title: Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor.
abstract: The clinical application of a bioartificial liver (BAL) depends on the availability of a human cell source with high hepatic functionality, such as the human hepatoma cell line HepaRG. This cell line has demonstrated high hepatic functionality, but the effect of BAL culture on its functionality in time is not known. Therefore, we studied the characteristics of the HepaRG-AMC-BAL over time, and compared the functionality of the HepaRG-AMC-BAL with monolayer cultures of HepaRG cells, normalized for protein (bioactive mass) and DNA (cell number). Histological analysis of 14-day-old BALs demonstrated functional heterogeneity similar to that of monolayer cultures. Hepatic functionality of the HepaRG-AMC-BALs increased during 2-3 weeks of culture. The majority of the measured protein-normalized hepatic functions were already higher in day 14 BAL cultures compared to monolayer cultures, including ammonia elimination (3.2-fold), urea production (1.5-fold), conversion of (15)N-ammonia into (15)N-urea (1.4-fold), and cytochrome P450 3A4 activity (7.9-fold). Lactate production in monolayer cultures switched into lactate consumption in the BAL cultures, a hallmark of primary hepatocytes. When normalized for DNA, only cytochrome P450 3A4 activity was 2.5-fold higher in the BAL cultures compared to monolayer cultures and lactate production switched to consumption, whereas urea production and (15)N-urea production were 1.5- to 2-fold lower. The different outcomes for protein and DNA normalized functions probably relate to a smaller cell volume of HepaRG cells when cultured in the AMC-BAL. Cell damage was 4-fold lower in day 14 BAL cultures compared to monolayer cultures. Transcript levels of cytochrome P450 1A2, 2B6, 3A4 and 3A7 genes and of regulatory genes hepatic nuclear factor 4α and pregnane X receptor increased in time in BAL cultures and reached higher levels than in monolayer cultures. Lastly, metabolism of amino acids, particularly the alanine consumption and ornithine production of HepaRG-AMC-BALs more resembled that of primary hepatocytes than monolayer HepaRG cultures. We conclude therefore that BAL culture of HepaRG cells increases its hepatic functionality, particularly when normalized for biomass, both over time, and compared to monolayer, and this is associated with a reduction in cell damage, upregulation of both regulatory and structural hepatic genes, and changes in amino-acid metabolism. These results underline the potential of HepaRG cells for BAL application.
authors.affiliation: Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Geert A A
authors.initials: GA
authors.lastname: Nibourg
authors.forename: Ruurdtje
authors.initials: R
authors.lastname: Hoekstra
authors.forename: Tessa V
authors.initials: TV
authors.lastname: van der Hoeven
authors.forename: Mariëtte T
authors.initials: MT
authors.lastname: Ackermans
authors.forename: Theodorus B M
authors.initials: TB
authors.lastname: Hakvoort
authors.forename: Thomas M
authors.initials: TM
authors.lastname: van Gulik
authors.forename: Robert A F M
authors.initials: RA
authors.lastname: Chamuleau
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000378:metabolism; D019149:Bioreactors; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000502:physiology*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology*; D019164:Liver, Artificial; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase; D001219:Aspartate Aminotransferases
keywords: 6β-OHT; 6β-hydroxytestosterone; ALB; AMC-BAL; ANOVA; ARG1; AST; BAL; Bioartificial liver; CPS; CYP; DMSO; Differentiation; GS; HNF4α; HepaRG; Hepatic functionality; LDH; PXR; albumin; arginase 1; aspartate aminotransferase; bioartificial liver; carbamoyl phosphate synthetase; cytochrome P450; dimethyl sulfoxide; glutamine synthetase; hepatic nuclear factor 4α; lactate dehydrogenase; one-way analysis of variance; pregnane X receptor