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Fully differentiated HepaRG cells organized in well-delineated trabeculae with many bright canaliculi-like structures.

HepaRG cell line is a human hepatic in vitro line used in liver biology research and for assessing liver pathology, hepatotoxicity, and drug-induced injury. The HepaRG model is considered a surrogate for Primary Human Hepatocytes, which are the most pertinent model to reproduce the human liver functioning as they express 99% of the same genes.

In contrast to the historic hepatic line HepG2, HepaRG cells preserved various liver-specific functions, including the expression of CYP enzymes and transporters, the formation of bile canaliculi, and, the ability to be applied in 2D and 3D configuration.

History

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In 1999, Dr. Christiane Guguen-Guillouzo and Dr. Christian Trépo collaborated on a medical and scientific project. During the course of their research, a tumor sample from a patient with cholangiocarcinoma and HCV was given to Sylvie Rumin to study hepatitis infection. Rumin observed a group of cells that resembled hepatic cells and gradually lost HCV infection markers. Philippe Gripon later developed these cells, finding that they had the ability to undergo complete hepatocyte differentiation while retaining all liver-specific functions. As a way to honor the contributions of Rumin and Gripon, the cells were named HepaRG, using the first letter of their last names. Since their discovery, many scientists characterized and used the HepaRG model in their studies.[1][2][3][4]

Characterization

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HepaRG cells are bipotent progenitors, capable of differentiating into both biliary and hepatocyte lineages. In culture, they are organized in well-delineated trabeculae with many bright canaliculi-like structures under 2D and 3D configurations. They are polarized cells that breathe aerobically, consume lactate, and contain as many mitochondria as the human hepatocytes. The cell line has the potential to express major properties of stem cells including high plasticity & transdifferentiation capacity.

The HepaRG cells have been found to express major nuclear receptors,[5] as well as drug and bile acids transporters,[6] and key hepatic nuclear factors.[7][8] They also possess functional levels of phase I (CYP (CYP1A1/2, CYP2B6, CYP2Cs, CYP3A4, etc.) and II (UGT1A1, GSTA1, GSTA4, GSTM1) drug metabolizing enzymes.[9] Additionally, the HepaRG cells have functional mitochondria, hepatokine secretion abilities, and a suitable response to insulin.[10][11]

One unique characteristic of HepaRG cells is that they can survive up to four weeks in culture, making them useful for long-term studies and repeated exposures to drugs and chemicals, unlike primary human hepatocytes. Moreover, the cells can be infected by HBV and HCV and support viral replication.

Undifferentiated hepatocyte-like cells appear in small, individualized, colonies

The cells are available as undifferentiated growth-stage cells that can be grown in-house with the possibility of cell manipulation and amplification; or as fully differentiated cells that are ready and easy-to-use cells with high inter-assay reproducibility and proven functionality across multiple applications. They can also be used as spheroids and co-culture.

Use in research

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HepaRG cells are considered fit-for-all-purpose cells as they were used in many applications ranging from drug development, drug metabolism and interaction assessment, chemical testing, assay validation, hepatotoxicity assessments, liver Biology, liver disease characterization, and virology studies.

Basic research

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HepaRG cells are a versatile tool for modeling various viral and parasitic infections such as hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and hepatitis delta virus (HDV).[12][13]

They are also useful for studying the interconnected pathways involved in carbohydrate homeostasis and lipid metabolism. In contrast to many other cell lines, HepaRG cells are capable of regulating glycogenolysis and gluconeogenesis at levels similar to primary human hepatocytes, as well as retaining the response mechanisms associated with lipid-metabolizing enzymes.[14][15]

Medical applications

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HepaRG cells have shown potential in the field of regenerative medicine, specifically in the development of bio-artificial livers and liver-assisting devices due to their ability to be cultured in 3D bioreactors.

A study demonstrated that HepaRG cells can be incorporated into a microfluidic system to form a functional bio-artificial liver capable of detoxifying ammonia and other harmful substances.[16] In another study, a modular extracorporeal liver support system, which combines HepaRG cells with a bioartificial scaffold was created to support liver function.[17] This system was shown to be effective in removing toxins from the blood and supporting liver function in animal models.

