Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge

School of Biological and Biomedical Science, Durham University, South Road, Durham DH1 3LE, UK.
Journal of Anatomy (Impact Factor: 2.1). 11/2007; 211(4):567-76. DOI: 10.1111/j.1469-7580.2007.00778.x
Source: PubMed


Cultured cells are dramatically affected by the micro-environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3-D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2-D) flat plastic surfaces. To enable 3-D growth of HepG2 cells in vitro, we cultured cells on 3-D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3-D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2-D surfaces. The function of liver cells grown on 3-D supports was significantly enhanced compared to activity of cells grown on 2-D standard plasticware. Unlike their 2-D counterparts, 3-D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3-D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2-D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3-D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2-D culture systems. This technology has potential applications for toxicity testing and drug screening.

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    • "A comparison of HepG2 cells grown in monolayer and 3D culture revealed improved albumin production in 3D culture (Chang and Hughes- Fulford, 2009; Mueller et al., 2011). Additionally, 3D culturing significantly improved formation of bile canaliculi (Bokhari et al., 2007). "
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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.
    Journal of Biotechnology 09/2015; 212:134-143. DOI:10.1016/j.jbiotec.2015.08.012 · 2.87 Impact Factor
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    • "For natural ECM, a decrease in the matrix stiffness by altering collagen crosslinking delayed the malignant growth and tumor development [16]. Moreover, the use of 3D porous polystyrene support as a substrate for cell cultures resulted in a lesser susceptibility to methotrexate cytotoxicity compared to 2D cultures [17]. Coculturing of hepatic cell line (HepG2) on 3D alginate-based hydrogels with MCF-7 was associated with an increase in the hepatic susceptibility and toxicity to chemical entities as acetaminophen and diclofenac compared to cells cultured in 2D flat surface [18]; these alterations in the cellular toxicities were well correlated with reported in vivo responses to these drugs [19]. "
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    ABSTRACT: In drug discovery programs, the alteration between in vivo and in vitro cellular responses to drug represents one of the main challenges. Since the variation in the native extracellular matrix (ECM) between in vivo and 2D in vitro conditions is one of the key reasons for such discrepancies, thus the utilization of substrate that likely mimics ECM characteristics (topography, stiffness, and chemical composition) is needed to overcome such problem. Here, we investigated the role of substrate nanotopography as one of the major determinants of hepatic cellular responses to a chemotherapeutic agent “cisplatin.” We studied the substratum induced variations in cisplatin cytotoxicity; a higher cytotoxic response to cisplatin was observed for cells cultured on the nanopattern relative to a flat substrate. Moreover, the nanofeatures with grating shapes that mimic the topography of major ECM protein constituents (collagen) induced alterations in the cellular orientation and chromatin condensation compared to flat surfaces. Accordingly, the developments of biomimetic substrates with a particular topography could have potentials in drug development analyses to reflect more physiological mimicry conditions in vitro.
    BioMed Research International 06/2015; 2015. DOI:10.1155/2015/925319 · 1.58 Impact Factor
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    • "For HepG2, which have been previously shown to form compact 3D spheroids in 3D cell-culture systems [27]–[30], we also studied the effects of lipid-reduced conditions in a 3-dimensional (3D) cell culture system that mimics natural tissues and organs more closely than 2-dimensional (2D) cell culture system. In normal growth conditions a time-dependent increase in size of spheroids was observed (Figure 2a). "
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    ABSTRACT: Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between de novo lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines - lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24) - raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate de novo lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on de novo lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates de novo lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell's lipid metabolism.
    PLoS ONE 09/2014; 9(9):e106913. DOI:10.1371/journal.pone.0106913 · 3.23 Impact Factor
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