A novel 3D liver organoid system for elucidation of hepatic glucose metabolism

Abstract

Hepatic glucose metabolism is a key player in diseases such as obesity and diabetes as well as in antihyperglycemic drugs screening. Hepatocytes culture in two-dimensional configurations is limited in vitro model for hepatocytes to function properly, while truly practical platforms to perform three-dimensional (3D) culture are unavailable. In this work, we present a practical organoid culture method of hepatocytes for elucidation of glucose metabolism under nominal and stress conditions. Employing this new method of culturing cells within a hollow fiber reactor, hepatocytes were observed to self-assemble into 3D spherical organoids with preservation of tight junctions and display increased liver-specific functions. Compared to both monolayer culture and sandwich culture, the hepatocyte organoids displayed higher intracellular glycogen content, glucose consumption, and gluconeogenesis and approached the in vivo values, as also confirmed by gene expression of key enzymes. Moreover, hepatocyte organoids demonstrated more realistic sensitivity to hormonal challenges with insulin, glucagon, and dexamethasone. Finally, the exposure to high glucose demonstrated toxicities including alteration of mitochondrial membrane potential, lipid accumulation, and reactive oxygen species formation, similar to the in vivo responses, which was not captured by monolayer cultures. Collectively, hepatocyte organoids mimicked the in vivo functions better than hepatocyte monolayer and sandwich cultures, suggesting suitability for applications such as antihyperglycemic drugs screening.

Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease

“…Our model utilised a combination of palmitic and oleic acids, which are common dietary long-chain FFAs found to accumulate in excess in human livers with steatosis[32,33]. When cultured in vitro , hepatocytes are also known to accumulate fat when exposed to high levels of glucose[34]. Therefore the media conditions used in this model were selected to have lower, more physiologically relevant, glucose and insulin concentrations when compared to standard hepatocyte culture media[35].…”

Experimental models of hepatitis B and C — new insights and progress

“…This approach includes the ability to facilitate the organization of all three of the major nonparenchymal cells in the liver (for example, hepatic stellate cells, Kupffer cells and endothelial cells) in self-assembled networks with hepatocytes and to allow the laminar-flow of fresh media through channels, using sophisticated perfusion apparatuses, over cells that are plated in scaffolds. The design and optimization of these devices, also called bioreactors, represent an exciting area of future use for models of viral hepatitis 106,107 . In addition, traditional approaches to immortalize primary hepatocytes by introducing oncogenes, such as human papilloma virus encoded E6 and E7, continue to be utilized with increasing success to extend the length of utility of PHHs in culture 108 .…”

Automation of 3D Cell Culture Using Chemically Defined Hydrogels

“…1,2 Therefore, researchers have begun to study cells in 3D to obtain more relevant results with in vitro cultures. In consequence, cell-based assays in which cells are grown in 3D are also increasingly gaining importance for drug development because the effects of drugs are reflected in such cultures much more closely to those found in the human body than they are in 2D cultures.…”

“…In consequence, cell-based assays in which cells are grown in 3D are also increasingly gaining importance for drug development because the effects of drugs are reflected in such cultures much more closely to those found in the human body than they are in 2D cultures. 2 Thus, the advantages of such cultures are (1) drugs that have been identified as false-negatives would be identified as effective and (2) late drug failure and possibly even drug removal from the market could be avoided, and thus a lot of money could be saved. 3 Three-dimensional cell culture opens new dimensions in cell-based assays.…”