Investigating the sustained impact of the physical cellular microenvironment on the structure and function of a hepatocellular carcinoma cell line.

Mechanotransduction defines the functional activity of cells by altering gene expression in accordance with cellular structure. With this in mind, a novel in vitro model has been developed where cells are ‘primed’ to a three-dimensional (3D) phenotype through growth on a biologically inert 3D substrate before liberation and reseeding into a secondary culture. To characterise this protocol, the structural and functional effects of 3D priming were evaluated on the HepG2 hepatocellular carcinoma cell line, frequently used for in vitro liver research on drug discovery and pathology. It was found that through using a 3D priming model, cells adopted a mechanical and functional memory based on the historical physical microenvironment. HepG2 cells primed in 3D demonstrated altered cytoskeletal organisation and cell morphology after reseeding, with cell populations more readily forming 3D structures compared to cells reseeded from conventional two-dimensional (2D) substrates. Global gene expression was significantly altered in the priming model, with enrichment of mechanical and structural genes occurring in 2D HepG2 cells compared to enrichment in key genes involved in hepatic metabolism and biosynthesis in the 3D primed HepG2 cells. 3D priming also resulted in enhanced production of the liver specific biomarkers albumin and urea; even within secondary 3D spheroid cultures. Metabolic activity was significantly altered through priming, with the 3D priming model showing decreased sensitivity to xenobiotic toxicity, though this difference balanced out in the secondary models. Overall, this study has shown that changing the substrate geometry impacts directly on cell structure and results in an altered transcriptional state in HepG2 cells that gives rise to a more physiologically relevant phenotype that is sustained even after enzymatic and mechanical disruption. This holds potential for improving the functionality of in vitro models and further elucidates the role of mechanotransduction in directing cell biology.