Discussion

Formation of three-dimensional structures resembling embryonic liver buds in vitro has recently been described using stem cell-derived cell types (Takebe et al., 2013). Such pre- formed structures can be transplanted into animals (or even patients) but may also be useful for other applications like ex vivo drug toxicity tests. In this study we used differentiated, human upcyte® cells to form liver buds in vitro. upcyte® cells are derived from primary human cells that underwent targeted genetic modification (upcyte® process) in order to transiently induce or extend cell proliferation resulting in mortal, but expendable, differentiated cells. Proliferating upcyte® cells can undergo up to 40 population doublings, and when contact-inhibited, they differentiate into functional cells while maintaining of most of their cell type specific characteristics.

We used a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) that spontaneously formed liver buds in vitro which were then cultured for up to 30 days in a matrigel-coated bioreactor (kirkstall® quasi vivo system). These self-organized, liver-like organoid structures harbour healthy, living cells showing typical functional characteristics of liver parenchyme, including basal as well as drug-induced activity of several Cytochrome P450 enzymes. Within the bud the cells formed a typical “liver-like” architecture built up by polarized hepatocytes.

In summary we hereby describe that 3-dimensional, functional liver structures can be generated in vitro using upcyte® cells and that these “mini-livers” are useful models to study human liver functions ex vivo for at least 30 days.