Human hepatocyte systems for in vitro toxicology analysis
Abstract
Drug induced liver injury (DILI) is still the leading single cause of drug failure during clinical phases and after market approval. Currently, many laboratories aim to develop appropriate in vitro systems to predict drug hepatotoxicity. Primary human hepatocytes are still the gold standard, but they have substantial disadvantages such as rapid dedifferentiation in vitro and lack of cell proliferation. In addition to primary human hepatocytes, liver cancer-derived cell lines such as HepG2, cytochrome P450 (CYP450) overexpressing HepG2 cell clones and HepaRG were studied intensively. In contrast to HepG2, HepaRG show promising characteristics of differentiated primary human hepatocytes, but they represent only one donor. There is some hope that this lack of donor variability can be solved by the use of iPS-derived hepatocytes. However, iPS technology still seems to need some improvement to produce physiologically relevant hepatocytes. Upcyte hepatocytes represent the most recent technical advancement combining some features of primary human hepatocytes such as physiological activity and donor variability with the ability of cell lines for extended proliferation. Altogether, more work is needed to develop and validate appropriate in vitro systems for precise prediction of DILI risk.
Abbreviations
DILI | drug-induced liver injury |
ESCs | embryonic stem cells |
iPS | induced pluripotent stem cells |
pHHs | primary human hepatocytes |
1Background
With a worldwide estimated annual incidence of 13.9–24 per 100,000 persons, drug induced liver injury (DILI) remains an economical challenge for public health systems and is the leading cause of acute liver failure in US, Europe and industrialized Asiatic states [1]. Hyman Zimmermann (1914–1999), who was an US American pioneer of clinical hepatology, observed a general rule for detecting DILI in patients. This so-called “Hy’s law” defines DILI as a combination of jaundice with hepatocellular injury detected by elevated serum levels of liver enzymes such as ALT and AST, but without existing cholestasis. In addition, patients must be negative for preexisting liver diseases that also can cause jaundice such as virus-induced hepatitis, liver damages by chronic alcohol abuse or non-alcoholic steatohepatitis (NASH). There are many studies showing that DILI patients have to face a mortality or liver transplantation rate of at least 5–10% [2, 3]. Moreover, in past 50 years DILI was the leading single cause of drug failure during clinical phases and after market approval [4, 5]. Therefore, regulatory authorities such as the Food and Drug Administration (FDA) recommended an industry guidance to detect DILI potentials of drug candidates as early as possible [6]. A major challenge is that DILI can occur in very small numbers of susceptible individuals at doses which normally are well tolerated. Very likely, this idiosyncratic DILI depends on individual genetic predispositions and multifactorial mechanisms.
In order to improve DILI prediction, numerous attempts have been undertaken such as to develop tools for risk assessment based on biomarkers [2, 7, 8] and to integrate experimental data into mathematical models [9]. Currently, many research groups are focusing on in vitro DILI models with human hepatocytes [10] because especially the risk for idiosyncratic DILI turned out to be almost undetectable in preclinical animal experiments [11, 12]. In order to assess the effect of liver biotransformation on drug hepatotoxicity, six different cytochrome P450 (CYP) isoenzymes are in focus. These are CYP1A2, –2C9, –2C19, –2D6, –2E1 and –3A4 which are responsible for more than 90% of oxidative biotransformation activity on human drugs [13]. Here we will compare the pros and cons of used hepatocyte models with respect to their biotransformation activity.
