In vitro expansion and functional recovery of mature hepatocytes from mouse adult liver

Abstract

Mature hepatocytes retain the ability to regenerate the liver lobule fully in vivo following injury. Several cytokines and soluble factors (hepatocyte growth factors, epidermal growth factors, insulin and nicotinamide) are known to be important for proliferation of mature hepatocytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradually lost their specific functions, particularly those in drug metabolism. We have explored and established a new culture system for expansion of functional hepatocytes. We evaluated two approaches for efficient expansion of mature hepatocytes: (i) Co-culture with mouse embryonic fibroblasts (MEF); (ii) Addition to culture of inhibitors of cell signals involved in liver regeneration. After expansion steps, 3-dimensional spheroid-forming culture was used to re-induce mature hepatocellular function. The addition of inhibitors for tumour growth factor (TGF) β and glycogen synthase kinase (GSK) 3β efficiently induced in vitro expansion of mature hepatocytes. Although expression of hepatocellular functional genes decreased after expansion in monolayer culture, their expression and the activity of cytochrome P450 enzymes significantly increased with spheroid formation. Furthermore, when hepatocytes were co-cultured with MEF, addition of a MAPK/ERK kinase (MEK) inhibitor at the spheroid formation step enhanced drug-metabolism-related gene expression. Combination of the MEF co-culture system with the addition of inhibitors of TGFβ and GSK3β induced in vitro expansion of hepatocytes. Moreover, expression of mature hepatocellular genes and the activity of drug-metabolism enzymes in expanded hepatocytes were re-induced after spheroid culture.