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, 15 (3), 559-67

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

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

Tammy T Chang et al. Tissue Eng Part A.

Abstract

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.

Figures

FIG. 1.
FIG. 1.
Human liver hepatocellular carcinoma cell line (HepG2) cells form cellular spheroids with cortical actin organization when cultured in the three-dimensional environment of rotating wall vessels (RWVs). HepG2 cells were cultured on tissue culture dishes (TCDs) (A, C, E, G) or in RWVs (B, D, F, H). Light-field microscopy was performed after 3 days (A, B) or 7 days (C, D). Cells grown on TCDs had spread cytoplasm and formed a monolayer by day 7. Spheroids in RWVs were up to 100 μm in diameter by 3 days and 500 μm by 7 days. Cells were stained with Hoechst dye for nuclei (E, F) and rhodamine phalloidin for actin (G, H) at day 3 of culture and visualized using fluorescence microscopy. Whereas cells in monolayers formed F-actin stress fibers, cells in spheroids had cortical actin organization. Magnification 30×.
FIG. 2.
FIG. 2.
Spheroid cellular architecture is dependent on continued culture in rotating wall vessels (RWVs). Human liver hepatocellular liver carcinoma cell line cells were cultured for 6 weeks in the three-dimensional (3D) environment of the RWV bioreactor. Formed cellular spheroids were subcultured in tissue culture dishes (TCDs) or RWVs for 7 days. Light microscopy showed disintegration of the spheroids in TCDs (A), compared with intact spheroids that remained in the 3D environment of RWVs (B). Magnification 20×.
FIG. 3.
FIG. 3.
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) of selected genes shows that metabolic and synthetic genes are upregulated in spheroids, whereas structural genes and certain oncogenes are upregulated in monolayers. qRT-PCR was performed on day 3 cultures of monolayers and spheroids. Gene expression was normalized to the housekeeping gene cyclophilin, and fold increase in one culture condition was calculated relative to the other condition. Figure represents pooled data from three independent experiments with three independent biological samples for each condition (n = 9). Gene symbols: CYP1A1, cytochrome P450 1A1; AKR1C1, aldo-keto reductase 1C1; EPHX1, epoxide hydrolase 1; LTB4DH, leukotriene B4 12-hydroxydehydrogenase; LDLR, low-density lipoprotein receptor; HMGCR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; GSTA1, glutathione S-transferase A1; GCLM, glutamate-cysteine ligase; ALB, albumin; ATP5I, adenosine triphosphate synthase; NDUFA3, nicotinamide adenine dinucleotide dehydrogenase; COL1A1 – collagen type I, alpha 1; CSPG2, chondroitin sulfate proteoglycan 2 (versican); CDH1, E-cadherin; CLDN6, claudin 6. *p < 0.001; **p < 0.005.
FIG. 4.
FIG. 4.
Overall proliferation is comparable between human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers and spheroids. HepG2 cells were grown in tissue culture dishes or rotating wall vessels for 6 h, 24 h, 72 h, or 6 days. At each time point, Cyquant DNA quantification assays were performed to determine cell number. Data are representative of three independent experiments. *p < 0.05.
FIG. 5.
FIG. 5.
Human liver hepatocellular carcinoma cell line spheroids cultured in rotating wall vessels demonstrate better liver-specific metabolic and synthetic functions than in monolayers. (A) Cytochrome P450 activity is greater in spheroids. After 7 days of culture as monolayers or spheroids, 7-ethoxyresorufin-o-dealkylase assays were performed to measure cytochrome P450 1A1 activity. Cyquant assays determined cell numbers in order to present data as amount of resorufin product per million cells. (B) Albumin production was greater in spheroids. After 7 days of culture, culture supernatants were collected and albumin quantified according to enzyme-linked immunosorbent assay. Cyquant assays were performed to determine cell numbers. Data represent concentration of albumin in culture supernatant per million cells. Data are representative of three independent experiments. *p < 0.001.
FIG. 6.
FIG. 6.
Spheroids lose liver-specific functions when placed in tissue culture dishes (TCDs). Human hepatocellular liver carcinoma cell line cells were cultured for 6 to 10 weeks in the rotating wall vessels (RWVs). Formed cellular spheroids were subcultured in TCDs or RWVs for 7 days. Cytochrome P450 activity as measured according to resorufin formation (A) and albumin production (B) was determined. Data were adjusted per million cells as determined using the Cyquant assay. *p < 0.005, **p < 0.001.

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