2Primary human hepatocytes
Freshly isolated primary human hepatocytes (pHHs) are the gold standard for investigations on biotransformation of drugs and their potential hepatotoxicity. Most protocols for pHH isolation involve usage of collagenase preparations from Clostridium histolyticum which, however, might contain bacterial LPS endotoxin contaminations that potentially can cause activation of cytokines leading to an inflammatory response [14, 15]. This can be one reason for substantial and fast dedifferentiation processes observed during primary cultivation of human hepatocytes. Subsequently, enzyme mixtures with better defined collagenase preparations were developed [16]. Further factors that might cause observed dedifferentiation are (I) loss of normal cell polarity during tissue disruption [14, 17] and (II) downregulation of liver-specific transcription factors with consequent diminution of most phase I and phase II protein expressions [18, 19]. Consequently, strategies to improve pHH performance focused on the application of new cell culture surfaces to mimic extracellular matrix and to allow for repolarization [17, 18, 20]. In addition, better-defined cell culture media [21, 22] and cell exposure to chemicals that influence gene expression or epigenetic procedures [17, 23] were shown to further improve liver-specific characteristics of cultured pHHs. In vivo, hepatocytes embed into a complex 3D tissue structure and do show polarity with one cell side faced to the bile canaliculi and the other one oriented to liver sinusoidal endothelial cells. It is thus not surprising that pHH performance turned better upon co-culturing with endothelial cells [24, 25] and through formation of 3D organizations in spheroids [26] or by employing micro-patterned structures [27]. Despite all these attempts, use of pHHs still is limited by problems such as scarcity of tissue material, donor variability [28], and lack of cell proliferation [14].
3HepG2 cells
Until now, there are far more than ten thousand scientific reports in PubMed on features and applications of HepG2 in combination with liver. HepG2 cells were established in 1979 from the liver tissue of a 15-year old Caucasian male [29]. In this original paper and in far most scientific reports thereafter, HepG2 cells were attributed as human hepatocellular carcinoma (hepatoma) cells. However, a reevaluation of the original histological data combined with new molecular genetic investigations provided strong evidence that HepG2 cells indeed were derived from a hepatoblastoma [30]. In contrast to cell cycle-arrested non-transformed pHHs, HepG2 cells are highly capable for proliferation. Despite of their cancer origin, HepG2 cells retained some interesting features of differentiated hepatocytes such as albumin secretion [31], insulin-stimulated glycogen synthesis [32] and glutathione-based detoxification [33, 34], making them a popular tool for studies on liver functions. This includes in vitro study on DILI [35].
Unfortunately, some important metabolic activities of mature hepatocytes are missing such as urea formation due to lack of activity of certain enzymes of the ammonia detoxification cycle [36]. Because of this deficiency and because of their cancerous nature, HepG2 cells do not give promise as cell source for bioartificial liver devices. A further disadvantage of HepG2 cells is the very low or even the lack of functional expression of almost all relevant human liver phase I (except CYP2B6) and phase II enzymes [37–40].
Since expressions of most CYP450 enzymes are downregulated in HepG2 cells, there have been many attempts to overexpress human CYP450 cDNAs recombinantly. This had been accomplished by using stable plasmid transfection [41], or transient overexpression by using adenoviral vectors [42, 43]. Both approaches do have advantages and disadvantages: Using plasmid transfection, stable cell clones can be generated, but recombinant gene expression levels are largely dependent on plasmid integration sites and DNA interaction with the nuclear matrix [44, 45]. Since recombinant adenoviral genomes usually stay extra-chromosomally due to the virus’ life cycle, gene expression initially is strong, but then attenuates along with cell proliferation [46, 47]. More recently, some groups employed lentiviral vectors for both strong and long-term gene expression of recombinant CYP450 enzymes [48, 49].
4HepaRG cells
Using a selection process on isolated liver tumor cells from a Hepatitis C virus infected female, Gripon and colleagues established the bipotent cell line HepaRG [50]. The authors showed that HepaRG cells proliferate as progenitor cell type by using a fetal calf serum-containing growth medium, but can differentiate into hepatocyte-like cells upon incubation with growth medium plus 2% DMSO for two weeks. Differentiated HepaRG form colonies of cells with typical hepatocyte morphology and bile canaliculus-like structures surrounded by biliary epithelial-like cells [51]. Interestingly, differentiated HepaRG cells display activities of relevant phase I enzymes, most of them within the range shown for pHHs. It should be noted, however, that different laboratories obtained disparate results for CYP1A2 and –3A4 enzyme activities (Table 1), although HepaRG cells were derived from only one donor. Differentiated HepaRG cells also showed expression of relevant phase II genes which were only slightly lower (GSTA4, GSTM1) or even higher (GSTA1/2, UGT1A1) than that of freshly isolated pHHs. These observed biotransformation activities made the authors conclude that HepaRG cells should be “a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies” [51]. Indeed, whole genome expression analyses upon exposure to known cytotoxic agents revealed that HepaRG resembled freshly isolated pHHs much more as was the case for HepG2 cells [52]. Interestingly, using 38 drugs with classified DILI risk according to FDA standard and a drug concentration for cell culture experiments set at 100-fold the therapeutic maximum plasma concentration, HepaRG were shown to predict the DILI risk with a sensitivity of 87% sensitivity and 87% specificity [53]. The following disadvantages are known for HepaRG: First, the cell line was derived from an individual with poor metabolizer alleles for CYP2D6, CYP3A5 and, to a lesser extent, also CYP2C9 [50, 54, 55]. Next to CYP3A4, CYP2D6 is the second most important phase I enzyme metabolizing about 30 % of all approved drugs [13]. Second, monolayer cultures of HepaRG cells have no detectable urea production [56]. Some ammonia detoxification function, however, could be rescued if HepaRG cells are maintained as three-dimensional cultures [57]. Third, HepaRG do represent the liver metabolizing function of only one single donor. Last, HepaRG cells were derived from a hepatocellular carcinoma. Thus, they are cancer cells supposed to be genetically instable, which excludes some studies on malignant transformation of normal hepatocytes.
Table 1
CYP450 enzyme activities of in vitro systems
| Specific activity1 | CYP1A2 | CYP2B6 | CYP2C9 | CYP2C19 | CYP2D6 | CYP3A4 | Reference2 |
| Freshly isolated pHHs | 9.2–68 | 283–395 | 212–1970 | [73, 74] | |||
| Cryopreserved pHHs | 2–20 | 1–2.6 | 1.8–46.6 | 0.8–2.5 | 0.18–0.9 | 10–104 | [62, 66, 75, 76] |
| HepG2 | 0.75–1.8 | 0.6 | 0.01 | 0.015–4.9 | [62, 73] | ||
| HepG2-1A2 | 120 | JHK3, unpublished | |||||
| HepG2-2C19 | 85 | (Steinbrecht et al., manuscript submitted) | |||||
| HepG2-3A4 | 600 | [48] | |||||
| HepaRG | 17.5–154.6 | 37.6–50 | 13.1–20 | 10 | 3.5 | 100–1160 | [51, 73, 76] |
| iPS-derived hepatocytes | 0.2–2 | 0.001–0.009 | 0.0075–4 | 0.04–0.13 | 0.15–0.4 | 0.5–5.5 | [61, 62] |
| Human ESC-derived hepatocytes | 0.15 | 0.0015 | 0.01 | 0.1 | [62, 77] | ||
| Upcyte hepatocytes | 0.7–3.4 | 20.9–161 | 4.8–29.1 | 0.1–16 | 21.4–225 | [66, 71] |
1pmol/mg protein/min. 2data show values from optimal conditions of each reference. 3Jan-Heiner Küpper, BTU Cottbus-Senftenberg.
5Pluripotent stem cell-derived hepatocytes
A hallmark of pluripotent stem cells is their intrinsic proliferation capacity and their potential to divide into cell types of all three germ layers. It is thus not surprising that there have been numerous attempts to generate functional hepatocytes from embryonic (ESCs) and induced pluripotent stem cells (iPS) [58–62]. ESCs represent a natural pluripotent stem cell type and their differentiation potential is thought to be superior to iPS cells [63]. Comparison of specific phase I enzyme activities reveals that hepatocytes derived from both pluripotent stem cell types perform much less well than HepaRG and Upcyte cells (Table 1). A major problem with ESCs is that for ethical reasons many countries released laws such as the so-called Embryo Protection Act in Germany which do not allow usage of ESC cell lines generated after a defined key date nor do they allow the generation of domestic ESC lines at all. Use of pluripotent stem cells generally is hampered by large cell line variabilities with respect to hepatic differentiation efficiency. An improvement of this limitation was that human pluripotent stem cell-derived hepatoblasts were shown to attach on recombinant human laminin-111 (LN111)-coated dishes to allow for their selective expansion [64]. Based on this procedure, Takayama and colleagues reproducibly reprogrammed pHHs into iPS which were differentiated into hepatocyte-like cells via hepatoblasts. They showed that pHH donor variability with respect to biotransformation capacity and hepatotoxicity was represented by the matched population of iPS-derived hepatocytes [65]. Since these authors did not disclose absolute enzyme activities, a quantitative comparison with the data shown in Table 1 is not possible. Altogether, it seems that up to date in vitro attempts failed to generate mature and fully differentiated hepatocytes from iPS.
