Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application

Three-dimensional Numerical Modeling and Computational Fluid Dynamics Simulations to Analyze and Improve Oxygen Availability in the AMC Bioartificial Liver

A numerical model to investigate fluid flow and oxygen (O(2)) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO(2)) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O(2) consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO(2) of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL

Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs) - integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications