Liver bud formation using differentiated human upcyte cells
Abstract: Liver organoids (LOs) are of interest in tissue replacement, hepatotoxicity and pathophysiological studies. For the in vitro generation of liver organoids, we used differentiated adult human upcyte liver cells. Upcyte cells are derived from primary human cells that underwent targeted genetic modification (upcyte process) in order to transiently induce cell proliferation resulting in expandable cells that maintain their differentiation potential. 
By co-culturing a defined mixture of differentiated human upcyte cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) on a layer of Matrigel, the cells self-organized to form liver bud-like structures within 24 hours. 
We found that liver organoid formation is modulated by the nature of the substrate - specifically by the interplay between stiffness and biochemical ligands - and that a high initial oxygen consumption rate of LOs may lead to hypoxia within the core unless a continuous flux of nutrients is supplied through a fluidic system. When cultured for another 10 days in a bioreactor, these liver buds revealed typical functional characteristics of liver cells including basal and induced CYP3A4 enzyme activities. 
Our results further indicate marked differences in enzyme modulation between liver organoids and monolayer cultures. Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in many polyvinylchloride medical devices and is washed out easily. Interestingly, DEHP modulated the expression of xenobiotic metabolizing enzymes, reduced the formation of bile canaliculi and cell polarity and inhibited CYP activity in hepatocytes. DEHP had a toxic effect on LX-2 and induced the fibrogenic activation of HSC. The mode of action of DEHP was different in monolayer cultures compared to 3D-liver organoids, which were more sensitive to DEHP. 
In conclusion, we describe the generation of 3D functional liver structures composed of primary human upcyte cells. These liver buds can be cultured for a prolonged period of time, and potentially represent an ex vivo model to study liver functions. We postulate that 3D tissue-like structures simulate the in vivo situation much better than conventional monolayer settings and should therefore preferentially be used for toxicity studies in vitro in the future.