6Upcyte / HepaFH cells
In order to circumvent some limitations of above mentioned in vitro hepatocyte systems, an additional approach was developed recently. Using the Upcyte / HepaFH protocol it is possible to directly generate proliferation-competent human hepatocytes by lentivirus-mediated transfer of coding sequences for defined proliferation genes (Upcyte® factors) into normal primary hepatocytes [66, 67].
The authors could demonstrate significant expression levels of relevant phase I and phase II enzymes, their inducibility and biotransformation activity. In addition, Upcyte hepatocytes, which represent non-transformed cells genetically stable over many passages, should be an interesting tool to study hepatotoxicity and clearance of xenobiotics [68–70]. More specifically, a panel of 11 chemicals known to induce or non-induce CYP3A4 and CYP2B6 were run on second generation Upcyte hepatocytes which proved to be a good prediction model for those CYP enzymes [71]. Mechanistically, expansion of Upcyte hepatocytes is dependent on upregulation of oncostatin M receptor during the growth phase, and differentiation into functional polarized hepatocytes upon withdrawal of oncostatin M [72]. Low expression of proliferation genes and medium components seem to allow for successful inhibition of epithelial-to-mesenchymal transition during both growth and differentiation phases. The authors could further show that Upcyte hepatocytes can be used as a cell culture model for Hepatitis C infection.
As is the case for hepatocytes derived from iPS, Upcyte hepatocytes are genetically engineered which might limit their use for therapeutic applications. However, the Upcyte technology is a good step forward towards physiologically relevant substitutes of pHHs. Upcyte hepatocytes are derived from normal primary hepatocytes, which allows for the generation of cell banks with representative donor variabilities. Physiological activities of CYP enzymes were reported to be in the range of pHHs and HepaRG outperforming those of HepG2 and pluripotent stem cell-derived hepatocytes (Table 1). In contrast to HepaRG, monolayer Upcyte hepatocytes are capable for urea production [66, 67].
7Conclusions
Freshly isolated pHHs are still the most physiological system to test hepatotoxicity of drugs. However, they have major disadvantages such as limited tissue availability, lack of proliferation and rapid dedifferentiation in vitro. As is known for human liver, pHHs show substantial donor variability. Use of liver cell lines such as HepG2 and, more recently, HepaRG circumvent the problem of limited availability. Especially HepaRG are a promising in vitro surrogate of pHHs as they display a good panel of liver-specific functions except urea formation. A disadvantage is that they represent only one donor and that they are cancer cells. The problem of donor variability can be solved by iPS-derived hepatocytes. This cell system, however, still seems to require improvement in order to catch up to pHHs, HepaRG and Upcyte / HepaFH cells. The latter cell system combines features of differentiated mature hepatocytes such as known for pHHs and HepaRG with the ability to proliferate as is the case for HepG2 and HepaRG. Furthermore, Upcyte / HepaFH cells can be generated from different donors as is the case for iPS-derived hepatocytes. Currently, Upcyte hepatocytes seem to be the most promising in vitro cell system for prediction of DILI.
Acknowledgments
This work was supported by the European Fonds for Regional Development (EFRE, Brandenburg, Germany; project “Entwicklung eines physiologisch relevanten Testsystems zur In-vitro-Erfassung von Hepatotoxizität im Hochdurchsatz” (project number: 85009748).
