Monolayer and Spheroid Culture of Human Liver Hepatocellular Carcinoma Cell Line Cells Demonstrate Distinct Global Gene Expression Patterns and Functional Phenotypes

Department of Surgery, University of California, San Francisco, San Francisco, California 94121, USA.
Tissue Engineering Part A (Impact Factor: 4.7). 09/2008; 15(3):559-67. DOI: 10.1089/ten.tea.2007.0434
Source: PubMed


Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.

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Available from: Millie Hughes-Fulford,
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    • "Like other hepatocyte cell lines, HepG2 cells retain a partially differentiated adult phenotype (Castell et al., 2006). 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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    • "In comparison, hESCs or DE cells cultured on ECM arrays without GFs retained a twodimensional (2D) monolayer morphology (data not shown). The organization of hESCs into spheroids on GF spots may be one reason for the enhanced hepatic function , as a number of recent studies have suggested that hepatocytes in three-dimensional (3D) culture are more functional than those in standard monolayer cultures (Chang and Hughes-Fulford, 2009; Curcio et al., 2007; Nakazawa et al., 2006). We previously observed a similar transition from monolayer to a spheroid configuration in primary hepatocytes cultured on HGF spots (Jones et al., 2009), and hypothesize that spheroid formation may be triggered by a high localized concentration of HGF, which is known to promote cell motility. "
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    ABSTRACT: Embryonic stem cells (ESCs) hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. Hepatocytes derived from ESCs will be useful for therapies, bioartificial liver assistance devices and drug discovery. In traditional stem cell cultivation/differentiation experiments, growth factors (GFs) are added in soluble form in order to provide signals for tissue-specific differentiation. In contrast, we investigated differentiation of hESCs cultured on top of GFs. In these experiments, glass substrates were imprinted with a mixture of ECM and GF molecules to form 500 µm diameter spots. hESCs were cultured onto these GF-containing ECM spots for up to 12 days to induce differentiation towards the hepatic lineage. The dynamics of differentiation were examined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. Stem cells cultured on GF-containing surfaces stained positive for the endoderm markers SOX17 and FOXA2, as well as early liver markers such as α-fetoprotein and albumin. qRT-PCR confirmed that pluripotency, endoderm and liver gene expression of hESCs cultured on GF-containing surfaces was consistent with hepatic differentiation. In comparison, hESCs cultured on ECM spots without GFs showed less pronounced loss of pluripotency and lower levels of liver gene expression. In summary, our study demonstrates that hESCs receive differentiation-inducing signals from GF-containing surfaces and may be pushed along the hepatic lineage when cultured on such surfaces. Compared to traditional approaches, the advantages of GF immobilization include reduction in the cost of experiments, stronger and longer stimulation and the possibility of screening GF-stem cell interactions in a multiplexed manner. Copyright © 2012 John Wiley & Sons, Ltd.
    Journal of Tissue Engineering and Regenerative Medicine 11/2014; 8(11). DOI:10.1002/term.1595 · 5.20 Impact Factor
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    • "Therefore, it is important to systematically explore the advantages of this new research opportunity. Different space flights have already demonstrated a 3D cell growth (Table 2) and similar results have been detected with the help of devices simulating μg in ground-based laboratories [34, 44, 46, 172–174]. "
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    ABSTRACT: How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering.
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