Phase 1 and Phase 2 Drug Metabolism and Bile Acid Production of HepaRG Cells in a Bioartificial Liver in Absence of Dimethyl Sulfoxide

Abstract

The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDPglucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver

“…We have also optimized the techniques to cold preserve this biocomponent to ensure its continuous availability, which is essential for any BAL to become a useful therapeutic tool for patients with ALF. As future prospects, these results encourage us to study other important liver functions, as transcription of albumin and clotting factors during reperfusion and to challenge it to treat acute liver failure of small animal models which will allow the measurement of bilirubin conjugation, blood clotting functions or intracranial pressure all important clinical prognostic predictors for ALF patients[28-30]. Also, to scale this MBR up and evaluate it in big animal models of ALF such as pigs.…”

Bioreactor technologies to support liver function in vitro

“…The scale is unequivocally enormous – tens to hundreds of grams of cells – a scale that presents an almost insurmountable barrier to using primary human hepatocytes on a routine basis, especially when the use of reactors is on an emergency and not pre-scheduled basis. Hence, of five reactor systems that progressed to clinical trials in the US and Europe, four were developed for use with primary porcine cells, one with the human HepG/C3a cell line [115] and nascent incorporation of HepaRG cells [116]. Translation to the clinic has been arduously slow, hampered by the high cost, difficulty in obtaining clear-cut results in clinical trials, and competition for subsets of patients by a cellular reactors that remove toxins [115].…”

“…An alternate hollow fiber design combined fiber matrix sheets interspersed with hollow fibers to similarly address the high oxygen demands of hepatocytes by providing a set of hollow fibers for gas exchange in the context of adhesion to the fibers was developed by Chamuleau and coworkers for clinical application and has also been scaled down from a clinical version to a version suitable for pharmacological studies [125– 127][128,129][116,130,131]. The large clinical-scale version of this reactor system was showed comparable to the Gerlach 3-fiber system in maintaining liver-specific function of primary porcine cells over 7 days in culture, and both maintained >90% function of the 10 billion cells seeded [132].…”

“…The authors suggest that combinations of substrate and autocrine factors may contribute to this zonation pattern [127]. This reactor system has also been optimized for stable differentiation and maintenance of HepaRG cells in long-term culture in the absence of DMSO for use in drug metabolism studies [116,130,131]. …”

A strategy for integrating essential three-dimensional microphysiological systems of human organs for realistic anticancer drug screening

“…This will filter drug compounds accordingly and secrete the appropriate factors into the tissue microenvironment. Liver tissue is complex and consists of different zones with specific enzyme activity (Phase I and Phase II enzymes) [91]. A major factor that regulates the enzyme activity in these zones is oxygen tension (i.e.…”