Induction of in vitro Metabolic Zonation in Primary Hepatocytes Requires Both Near-Physiological Oxygen Concentration and Flux

Abstract

Pre-clinical drug screening is an important step in assessing the metabolic effects and hepatic toxicity of new pharmaceutical compounds. However, due to the complexity of the liver microarchitecture, simplified in vitro models do not adequately reflect in vivo situations. Especially spatial heterogeneity, known as metabolic zonation, is often lost due to limitations introduced by typical culture conditions. By culturing primary rat hepatocytes in varied ambient oxygen levels on either gas-permeable or non-permeable culture plates, we highlight the importance of biomimetic oxygen supply for the targeted induction of zonation-like phenotypes. Resulting cellular profiles illustrate the effect of pericellular oxygen concentration and consumption rates on hepatic functionality in terms of zone-specific metabolism and β-catenin signaling. We show that modulation of ambient oxygen tension can partially induce metabolic zonation in vitro when considering high supply rates, leading to in vivo-like drug metabolism. However, when oxygen supply is limited, similar modulation instead triggers an ischemic reprogramming, resembling metabolic profiles of hepatocellular carcinoma and increasing susceptibility toward drug-induced injury. Application of this knowledge will allow for the development of more accurate drug screening models to better identify adverse effects in hepatic drug metabolism.

Hierarchical Coculture of Hepatocyte and Hepatic Non-Parenchymal Cells for Liver Fibrosis Studies in vitro

“…Therefore, CYP P450 activity in the central vein is higher in vivo compared to the higher oxygen area, the portal vein. CYP3A4 activity showed oxygen tension-dependent behavior thanks to the continuous oxygenation even in hypoxic oxygen tension [43,44].…”

The Role of Liver Zonation in Physiology, Regeneration, and Disease

“…Extracellular glucose levels and glucose-6-phosphate (G6PC) gene expression were assessed and supported increased gluconeogenesis in periportal-like cultures (high oxygen). (Adapted from Scheidecker et al 95 ). (C) Flat-plate bioreactor to subject hepatocyte cultures to an oxygen gradient via flowing culture medium.…”

Accurate Evaluation of Hepatocyte Metabolisms on a Noble Oxygen-Permeable Material With Low Sorption Characteristics

“…Microphysiological systems (MPS), also known as organ-on-a-chip technology, have been proposed to recapitulate the in vivo physiological environment ( Ribeiro et al, 2019 ; Baudy et al, 2020 ). Hepatocytes require a relatively greater amount of oxygen among the various cell types, and if cultured on gas-impermeable materials, they are found to adapt to hypoxic conditions by altering metabolisms ( Scheidecker et al, 2020 ). Therefore, sufficient oxygen supply is critical to improve not only the hepatic function, but also various aspects of the hepatocyte culture, including initial cell attachment, survival rate, cell density, and interaction with non-parenchymal cells, and in unison, those factors can further ameliorate hepatic function ( Scheidecker et al, 2020 ).…”

“…Hepatocytes require a relatively greater amount of oxygen among the various cell types, and if cultured on gas-impermeable materials, they are found to adapt to hypoxic conditions by altering metabolisms ( Scheidecker et al, 2020 ). Therefore, sufficient oxygen supply is critical to improve not only the hepatic function, but also various aspects of the hepatocyte culture, including initial cell attachment, survival rate, cell density, and interaction with non-parenchymal cells, and in unison, those factors can further ameliorate hepatic function ( Scheidecker et al, 2020 ). Perfusion of culture medium in both a conventional culture and MPS devices is known to facilitate the supply of oxygen and other nutrients to cultured hepatocytes.…”