Microphysical space of a liver sinusoid device enables simplified long-term maintenance of chimeric mouse-expanded human hepatocytes.

Steven P Maher,Dennis E Kyle,Emilee C Bannister,Francy L Sinatra,Wajeeh M Saadi,Anil Kumar H Achyuta,Amy J Conway,Richard B Crouse,John H Adams,Amy Y Clark,Joseph D Cuiffi

doi:10.1007/s10544-014-9877-x

Steven P Maher, Dennis E Kyle + Show 9 more

Open Access

https://doi.org/10.1007/s10544-014-9877-x

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Abstract

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as co-culture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary hepatocyte culture systems suffer from reproducibility issues due to phenotypic variation and expensive, limited supplies of donor lots. Here we describe a microfluidic bilayer device that sustains primary human hepatocyte phenotypes, including albumin production, factor IX production, cytochrome P450 3A4 drug metabolism and bile canaliculi formation for at least 14 days in a simple monoculture format with static media. Using a variety of channel architectures, we describe how primary cell phenotype is promoted by spatial confinement within the microfluidic channel, without the need for perfusion or co-culture. By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRG™-KO) humanized mouse model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-014-9877-x) contains supplementary material, which is available to authorized users.

Highlights

The liver is involved in the course of many infectious and noninfectious diseases as it maintains hundreds of biological functions and is the primary organ responsible for activation and clearance of most therapeutic drugs
By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRGTM-KO) humanized mouse model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research
To address the challenges of integrating multiple mechanisms into one system for long-term, stable culture of human hepatocytes, we have designed a microfluidic bilayer device (MBD) featuring two microfluidic channels separated by a polydimethylsiloxane (PDMS) membrane. While this MBD is capable of media perfusion, zonal deposition of extracellular matrix, and cell co-culture, we describe how static culture of hepatocytes in the MDB is sufficient to maintain hepatocyte phenotype for three weeks, without the need for perfusion or co-culture

Summary

Introduction

The liver is involved in the course of many infectious and noninfectious diseases as it maintains hundreds of biological functions and is the primary organ responsible for activation and clearance of most therapeutic drugs While some studies, such as hepatic drug metabolism and hepatotoxicity screening, can utilize short-term hepatocyte cultures or liver microsomes, long-term culture technologies are needed to model most liver diseases and their potential therapies (Donato et al 2008). Previous liver culture studies with the causative agents of viral hepatitis and malaria have demonstrated that such models require at least three weeks of continuous host cell culture (Mazier et al 1984, Ploss et al 2010, March et al 2013) Despite their limited availablity and lot-to-lot variation, these and other studies have found human hepatocytes essential in order to properly model both the disease and the human response to potential therapeutics. A complete infectious disease liver model for drug discovery should incorporate a renewable source of human hepatocytes in a simple, long term, and flexible culture system.

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