References
[1] | Suk KT , Kim DJ . Drug-induced liver injury: Present and future. Clin Mol Hepatol. (2012) ;18: (3):249–57. |
[2] | Bjornsson E , Olsson R . Outcome and prognostic markers in severe drug-induced liver disease. Hepatology. (2005) ;42: (2):481–9. |
[3] | Robles-Diaz M , Lucena MI , Kaplowitz N , Stephens C , Medina-Caliz I , Gonzalez-Jimenez A , et al. Use of Hy’s law and a new composite algorithm to predict acute liver failure in patients with drug-induced liver injury. Gastroenterology. (2014) ;147: (1):109–18 e5. |
[4] | Chan R , Benet LZ . Evaluation of DILI Predictive Hypotheses in Early Drug Development. Chem Res Toxicol. (2017) ;30: (4):1017–29. |
[5] | Regev A . Drug-induced liver injury and drug development: Industry perspective. Semin Liver Dis. (2014) ;34: (2):227–39. |
[6] | FDA. Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation. (2009) . |
[7] | Chen M , Suzuki A , Borlak J , Andrade RJ , Lucena MI . Drug-induced liver injury: Interactions between drug properties and host factors. J Hepatol. (2015) ;63: (2):503–14. |
[8] | Schadt S , Simon S , Kustermann S , Boess F , McGinnis C , Brink A , et al. Minimizing DILI risk in drug discovery - A screening tool for drug candidates. Toxicol In Vitro. (2015) ;30: (1 Pt B):429–37. |
[9] | Howell BA , Yang Y , Kumar R , Woodhead JL , Harrill AH , Clewell HJ 3rd , et al. In vitro to in vivo extrapolation and species response comparisons for drug-induced liver injury (DILI) using DILIsym: A mechanistic, mathematical model of DILI. J Pharmacokinet Pharmacodyn. (2012) ;39: (5):527–41. |
[10] | Albrecht W . Highlight report: Prediction of drug induced liver injury (DILI) with human hepatocytes in vitro. Arch Toxicol. (2017) ;91: (12):4021–2. |
[11] | Funk C , Roth A . Current limitations and future opportunities for prediction of DILI from in vitro. Arch Toxicol. (2017) ;91: (1):131–42. |
[12] | Robles-Diaz M , Medina-Caliz I , Stephens C , Andrade RJ , Lucena MI . Biomarkers in DILI: One More Step Forward. Front Pharmacol. (2016) ;7: :267. |
[13] | Arimoto R . Computational models for predicting interactions with cytochrome p450 enzyme. Curr Top Med Chem. (2006) ;6: (15):1609–18. |
[14] | Elaut G , Henkens T , Papeleu P , Snykers S , Vinken M , Vanhaecke T , et al. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Current drug metabolism. (2006) ;7: (6):629–60. |
[15] | Paine AJ , Andreakos E . Activation of signalling pathways during hepatocyte isolation: Relevance to toxicology in vitro. Toxicol In Vitro. (2004) ;18: (2):187–93. |
[16] | Gramignoli R , Green ML , Tahan V , Dorko K , Skvorak KJ , Marongiu F , et al. Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation. Cell Transplant. (2012) ;21: (6):1245–60. |
[17] | Luttringer O , Theil FP , Lave T , Wernli-Kuratli K , Guentert TW , de Saizieu A . Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem Pharmacol. (2002) ;64: (11):1637–50. |
[18] | Borlak J , Singh PK , Rittelmeyer I . Regulation of Liver Enriched Transcription Factors in Rat Hepatocytes Cultures on Collagen and EHS Sarcoma Matrices. PLoS One. (2015) ;10: (4):e0124867. |
[19] | Henkens T , Vinken M , Vanhaecke T , Rogiers V . Modulation of CYP1A1 and CYP2B1 expression upon cell cycle progression in cultures of primary rat hepatocytes. Toxicol In Vitro. (2007) ;21: (7):1253–7. |
[20] | Zeisberg M , Kramer K , Sindhi N , Sarkar P , Upton M , Kalluri R . De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane. Mol Cell Biochem. (2006) ;283: (1-2):181–9. |
[21] | Lubberstedt M , Muller-Vieira U , Biemel KM , Darnell M , Hoffmann SA , Knospel F , et al. Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies. J Tissue Eng Regen Med. (2015) ;9: (9):1017–26. |
