UNLABELLED: Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e., personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECMs) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned coculture (iMPCC) platform in a multiwell format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for at least 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel). We assessed iHep maturity by global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal cytochrome P450 (CYP450) activities, phase II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. CONCLUSION: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multiorgan responses to compounds.