Industrial applications

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HepaRG is owned by the French National Institute of Health and Medical Research (INSERM) and since 2003, Biopredic International acquired the license of the cells and settled both the master and the working banks for preserving the stability of the line.

In vitro-ADME applications

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HepaRG cells have many applications in in-vitro ADME (absorption, distribution, metabolism, and excretion) studies. They were used to study drug metabolism and toxicity, including phase I and phase II enzyme metabolism, induction, and inhibition.[18][19][20] Moreover, HepaRG cells have been used to study drug transporters, measure compound clearance, and predict metabolic stability as well as drug-drug interactions. Additionally, HepaRG has been used to evaluate acute and chronic drug toxicity, genotoxicity, and hepatotoxicity.[21][22]

The high reproducibility of the drug-induced metabolic enzyme levels between batches enables routine high-throughput analysis of compound clearance.[23][24]

Hepatotoxicity screening and mechanistic testing

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HepaRG cells have been utilized for assessing drug-induced liver injury (DILI), including steatosis, cholestasis, and phospholipidosis, as well as for evaluating genotoxicity and carcinogenicity.[25][26][27][28] Additionally, they have been employed for studying drug-induced mitochondrial toxicity, apoptosis, and inflammation.[29] HepaRG cells are advantageous for uptake and biliary secretion studies due to their expression of various uptake and efflux drug transporters, and the formation of tight junctions and bile canaliculi.

For instance, HepaRG cells have been used to evaluate the hepatotoxicity of compounds in drug development, such as acetaminophen, troglitazone, and valproic acid, as well as environmental toxins like aflatoxin B1 and ethanol.[30][31][32]

