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

Benedikt Scheidecker,Masahiro Sugimoto,Masaki Nishikawa,Mathieu Danoy,Marie Shinohara,Yasuyuki Sakai

doi:10.3389/fbioe.2020.00524

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.

Highlights

The development of pharmaceuticals requires strict compliance with regulatory requirements
Gas-permeable culture vessels allow for precise regulation of cellular oxygen levels, they are generally not used in routine applications involving hepatocyte cultures
By blocking the bottom membrane of selected cultures, oxygenation similar to conventional tissue culture plates was achieved, limiting the oxygen supply to diffusion through the aqueous medium—while ensuring equal cellular setups between conditions in terms of cell density and physical quality of materials. These setups were subsequently cultured in incubators with ambient oxygen levels ranging between 20 and 2.5% (Figures 1A, S1a) with the intent of generating defined, oxygen-dependent phenotypes mimicking zonation-like metabolic patterns

Summary

Introduction

The development of pharmaceuticals requires strict compliance with regulatory requirements. Efficacy and toxicity of drugs are essential parameters that need to be considered before further testing in clinical trials. In these pre-clinical studies, the safety of pharmaceutical compounds is assessed through a myriad of in vivo and in vitro assays regarding their absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox). Even though these functions require the interaction of multiple organs, the focus of research often centers on the liver as the main site of metabolism. While in vivo models do represent the metabolism of an organism adequately, utilization of animal models faces multiple issues ranging from the ethical questions regarding animal use to the applicability of results due to interspecies differences (Begley and Ellis, 2012).

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Conclusion