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
    • "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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    • "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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