A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Design and Fabrication of the Vertical-Flow Bioreactor for Compaction Hepatocyte Culture in Drug Testing Application

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.

A Versatile Microfluidic Tool for the 3D Culture of HepaRG Cells Seeded at Various Stages of Differentiation

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of HepaRG cells at various stages of differentiation. To prevent hepatoblast-like cells from migrating out of the chambers, the slit height was decreased and the medium flow rate increased. Maintaining already differentiated and less adherent cells within the chambers required desensitisation of the system to pressure variations. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Previous studies have suggested that primary hepatocytes cultured in vitro undergo a rapid loss of function. On the other hand, in the clinic, drug induced liver injury typically manifests 5 days to 3 months after starting a medication. Thus, novel approaches that can maintain the function of primary human hepatocytes for longer durations of time may enable the development of improved in vitro assays for detecting hepatotoxicity. Previous studies have demonstrated that two-dimensional micro-patterning of hepatocytes with fibroblasts leads to improved maintenance of the hepatocyte phenotype relative to hepatocyte monocultures, in serum containing medium. Additionally, we, and others, have shown that three-dimensional culture of hepatocytes leads to enhanced function (in serum-free medium). In this study we wanted to (i) examine the effect of combining the above two approaches on hepatocyte function, and (ii) to further delineate the effect of serum on hepatocyte function. We developed a user-friendly and inexpensive approach for constructing layered spheroids. Similar to previous results in two-dimensional (2d) culture, we observed that 3d culture of hepatocytes alone (i.e. monoculture) in serum-containing medium led to an increase in the urea production rate, but near-complete loss of cytochrome activity in both lots of primary human hepatocytes (PHH) tested. In serum-free sandwich culture, cytochrome activity was maintained at the level observed in freshly thawed PHH for one lot, but almost completely lost in another lot. Spheroid culture of both lots of PHH in serum-free medium led to maintenance of CYP3A4 and CYP1A2 activity at the fresh thaw level, though CYP2B6 activity was reduced. In contrast to PHH monoculture, co-cultures of PHH with NIH 3T3 fibroblast cells benefitted from the presence of serum, and led to 3-5-fold increases in CYP activity relative to even serum-free spheroid monocultures. Layering of the fibroblasts did not result in improvements over mixed co-cultures. These results indicate the importance of appropriate serum-free monoculture control experiments in the evaluation of novel biomaterials and techniques for hepatocyte co-culture. Further, urea production and cytochrome production are decoupled; therefore, urea production is an insufficient readout when developing models for pharmaceutical applications.