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Development of 2D organotypic rat liver co-culture models for assessing hepatotoxicant modes of action

ABSTRACT: In both the regulatory and commercial arenas, the goal is to move away from time consuming, costly in-life rodent studies and towards safety assessment strategies that rely on testing species-relevant cells in vitro. The liver has been a major focus of these efforts, yet there are currently no in vitro alternatives for hepatotoxicity testing accepted by regulators, and the assays that do exist typically utilize hepatocyte monolayer culture. While hepatocytes have been the primary component of in vitro hepatotoxicity assay development, the non-parenchymal cells (NPCs) (i.e., hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells) also play a critical role in the progression of liver pathologies. The goal of this study was to develop an organotypic co-culture system that could read out the various mechanisms of action observed in vivo, as well as maintain metabolic capability and extended viability to allow for repeat dosing. We developed a two-dimensional 96-well plate-based co-culture system that includes primary rat hepatocytes, stellate, Kupffer, and endothelial cells that supports hepatocyte viability and phenotype stability for up to eight days. Importantly, markers of hepatocyte differentiation and polarity are maintained in co-culture, but hepatocyte monocultures with no other cell types lose phenotypic markers after prolonged culture. This co-culture model was leveraged to assess the effects of known hepatotoxic stimuli, and the co-culture model resulted in responses that were more representative of the in vivo phenotypes.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE133717 | GEO | 2021/07/01

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.

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Development of 2D organotypic rat liver co-culture models for assessing hepatotoxicant modes of action

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