In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System
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International Journal of Molecular Sciences Article In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System Fanny Knöspel 1, Frank Jacobs 2, Nora Freyer 1, Georg Damm 3, An De Bondt 2, Ilse van den Wyngaert 2, Jan Snoeys 2, Mario Monshouwer 2, Marco Richter 1, Nadja Strahl 1, Daniel Seehofer 3 and Katrin Zeilinger 1,* 1 Bioreactor Group, Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin 13353, Germany; [email protected] (F.K.); [email protected] (N.F.); [email protected] (M.R.); [email protected] (N.S.) 2 Janssen Research & Development, Beerse 2340, Belgium; [email protected] (F.J.); [email protected] (A.D.B.); [email protected] (I.W.); [email protected] (J.S.); [email protected] (M.M.) 3 Department for General, Visceral and Transplantation Surgery, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin 13353, Germany; [email protected] (G.D.); [email protected] (D.S.) * Correspondence: [email protected]; Tel.: +49-30-450-552-501 Academic Editor: Rolf Teschke Received: 29 February 2016; Accepted: 12 April 2016; Published: 16 April 2016 Abstract: Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro. Keywords: primary human hepatocytes, in vitro hepatotoxicity model; three-dimensional (3D) bioreactor; diclofenac 1. Introduction To date, there is an unmet need towards innovative in vitro liver models enabling clinical translation of laboratory research. Each new drug has to undergo extensive preclinical testing before entering clinical trials, and on average only one out of nine developed compounds receives approval by the regulatory authorities [1]. Animal models and in vitro models are both currently used to investigate pharmacodynamics and pharmacokinetics of new drugs. A major limitation of animal models is Int. J. Mol. Sci. 2016, 17, 584; doi:10.3390/ijms17040584 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 584 2 of 21 Int. J. Mol. Sci. 2016, 17, 584 2 of 20 themodels insufficient is the insufficient prediction prediction of human of drug human effects drug due effects to species-dependent due to species-dependent differences differences in hepatic in metabolismhepatic metabolism and different and different sensitivity sensitivity to toxic effectsto toxic [2 ].effectsIn vitro [2].culture In vitro models culture provide models an provide option an to addressoption to this address limitation this bylimitation using human by using liver huma cells.n Humanliver cells. hepatoma Human cell hepatoma lines were cell suggested lines were as asuggested potential as human a potential cell source human for cellin source vitro studies for in vitro due studies to their due high to proliferation their high proliferation capacity and capacity good availabilityand good availability [3,4], but aberrations [3,4], but aberrations in specific metabolicin specific enzymes metabolic can enzymes alter bioactivation can alter bioactivation processes in pharmacologicalprocesses in pharmacological studies. The studies. generation The of generation hepatocytes of hepatocytes from human from stem human cells, such stem as cells, embryonic such as orembryonic induced pluripotentor induced stempluripotent cells, holds stem large cells, potential holds large for futurepotential applications for future but applications currently lacks but robustcurrently protocols lacks robust for generating protocols hepatocyte-likefor generating hepatocyte-like cells with a sufficiently cells with mature a sufficiently hepatic mature phenotype hepatic [5]. Thus,phenotype primary [5]. Thus, human primary hepatocytes human (pHH) hepatocytes are currently (pHH) are deemed currently as the deemed gold standardas the gold to standard provide human-predictiveto provide human-predictive data on hepatic data metabolismon hepatic metabolism and toxicity and of new toxicity drugs of [new6]. drugs [6]. The commonlycommonly used used two-dimensional two-dimensional (2D) (2D) cultures cultures of pHH of pHH are characterized are characterized by a lack by of a cell–celllack of contactscell–cell andcontacts paracrine and paracrine signaling signaling resulting resulting in a rapid in loss a rapid of metabolic loss of metabolic functions [functions7,8]. To overcome [7,8]. To theovercome limitations the limitations of classical of 2D classical culture 2D systems, culture various systems, 2D various co-culture 2D and co-culture 3D culture and systems3D culture for cultivationsystems for of cultivation pHH were of developed pHH were to improvedeveloped the to long-term improve maintenancethe long-term and maintenance functionality and of thefunctionality cells in vitro of[ 9the–11 ].cells Our in approach vitro [9–11]. is based Our on approach a 3D multi-compartment is based on a 3D hollow-fiber multi-compartment bioreactor technologyhollow-fiber (Figure bioreactor1) for technology high-density (Figure perfusion 1) for culture high-density of pHH perfusion in co-culture culture with of non-parenchymalpHH in co-culture liverwith cellsnon-parenchymal [12]. Tissue-like liver cell cells reorganization [12]. Tissue-like and stablecell reorganization cell maintenance and overstable at cell least maintenance two weeks wereover observedat least two using weeks different were scalesobserved of the using technology different [13 scales]. In addition, of the technology stable preservation [13]. In addition, of cytochrome stable P450preservation (CYP) dependent of cytochrome enzyme P450 activities (CYP) under dependent serum-free enzyme conditions activities was under shown, serum-free using a miniaturized conditions bioreactorwas shown, variant using [a14 miniaturized,15]. bioreactor variant [14,15]. Figure 1.1. Schematic illustration of the three-dimensionalthree-dimensional (3D)(3D) multi-compartmentmulti-compartment hollow-fiberhollow-fiber bioreactorbioreactor technology.technology. The The bioreactor bioreactor consists consists of three of independentthree independent bundles ofbundles hollow fiberof hollow membranes fiber interwovenmembranes ininterwoven four layers. in Each four layerlayers. is composedEach layer ofis alternatelycomposed arrangedof alternately medium arranged and gas medium capillaries. and Thegas capillaries. two medium-carrying The two medium-carrying bundles (red and bundlesblue) are(red perfused and blue in) aare counter-current perfused in a waycounter-current to enhance massway to exchange. enhance mass The thirdexchange. bundle The (thirdorange bundle) serves (orange for gas) serves supply for gas of the supply cells of cultured the cells betweencultured thebetween capillaries. the capillaries. InIn this this study, study, the the metabolism metabolism and toxicity and toxicity of the non-steroidal of the non-steroidal anti-inflammatory anti-inflammatory drug diclofenac drug diclofenacwere investigated. were investigated. In humans, In diclofenac humans, diclofenacis mainly hydroxylated is mainly hydroxylated by cytochrome by cytochrome P450 2C9 (CYP2C9) P450 2C9 (CYP2C9)to form tothe form oxidative the oxidative metabolite metabolite 4′-OH-diclofenac 41-OH-diclofenac [16]. [ 16In]. Inaddi addition,tion, diclofenac diclofenac undergoesundergoes glucuronidationglucuronidation by uridineuridine diphosphatediphosphate (UDP)-glucuronosyltransferase-2B7(UDP)-glucuronosyltransferase-2B7 (UGT2B7) to form thethe unstableunstable diclofenac-acyl-glucuronide,diclofenac-acyl-glucuronide, which can be further hydroxylated by CYP2C8CYP2C8 leadingleading toto 441′-OH-diclofenac-acyl-glucuronide [[17]17] (Figure(Figure2 2).). DiclofenacDiclofenac can cause idiosyncratic drug-induced liver injury (DILI), which is barely predictablepredictable inin terms terms of of manifestation manifestation and and dose-dependency dose-dependency of effe ofcts effects [18]. In [ 18a study]. In awith study 180 patients, with 180 diclofenac patients, diclofenachepatotoxicity hepatotoxicity was observed was in observed 85% of cases in 85% within of cases six months within sixof therapy months of[19]. therapy Liver injury [19]. Liver was injurycharacterized