[22] | Nelson LJ , Treskes P , Howie AF , Walker SW , Hayes PC , Plevris JN . Profiling the impact of medium formulation on morphology and functionality of primary hepatocytes in vitro. Sci Rep. (2013) ;3: :2735. |
[23] | Henkens T , Papeleu P , Elaut G , Vinken M , Rogiers V , Vanhaecke T . Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes. Toxicol Appl Pharmacol. (2007) ;218: (1):64–71. |
[24] | Shulman M , Nahmias Y . Long-term culture and coculture of primary rat and human hepatocytes. Methods Mol Biol. (2013) ;945: :287–302. |
[25] | Ware BR , Durham MJ , Monckton CP , Khetani SR . A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells. Cell Mol Gastroenterol Hepatol. (2018) ;5: (3):187–207. |
[26] | Bell CC , Hendriks DF , Moro SM , Ellis E , Walsh J , Renblom A , et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep. (2016) ;6: :25187. |
[27] | Wang WW , Khetani SR , Krzyzewski S , Duignan DB , Obach RS . Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites. Drug Metab Dispos. (2010) ;38: (10):1900–5. |
[28] | Shimada T , Yamazaki H , Mimura M , Inui Y , Guengerich FP . Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther. (1994) ;270: (1):414–23. |
[29] | Aden DP , Fogel A , Plotkin S , Damjanov I , Knowles BB . Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature. (1979) ;282: (5739):615–6. |
[30] | Lopez-Terrada D , Cheung SW , Finegold MJ , Knowles BB . Hep G2 is a hepatoblastoma-derived cell line. Hum Pathol. (2009) ;40: (10):1512–5. |
[31] | Busso N , Chesne C , Delers F , Morel F , Guillouzo A . Transforming growth-factor-beta (TGF-beta) inhibits albumin synthesis in normal human hepatocytes and in hepatoma HepG2 cells. Biochem Biophys Res Commun. (1990) ;171: (2):647–54. |
[32] | Meier M , Klein HH , Kramer J , Drenckhan M , Schutt M . Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling. J Endocrinol. (2007) ;193: (1):45–51. |
[33] | Kade S , Herzog N , Schmidkte K-U , Küpper J-H . Chronic ethanol treatment depletes glutathione regeneration capacity in hepatoma cell line HepG2. Journal of Cellular Biotechnology. (2016) ;1: :13. |
[34] | Lamy E , Volkel Y , Roos PH , Kassie F , Mersch-Sundermann V . Ethanol enhanced the genotoxicity of acrylamide in human, metabolically competent HepG2 cells by CYP2E1 induction and glutathione depletion. Int J Hyg Environ Health. (2008) ;211: (1-2):74–81. |
[35] | Ghallab A . The rediscovery of HepG2 cells for prediction of drug induced liver injury (DILI). EXCLI J. (2014) ;13: :1286–8. |
[36] | Mavri-Damelin D , Eaton S , Damelin LH , Rees M , Hodgson HJ , Selden C . Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2. Int J Biochem Cell Biol. (2007) ;39: (3):555–64. |
[37] | Castell JV , Jover R , Martnez-Jimnez CP , Gmez-Lechn MJ . Hepatocyte cell lines: Their use, scope and limitations in drug metabolism studies. Expert Opinion on Drug Metabolism & Toxicology. (2006) ;2: (2):183–212. |
[38] | Rodríguez-Antona C , Donato MT , Boobis A , Edwards RJ , Watts PS , Castell JV , et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: Molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. (2002) ;32: (6):505–20. |
[39] | Westerink WMA , Schoonen WGEJ . Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicology in Vitro. (2007) ;21: (8):1581–91. |
[40] | Wilkening S , Stahl F , Bader A . Comparison of primary human hepatocytes and hepatoma cell line HEPG2 with regard to their biotransformation properties. Drug Metabolism and Disposition. (2003) ;31: (8):1035–42. |
[41] | Frederick DM , Jacinto EY , Patel NN , Rushmore TH , Tchao R , Harvison PJ . Cytotoxicity of 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) and analogues in wild type and CYP3A4 stably transfected HepG2 cells. Toxicology in Vitro. (2011) ;25: (8):2113–9. |