References

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  1. ^ Anthérieu, Sébastien; Chesné, Christophe; Li, Ruoya; Camus, Sandrine; Lahoz, Agustin; Picazo, Laura; Turpeinen, Miia; Tolonen, Ari; Uusitalo, Jouko; Guguen-Guillouzo, Christiane; Guillouzo, André (March 2010). "Stable Expression, Activity, and Inducibility of Cytochromes P450 in Differentiated HepaRG Cells". Drug Metabolism and Disposition. 38 (3): 516–525. doi:10.1124/dmd.109.030197. PMID 20019244. S2CID 2134987.
  2. ^ Gerets, H. H. J.; Tilmant, K.; Gerin, B.; Chanteux, H.; Depelchin, B. O.; Dhalluin, S.; Atienzar, F. A. (April 2012). "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. 28 (2): 69–87. doi:10.1007/s10565-011-9208-4. PMC 3303072. PMID 22258563.
  3. ^ Jossé, Rozenn; Aninat, Caroline; Glaise, Denise; Dumont, Julie; Fessard, Valérie; Morel, Fabrice; Poul, Jean-Michel; Guguen-Guillouzo, Christiane; Guillouzo, André (June 2008). "Long-Term Functional Stability of Human HepaRG Hepatocytes and Use for Chronic Toxicity and Genotoxicity Studies". Drug Metabolism and Disposition. 36 (6): 1111–1118. doi:10.1124/dmd.107.019901. PMID 18347083. S2CID 29337628.
  4. ^ Antoun, Joseph; Amet, Yolande; Simon, Brigitte; Dréano, Yvonne; Corlu, Anne; Corcos, Laurent; Salaun, Jean Pierre; Plée-Gautier, Emmanuelle (2006). "CYP4A11 is repressed by retinoic acid in human liver cells". FEBS Letters. 580 (14): 3361–3367. doi:10.1016/j.febslet.2006.05.006. PMID 16712844. S2CID 12620173.
  5. ^ Bu, Hai-Zhi; Kang, Ping; Zhao, Ping; Pool, William F.; Wu, Ellen Y. (October 2005). "A Simple Sequential Incubation Method for Deconvoluting the Complicated Sequential Metabolism of Capravirine in Humans". Drug Metabolism and Disposition. 33 (10): 1438–1445. doi:10.1124/dmd.105.005413. PMID 16006566. S2CID 9884327.
  6. ^ Bachour-El Azzi, Pamela; Sharanek, Ahmad; Abdel-Razzak, Ziad; Antherieu, Sebastien; Al-Attrache, Houssein; Savary, Camille C.; Lepage, Sylvie; Morel, Isabelle; Labbe, Gilles; Guguen-Guillouzo, Christiane; Guillouzo, André (September 2014). "Impact of Inflammation on Chlorpromazine-Induced Cytotoxicity and Cholestatic Features in HepaRG Cells". Drug Metabolism and Disposition. 42 (9): 1556–1566. doi:10.1124/dmd.114.058123. PMID 25002748. S2CID 1161113.
  7. ^ Tascher, Georg; Burban, Audrey; Camus, Sandrine; Plumel, Marine; Chanon, Stéphanie; Le Guevel, Remy; Shevchenko, Valery; Van Dorsselaer, Alain; Lefai, Etienne; Guguen-Guillouzo, Christiane; Bertile, Fabrice (21 February 2019). "In-Depth Proteome Analysis Highlights HepaRG Cells as a Versatile Cell System Surrogate for Primary Human Hepatocytes". Cells. 8 (2): 192. doi:10.3390/cells8020192. PMC 6406872. PMID 30795634.
  8. ^ Yokoyama, Yuichi; Sasaki, Yoshifumi; Terasaki, Natsuko; Kawataki, Taku; Takekawa, Koji; Iwase, Yumiko; Shimizu, Toshinobu; Sanoh, Seigo; Ohta, Shigeru (1 May 2018). "Comparison of Drug Metabolism and Its Related Hepatotoxic Effects in HepaRG, Cryopreserved Human Hepatocytes, and HepG2 Cell Cultures". Biological and Pharmaceutical Bulletin. 41 (5): 722–732. doi:10.1248/bpb.b17-00913. PMID 29445054.
  9. ^ Li, Jinpeng; Settivari, Raja S.; LeBaron, Matthew J.; Marty, Mary Sue (1 December 2019). "Functional Comparison of HepaRG Cells and Primary Human Hepatocytes in Sandwich and Spheroid Culture as Repeated-Exposure Models for Hepatotoxicity". Applied in Vitro Toxicology. 5 (4): 187–195. doi:10.1089/aivt.2019.0008. S2CID 213691760.
  10. ^ Iroz, Alison; Couty, Jean-Pierre; Postic, Catherine (August 2015). "Hepatokines: unlocking the multi-organ network in metabolic diseases". Diabetologia. 58 (8): 1699–1703. doi:10.1007/s00125-015-3634-4. ISSN 1432-0428. PMID 26032022. S2CID 7141228.
