A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation.

William J Mccarty,O Berk Usta,Martin L Yarmush

doi:10.1038/srep26868

Abstract

In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

Highlights

In vitro liver models are important tools that have been used in various forms for more than 40 years for basic science and translational academic research and preclinical toxicity screening by the pharmaceutical industry
The quantified viability across the width of the device (Fig. 5e) suggested a gradient in CYP activity (Supplemental Fig. 1c), demonstrating an in vivo-like gradient in drug conjugation metabolism (Fig. 5f). These results demonstrate the induction of gradients in a variety of hepatocyte metabolisms during in vitro culture within a microdevice
We modified hepatocyte metabolism through short-term (24 hr) concentration gradient induction with hormones or a chemical (3-MC) (Fig. 1). This induction produced a change from zone 1-like to zone 3-like hepatocyte metabolisms across approximately 25–40 cells, similar to the magnitude of change occurring along the sinusoid in vivo

Summary

Introduction

In vitro liver models are important tools that have been used in various forms for more than 40 years for basic science and translational academic research and preclinical toxicity screening by the pharmaceutical industry. Zone 3 hepatocytes are efficient at glucose uptake and storage as glycogen[10,17], glutamine formation from ammonia[10,11,18], alcohol degradation[19], and phase I drug conjugation of xenobiotic compounds via cytochrome P450 monooxygenases[14,20,21,22,23,24]. This change in metabolism occurs over the length of the sinusoid, which contains approximately 25 cells[2]. In addition to recapitulating the zonation of glucose storage and urea production, this in vitro model mimics the zonal toxicity responses of the liver to example environmental agents and pharmaceuticals

Methods

Results

Conclusion