[42] | Hosomi H , Fukami T , Iwamura A , Nakajima M , Yokoi T . Development of a Highly Sensitive Cytotoxicity Assay System for CYP3A4-Mediated Metabolic Activation. Drug Metabolism and Disposition. (2011) ;39: (8):1388–95. |
[43] | Tolosa L , Gómez-Lechón MJ , Pérez-Cataldo G , Castell JV , Donato MT . HepG2 cells simultaneously expressing five P450 enzymes for the screening of hepatotoxicity: Identification of bioactivable drugs and the potential mechanism of toxicity involved. Archives of Toxicology. (2013) ;87: (6):1115–27. |
[44] | Kupper JH , Muller M , Jacobson MK , Tatsumi-Miyajima J , Coyle DL , Jacobson EL , et al. trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N’-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon. Mol Cell Biol. (1995) ;15: (6):3154–63. |
[45] | Phi-Van L , von Kries JP , Ostertag W , Stratling WH . The chicken lysozyme 5’ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. (1990) ;10: (5):2302–7. |
[46] | Rauschhuber C , Noske N , Ehrhardt A . New insights into stability of recombinant adenovirus vector genomes in mammalian cells. Eur J Cell Biol. (2012) ;91: (1):2–9. |
[47] | Lai CM , Lai YK , Rakoczy PE . Adenovirus and adeno-associated virus vectors. DNA Cell Biol. (2002) ;21: (12):895–913. |
[48] | Herzog N , Katzenberger N , Martin F , Schmidkte K-U , Küpper J-H . Generation of cytochrome P450 3A4-overexpressing HepG2 cell clones for standardization of hepatocellular testosterone 6 beta-hydroxylation activity. Jounal of Cellular Biotechnology. (2015) ;1: :11. |
[49] | Xuan J , Chen S , Ning B , Tolleson WH , Guo L . Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity. Chem Biol Interact. (2016) ;255: :63–73. |
[50] | Gripon P , Rumin S , Urban S , Le Seyec J , Glaise D , Cannie I , et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A. (2002) ;99: (24):15655–60. |
[51] | Aninat C , Piton A , Glaise D , Le CT , Langouet S , Morel F , et al. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab Dispos. (2006) ;34: (1):75–83. |
[52] | Jennen DG , Magkoufopoulou C , Ketelslegers HB , van Herwijnen MH , Kleinjans JC , van Delft JH . Comparison of HepG2 and HepaRG by whole-genome gene expression analysis for the purpose of chemical hazard identification. Toxicol Sci. (2010) ;115: (1):66–79. |
[53] | Tomida T , Okamura H , Yokoi T , Konno Y . A modified multiparametric assay using HepaRG cells for predicting the degree of drug-induced liver injury risk. J Appl Toxicol. (2017) ;37: (3):382–90. |
[54] | Jackson JP , Li L , Chamberlain ED , Wang H , Ferguson SS . Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures. Drug Metab Dispos. (2016) ;44: (9):1463–79. |
[55] | Guillouzo A , Corlu A , Aninat C , Glaise D , Morel F , Guguen-Guillouzo C . The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact. (2007) ;168: (1):66–73. |
[56] | Lubberstedt M , Muller-Vieira U , Mayer M , Biemel KM , Knospel F , Knobeloch D , et al. HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J Pharmacol Toxicol Methods. (2011) ;63: (1):59–68. |
[57] | Nibourg GA , Hoekstra R , van der Hoeven TV , Ackermans MT , Hakvoort TB , van Gulik TM , et al. Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor. Int J Biochem Cell Biol. (2013) ;45: (8):1860–8. |
[58] | Freyer N , Knospel F , Strahl N , Amini L , Schrade P , Bachmann S , et al. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor. Biores Open Access. (2016) ;5: (1):235–48. |
[59] | Kang SJ , Lee HM , Park YI , Yi H , Lee H , So B , et al. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2. Cell Biol Toxicol. (2016) ;32: (5):403–17. |