  11. ^ Mallanna, Sunil K.; Duncan, Stephen A. (September 2013). "Differentiation of Hepatocytes from Pluripotent Stem Cells". Current Protocols in Stem Cell Biology. 26 (1): 1G.4.1–1G.4.13. doi:10.1002/9780470151808.sc01g04s26. PMC 3920294. PMID 24510789.
  12. ^ Lucifora, Julie; Xia, Yuchen; Reisinger, Florian; Zhang, Ke; Stadler, Daniela; Cheng, Xiaoming; Sprinzl, Martin F.; Koppensteiner, Herwig; Makowska, Zuzanna; Volz, Tassilo; Remouchamps, Caroline; Chou, Wen-Min; Thasler, Wolfgang E.; Hüser, Norbert; Durantel, David; Liang, T. Jake; Münk, Carsten; Heim, Markus H.; Browning, Jeffrey L.; Dejardin, Emmanuel; Dandri, Maura; Schindler, Michael; Heikenwalder, Mathias; Protzer, Ulrike (14 March 2014). "Specific and Nonhepatotoxic Degradation of Nuclear Hepatitis B Virus cccDNA". Science. 343 (6176): 1221–1228. Bibcode:2014Sci...343.1221L. doi:10.1126/science.1243462. PMC 6309542. PMID 24557838.
  13. ^ Tout, Issam; Gomes, Melissa; Ainouze, Michelle; Marotel, Marie; Pecoul, Timothee; Durantel, David; Vaccarella, Salvatore; Dubois, Bertrand; Loustaud-Ratti, Veronique; Walzer, Thierry; Alain, Sophie; Chemin, Isabelle; Hasan, Uzma (15 October 2018). "Hepatitis B Virus Blocks the CRE/CREB Complex and Prevents TLR9 Transcription and Function in Human B Cells". The Journal of Immunology. 201 (8): 2331–2344. doi:10.4049/jimmunol.1701726. ISSN 0022-1767. PMID 30185518. S2CID 52166654.
  14. ^ Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart (June 2013). "Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes". Molecular and Cellular Biology. 33 (11): 2202–2211. doi:10.1128/MCB.01004-12. PMC 3648076. PMID 23530060.
  15. ^ Madec, Stéphanie; Cerec, Virginie; Plée-Gautier, Emmanuelle; Antoun, Joseph; Glaise, Denise; Salaun, Jean-Pierre; Guguen-Guillouzo, Christiane; Corlu, Anne (October 2011). "CYP4F3B expression is associated with differentiation of HepaRG human hepatocytes and unaffected by fatty acid overload". Drug Metabolism and Disposition: The Biological Fate of Chemicals. 39 (10): 1987–1996. doi:10.1124/dmd.110.036848. ISSN 1521-009X. PMID 21778351. S2CID 25447159.
  16. ^ Schuessler, Teresa K.; Chan, Xin Yi; Chen, Huanhuan Joyce; Ji, Kyungmin; Park, Kyung Min; Roshan-Ghias, Alireza; Sethi, Pallavi; Thakur, Archana; Tian, Xi; Villasante, Aranzazu; Zervantonakis, Ioannis K.; Moore, Nicole M.; Nagahara, Larry A.; Kuhn, Nastaran Z. (1 October 2014). "Biomimetic Tissue–Engineered Systems for Advancing Cancer Research: NCI Strategic Workshop Report". Cancer Research. 74 (19): 5359–5363. doi:10.1158/0008-5472.CAN-14-1706. PMC 4184963. PMID 25095784.
  17. ^ Sauer, Igor M.; Gerlach, Joerg C. (August 2002). "Modular Extracorporeal Liver Support". Artificial Organs. 26 (8): 703–706. doi:10.1046/j.1525-1594.2002.06931_1.x. PMID 12139497.
  18. ^ Gripon, Philippe; Rumin, Sylvie; Urban, Stephan; Le Seyec, Jacques; Glaise, Denise; Cannie, Isabelle; Guyomard, Claire; Lucas, Josette; Trepo, Christian; Guguen-Guillouzo, Christiane (26 November 2002). "Infection of a human hepatoma cell line by hepatitis B virus". Proceedings of the National Academy of Sciences. 99 (24): 15655–15660. Bibcode:2002PNAS...9915655G. doi:10.1073/pnas.232137699. PMC 137772. PMID 12432097.
  19. ^ Aninat, Caroline; Piton, Amélie; Glaise, Denise; Le Charpentier, Typhen; Langouët, Sophie; Morel, Fabrice; Guguen-Guillouzo, Christiane; Guillouzo, André (January 2006). "EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS". Drug Metabolism and Disposition. 34 (1): 75–83. doi:10.1124/dmd.105.006759. PMID 16204462. S2CID 16310821.
  20. ^ Guillouzo, André; Corlu, Anne; Aninat, Caroline; Glaise, Denise; Morel, Fabrice; Guguen-Guillouzo, Christiane (May 2007). "The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics". Chemico-Biological Interactions. 168 (1): 66–73. doi:10.1016/j.cbi.2006.12.003. PMID 17241619.
