Molecular mechanisms underlying the enhanced functions of three-dimensional hepatocyte aggregates

Abstract

Three-dimensional (3D) culture of hepatocytes leads to improved and prolonged synthetic and metabolic functions, but the underlying molecular mechanisms are unknown. In order to investigate the role of 3D cell-cell interactions in maintaining hepatocyte differentiated functions ex vivo, primary mouse hepatocytes were cultured either as monolayers on tissue culture dishes (TCD) or as 3D aggregates in rotating wall vessel (RWV) bioreactors. Global gene expression analyses revealed that genes upregulated in 3D culture were distinct from those upregulated during liver development and liver regeneration. Instead, they represented a diverse array of hepatocyte-specific functional genes with significant over-representation of hepatocyte nuclear factor 4α (Hnf4a) binding sites in their promoters. Expression of Hnf4a and many of its downstream target genes were significantly increased in RWV cultures as compared to TCD. Conversely, there was concomitant suppression of mesenchymal and cytoskeletal genes in RWV cultures that were induced in TCDs. These findings illustrate the importance of 3D cell-cell interactions in maintaining fundamental molecular pathways of hepatocyte function and serve as a basis for rational design of biomaterials that aim to optimize hepatocyte functions ex vivo for biomedical applications.

Keywords: Bioreactor; Gene expression; Hepatocyte; Microarray; Spheroid; Three-dimensional cell culture.

Figure 1

Hepatocyte 3D aggregates generated in rotating wall vessels (RWVs) demonstrated improved hepatocyte-specific functions compared to monolayers in tissue culture dishes (TCDs). (A) Primary mouse hepatocytes were cultured in either TCDs or RWVs and phase-contrast photomicrographs were taken at 24h and day 3 of culture. Scale bar = 100μm. Total magnification 40x. (B) Albumin production was significantly increased in RWVs at 36h and day 3 of culture compared to TCDs. (C) Likewise, cytochrome P450 1a1 activity was significantly increased in RWVs at 4h, 24h, and day 3 of culture compared to TCDs. Data are representative of 3 independent experiments. *p<0.001. Error bars indicate standard deviation.

Figure 2

Primary hepatocytes cultured on TCDs have a slight growth advantage compared to those cultured in RWVs. (A) Cell numbers were enumerated by the Cyquant assay on day 3 of culture. The slight increases in cell numbers in TCD cultures compared to RWV were not statistically significant. Data are representative of 3 independent experiments. Error bars indicate standard deviation. (B) Expression of Pcna (proliferating cell nuclear antigen) in hepatocytes cultured on TCDs was significantly higher than in those cultured in RWV. Data are the average of 5 independent experiments. *p<0.005. Error bars represent standard error.

Figure 3

Microarray analysis of global gene expression in hepatocytes cultured in monolayers compared to 3D aggregates. (A) Principal component analysis of significant differentially expressed genes in freshly isolated hepatocytes (grey diamonds) and hepatocyte culture for 4h (squares) or 24h (circles) in TCD (red) and RWV (blue). Each symbol represents an independent sample from 3 independent experiments. (B) Hierarchical clustering of genes with 2-fold or greater expression difference between TCD and RWV culture conditions at either 4h or 24h. Each column represents the average expression values of 3 independent experiments. (C) Venn diagram that compares genes significantly upregulated by 2-fold or more in hepatocyte 3D aggregates compared to monolayers with genes significantly upregulated during liver development and liver regeneration. Numbers represent the number of genes either distinct in or shared by the 3 processes.

Figure 4

Metabolic and synthetic genes were upregulated in hepatocytes cultured as 3D aggregates in RWVs compared to monolayers in TCDs. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) demonstrated that expression levels of bile acid co-enzyme A (Baat), aldo-ketoreductase 1c19 (Akr1c19), glycogen synthase 2 (Gys2), factor VII (F7), hemochromatosis type 2 (Hfe2), and leptin receptor (Lepr) were significantly upregulated in RWV compared to TCD. Data are the average of 6 independent experiments. *p<0.005, **p<0.0001. Error bars indicate standard error.

Figure 5

Expression of Hnf4a was significantly increased in 3D aggregates in RWVs compared to monolayers in TCDs, as determined by qRT-PCR. Expression levels of Cebpa and Foxa2, two other liver-enriched transcription factors, were not significantly different between the two culture conditions. Data are the average of 6 independent experiments. *p<0.001. Error bars indicate standard error.

Figure 6

Mesenchymal and cytoskeletal genes were upregulated in hepatocytes cultured as monolayers in TCDs compared to 3D aggregates in RWVs. qRT-PCR demonstrated that expression levels of Snail (Snai1), vimentin (Vim), lysyl oxidase-like 2 (Loxl2), filamin A (Flna), α1 actin (Acta1), and β-tropomyosin (Tpm2) were significantly upregulated in TCD compared to RWV at 24h of culture. Data are the average of 6 independent experiments. *p<0.05, **p<0.005, ***p<0.001. Error bars indicate standard error.