[60] | Liu H , Ye Z , Kim Y , Sharkis S , Jang YY . Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes. Hepatology. (2010) ;51: (5):1810–9. |
[61] | Starokozhko V , Hemmingsen M , Larsen L , Mohanty S , Merema M , Pimentel RC , et al. Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering. J Tissue Eng Regen Med. (2018) ;12: (5):1273–84. |
[62] | Ulvestad M , Nordell P , Asplund A , Rehnstrom M , Jacobsson S , Holmgren G , et al. Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells. Biochem Pharmacol. (2013) ;86: (5):691–702. |
[63] | Feng C , Jia YD , Zhao XY . Pluripotency of induced pluripotent stem cells. Genomics Proteomics Bioinformatics. (2013) ;11: (5):299–303. |
[64] | Takayama K , Nagamoto Y , Mimura N , Tashiro K , Sakurai F , Tachibana M , et al. Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes. Stem Cell Reports. (2013) ;1: (4):322–35. |
[65] | Takayama K , Morisaki Y , Kuno S , Nagamoto Y , Harada K , Furukawa N , et al. Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes. Proc Natl Acad Sci U S A. (2014) ;111: (47):16772–7. |
[66] | Burkard A , Dahn C , Heinz S , Zutavern A , Sonntag-Buck V , Maltman D , et al. Generation of proliferating human hepatocytes using Upcyte(R) technology: Characterisation and applications in induction and cytotoxicity assays. Xenobiotica. (2012) ;42: (10):939–56. |
[67] | Herzog N , Hansen M , Miethbauer S , Schmidtke KU , Anderer U , Lupp A , et al. Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures. Cell Biol Int. (2016) ;40: (3):341–53. |
[68] | Norenberg A , Heinz S , Scheller K , Hewitt NJ , Braspenning J , Ott M . Optimization of upcyte(R) human hepatocytes for the in vitro micronucleus assay. Mutat Res. (2013) ;758: (1-2):69–79. |
[69] | Schaefer M , Schanzle G , Bischoff D , Sussmuth RD . Upcyte Human Hepatocytes: A Potent In Vitro Tool for the Prediction of Hepatic Clearance of Metabolically Stable Compounds. Drug Metab Dispos. (2016) ;44: (3):435–44. |
[70] | Tolosa L , Gomez-Lechon MJ , Lopez S , Guzman C , Castell JV , Donato MT , et al. Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing. Toxicol Sci. (2016) ;152: (1):214–29. |
[71] | Ramachandran SD , Vivares A , Klieber S , Hewitt NJ , Muenst B , Heinz S , et al. Applicability of second-generation upcyte(R) human hepatocytes for use in CYP inhibition and induction studies. Pharmacol Res Perspect. (2015) ;3: (5):e00161. |
[72] | Levy G , Bomze D , Heinz S , Ramachandran SD , Noerenberg A , Cohen M , et al. Long-term culture and expansion of primary human hepatocytes. Nat Biotechnol. (2015) ;33: (12):1264–71. |
[73] | Gerets HHJ , Tilmant K , Gerin B , Chanteux H , Depelchin BO , Dhalluin S , et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biology and Toxicology. (2012) ;28: (2):69–87. |
[74] | Gramignoli R , Tahan V , Dorko K , Venkataramanan R , Fox IJ , Ellis EC , et al. Rapid and sensitive assessment of human hepatocyte functions. Cell Transplant. (2014) ;23: (12):1545–56. |
[75] | Gomez-Lechon MJ , Castell JV , Donato MT . An update on metabolism studies using human hepatocytes in primary culture. Expert Opin Drug Metab Toxicol. (2008) ;4: (7):837–54. |
[76] | Yokoyama Y , Sasaki Y , Terasaki N , Kawataki T , Takekawa K , Iwase Y , et al. Comparison of drug metabolism and its related hepatotoxic effects in HepaRG, cryopreserved human hepatocytes, and HepG2 cell cultures. Biol Pharm Bull. (2018) . |
[77] | Kang SJ , Park YI , Hwang SR , Yi H , Tham N , Ku HO , et al. Hepatic population derived from human pluripotent stem cells is effectively increased by selective removal of undifferentiated stem cells using YM155. Stem Cell Res Ther. (2017) ;8: (1):78. |
