Article

Organotypic liver culture models: Meeting current challenges in toxicity testing

The Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA.
Critical Reviews in Toxicology (Impact Factor: 5.1). 05/2012; 42(6):501-48. DOI: 10.3109/10408444.2012.682115
Source: PubMed

ABSTRACT

Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment.

    • "They monitored the cellular activity for only 2 days [18]. However, hepatic models that utilize conventional 2D mono-and co-cultures of normal and diseased human cells fail to mimic the complex threedimensional (3D) in vivo microenvironment and extracellular matrix (ECM) support [16] [19] [20]. Thus, it can prevent normal cellular function and response to the tested drugs. "
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    ABSTRACT: The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.
    No preview · Article · Jan 2016 · Biofabrication
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    • "Another critical feature of the ideal hepatocyte spheroid model is the presence of fluid flow to improve mass transfer. Perfusion culture has been shown to improve the viability, life span and metabolic performance of primary hepatocytes [43] as a result of continuous nutrient supply and waste removal [23]. However, spheroids grown on low-adhesion or "
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    ABSTRACT: Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.
    Full-text · Article · Dec 2015 · Biomaterials
    • "Preclinical studies in animal models fail to fully predict drug toxicity in humans, due to interspecies variability in detoxification mechanisms. Hepatic immortalized cell lines are available, but display low activity levels of cytochrome P450 (CYP450) (LeCluyse et al., 2012). Hepatocyte-like cells (HLCs) derived from pluripotent stem cells (PSCs) show great potential for the prediction of hepatotoxicity, with the advantage of unlimited availability . "
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    ABSTRACT: The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short-term viability in two-dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co-cultured with human bone marrow mesenchymal stem cells (BM-MSCs) as spheroids in automated, computer-controlled, stirred-tank bioreactors with perfusion operation mode. A dual-step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM-MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co-cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated-dose toxicity testing. Moreover, the excretory activity was maintained in co-cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM-MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.
    No preview · Article · Oct 2015 · Journal of Tissue Engineering and Regenerative Medicine
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