In vitro models for liver toxicity testing

Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599, USA.
Toxicology Research (Impact Factor: 3.98). 03/2013; 2(1):23-39. DOI: 10.1039/C2TX20051A
Source: PubMed


Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system.

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Available from: Valerie Soldatow, Nov 06, 2015
    • "However, restricted access to human liver tissue and the very limited ability of adult differentiated hepatocytes to proliferate in vitro hinders widespread use of these cells. Therefore, cell lines such as HepG2 or HepaRG have been used, despite their tumorous nature and their differences to primary hepatocytes (Soldatow et al., 2013). "
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    ABSTRACT: Reconstituted three-dimensional (3D) liver models obtained by engrafting hepatic cells into an extracellular matrix (ECM) are valuable tools to study tissue regeneration, drug action and toxicology ex vivo. The aim of the present study was to establish a system for the functional investigation of a viral vector in a 3D liver model composed of human HepG2 cells on a rat ECM. An adeno-associated viral (AAV) vector expressing the Emerald green fluorescent protein (EmGFP) and a short hairpin RNA (shRNA) directed against human cyclophilin b (hCycB) was injected into the portal vein of 3D liver models. Application of the vector did not exert toxic effects, as shown by analysis of metabolic parameters. Six days after transduction, fluorescence microscopy analysis of EmGFP production revealed widespread distribution of the AAV vectors. After optimization of the recellularization and transduction conditions, averages of 55 and 90 internalized vector genomes per cell in two replicates of the liver model were achieved, as determined by quantitative PCR analysis. Functionality of the AAV vector was confirmed by efficient shRNA-mediated knockdown of hCycB by 70-90%. Our study provides a proof-of-concept that a recellularized biological ECM provides a valuable model to study viral vectors ex vivo.
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    • "One additional potential application for in vitro systems that maintain metabolic function over relatively long periods of time is the prediction of hepatic injury (Dash et al., 2009; Kratschmar, 2013; Yeon, 2010). In this case, co-cultures of hepatocytes with other cell types such as Kuppfer cells or other NPCs (stellate or sinusoidal endothelial cells) could be relevant (Soldatow et al., 2013). This illustrates that there remains considerable scope to explore the potential of this 3D microfluidic model, LiverChip TM system. "
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    ABSTRACT: 1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.
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    ABSTRACT: Liver extracellular matrix (ECM) composition, topography and biomechanical properties influence cell-matrix interactions. The ECM presents guiding cues for hepatocyte phenotype maintenance, differentiation and proliferation both in vitro and in vivo. Current understanding of such cell-guiding cues along with advancement of techniques for scaffold fabrication has led to evolution of matrices for liver tissue culture from simple porous scaffolds to more complex 3D matrices with microarchitecture similar to in vivo. Natural and synthetic polymeric biomaterials fabricated in different topographies and porous matrices have been used for hepatocyte culture. Heterotypic and homotypic cell interactions are necessary for developing an adult liver as well as an artificial liver. A high oxygen demand of hepatocytes as well as graded oxygen distribution in liver is another challenging attribute of the normal liver architecture that further adds to the complexity of engineered substrate design. A balanced interplay of cell-matrix interactions along with cell-cell interactions and adequate supply of oxygen and nutrient determines the success of an engineered substrate for liver cells. Techniques devised to incorporate these features of hepatic function and mimic liver architecture range from maintaining liver cells in mm-sized tailor-made scaffolds to a more bottoms up approach that starts from building the microscopic subunit of the whole tissue. In this review, we discuss briefly various biomaterials used for liver tissue engineering with respect to design parameters such as scaffold composition and chemistry, biomechanical properties, topography, cell-cell interactions and oxygenation.
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