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

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.