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A pumpless liver-on-a-chip for drug hepatotoxicity analysis

ABSTRACT: This study presents the development and validation of an innovative microfluidic liver-on-a-chip device utilizing gravity-driven perfusion for the evaluation of drug hepatotoxicity. This research involved the construction of a hydrogel-based coculture chip that integrates liver parenchymal and stellate cells within a tri-channel configuration. The assembly and operation of the liver-on-a-chip and its accompanying custom rocker were straightforward. The chip maintained high cell viability and continuous liver albumin synthesis over extended culture durations. Acetaminophen, a hepatic injury-inducing drug, was utilized as a positive control in hepatic toxicity assays on the chip. The liver chip exhibited hepatotoxic responses comparable to those observed in 2D models. Furthermore, in this study we evaluated the effects of two plant-derived natural compounds, aristolochic acid I (AA) and its ana-log aristolactam AII (AL), in both 2D cell models and the liver-on-a-chip system. Aristolochic acid I, known for its hepatorenal toxicity, was observed to cause hepatotoxicity in both the 2D models and on the chip. Flow cytometry and mRNA sequencing results confirmed the propensity of these compounds to induce liver cell apoptosis. Notably, aristolactam AII, previously consid-ered nontoxic, provoked a significant decrease in the hepatic functionality marker albumin exclusively in the liver chip but not in 2D models, indicating the liver chip's enhanced sensitivity to toxic substances. In summary, this pumpless liver-on-a-chip offers a simple yet powerful tool for drug hepatotoxicity studies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252399 | GEO | 2024/01/02

REPOSITORIES: GEO

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Project description: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 (ECM) 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 co-culture (iMPCC) platform in a multi-well format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for 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 via global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal 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 multi-organ responses to compounds. We used Affymetrix microarrays to profile the global gene expression of co-culture stabilized iHeps (iMPCCs) relative to freshly isolated and co-culture stabilized primary human hepatocytes (2 donors). To assess the transcriptomic stability of iHeps in iMPCCs, RNA was extracted following 9 and 21 days of culture for hybridization to Affymetrix microarrays. The hepatic maturation state of iHeps was assessed by comparing gene expression against microarrays containing data from two primary human hepatocyte donors, both following hepatocyte isolation (day 0) and after stabilization in the micropatterened co-culture platform (day 6 and day 42 MPCCs), as previously described.

Project description:In drug development, a system for predicting drug metabolism and drug-induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug-metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4-mediated drug metabolism and drug-induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells (HLCs) or intestinal epithelial-like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4-knockout (KO) iPS cell-derived HLCs and IECs for the evaluation of CYP3A4-mediated drug metabolism and drug-induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off-target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4-KO cells but were observed in CYP3A4 inhibitor-treated (e.g., ketoconazole) cells. To evaluate whether drug-induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4-KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4-mediated toxicity. Finally, we examined whether the therapeutic effects of an anti-hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4-KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti-viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4-mediated drug metabolism and drug-induced toxicity.

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A pumpless liver-on-a-chip for drug hepatotoxicity analysis

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