Influence of three-dimensional culture in an interwoven hollow fiber bioreactor on the gene expression profile of hepatic HepG2 cells

Purpose: In this study, we aimed to elucidate the specific molecular mechanisms that are activated when hepatic cells are cultured in the 3D environment of an interwoven hollow fiber bioreactor device. Methods: HepG2 human hepatocellular carcinoma cells were either cultured in an interwoven hollow fiber bioreactor or on a conventional two-dimensional (2D) surface. The cells were harvested and RNA sequencing was performed to identify the differentially expressed genes between the two culture conditions. Ingenuity Pathway Analysis (IPA) software was utilized to algorithmically generate gene networks and canonical pathways that were either activated or repressed as a result of the experimental conditions. Results: RNA sequencing analysis identified 5,711 genes that were differentially expressed (FDR < 0.05). Of these, 1,248 genes were biologically upregulated (FC > 2) and 629 genes were biologically downregulated (FC < -2). Several of these upregulated genes have known associations with metabolic activity, cellular proliferation, and apico-basal cell organization. IPA analysis confirmed the likely activation of metabolic pathways in 3D culture and suggest a strong relationship between hepatic metabolism, cell-cell interactions, and polarity markers Conclusions: Our results demonstrate the likely activation of metabolic pathways in 3D culture and suggest a strong relationship between hepatic metabolism, cell-cell interactions, and polarity markers. Further experimentation must be performed to further elucidate the connections between these pathways. cDNA libraries were generated from mRNAs extracted from HepG2 cells cultured in a conventional 2D culture and in a 3D bioreactor; these libraries were applied to deep sequencing followed by transcriptome analysis