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

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.