  21. ^ Le Vee, Marc; Jigorel, Emilie; Glaise, Denise; Gripon, Philippe; Guguen-Guillouzo, Christiane; Fardel, Olivier (May 2006). "Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line". European Journal of Pharmaceutical Sciences. 28 (1–2): 109–117. doi:10.1016/j.ejps.2006.01.004. PMID 16488578.
  22. ^ Turpeinen, Miia; Tolonen, Ari; Chesne, Christophe; Guillouzo, André; Uusitalo, Jouko; Pelkonen, Olavi (June 2009). "Functional expression, inhibition and induction of CYP enzymes in HepaRG cells". Toxicology in Vitro. 23 (4): 748–753. doi:10.1016/j.tiv.2009.03.008. PMID 19328226.
  23. ^ Gripon, Philippe; Rumin, Sylvie; Urban, Stephan; Le Seyec, Jacques; Glaise, Denise; Cannie, Isabelle; Guyomard, Claire; Lucas, Josette; Trepo, Christian; Guguen-Guillouzo, Christiane (26 November 2002). "Infection of a human hepatoma cell line by hepatitis B virus". Proceedings of the National Academy of Sciences. 99 (24): 15655–15660. Bibcode:2002PNAS...9915655G. doi:10.1073/pnas.232137699. PMC 137772. PMID 12432097.
  24. ^ Zanelli, Ugo; Caradonna, Nicola Pasquale; Hallifax, David; Turlizzi, Elisa; Houston, J. Brian (January 2012). "Comparison of Cryopreserved HepaRG Cells with Cryopreserved Human Hepatocytes for Prediction of Clearance for 26 Drugs". Drug Metabolism and Disposition. 40 (1): 104–110. doi:10.1124/dmd.111.042309. PMID 21998403. S2CID 49654.
  25. ^ Bachour-El Azzi, Pamela; Sharanek, Ahmad; Abdel-Razzak, Ziad; Antherieu, Sebastien; Al-Attrache, Houssein; Savary, Camille C.; Lepage, Sylvie; Morel, Isabelle; Labbe, Gilles; Guguen-Guillouzo, Christiane; Guillouzo, André (September 2014). "Impact of Inflammation on Chlorpromazine-Induced Cytotoxicity and Cholestatic Features in HepaRG Cells". Drug Metabolism and Disposition. 42 (9): 1556–1566. doi:10.1124/dmd.114.058123. PMID 25002748. S2CID 1161113.
  26. ^ Anthérieu, Sébastien; Rogue, Alexandra; Fromenty, Bernard; Guillouzo, André; Robin, Marie‐Anne (June 2011). "Induction of vesicular steatosis by amiodarone and tetracycline is associated with up‐regulation of lipogenic genes in heparg cells". Hepatology. 53 (6): 1895–1905. doi:10.1002/hep.24290. PMID 21391224. S2CID 8096472.
  27. ^ Rogue, Alexandra; Anthérieu, Sébastien; Vluggens, Aurore; Umbdenstock, Thierry; Claude, Nancy; de la Moureyre-Spire, Catherine; Weaver, Richard J.; Guillouzo, André (April 2014). "PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells" (PDF). Toxicology and Applied Pharmacology. 276 (1): 73–81. doi:10.1016/j.taap.2014.02.001. PMID 24534255. S2CID 5229406.
  28. ^ Anthérieu, Sébastien; Chesné, Christophe; Li, Ruoya; Guguen-Guillouzo, Christiane; Guillouzo, André (December 2012). "Optimization of the HepaRG cell model for drug metabolism and toxicity studies". Toxicology in Vitro. 26 (8): 1278–1285. doi:10.1016/j.tiv.2012.05.008. PMID 22643240.
  29. ^ McGill, Mitchell R.; Yan, Hui-Min; Ramachandran, Anup; Murray, Gordon J.; Rollins, Douglas E.; Jaeschke, Hartmut (March 2011). "HepaRG cells: A human model to study mechanisms of acetaminophen hepatotoxicity". Hepatology. 53 (3): 974–982. doi:10.1002/hep.24132. PMC 3073317. PMID 21319200.
  30. ^ Sison-Young, Rowena L. C.; Mitsa, Dimitra; Jenkins, Rosalind E.; Mottram, David; Alexandre, Eliane; Richert, Lysiane; Aerts, Hélène; Weaver, Richard J.; Jones, Robert P.; Johann, Esther; Hewitt, Philip G.; Ingelman-Sundberg, Magnus; Goldring, Christopher E. P.; Kitteringham, Neil R.; Park, B. Kevin (October 2015). "Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication". Toxicological Sciences. 147 (2): 412–424. doi:10.1093/toxsci/kfv136. PMC 4583060. PMID 26160117.
  31. ^ Tolosa, Laia; Gómez-Lechón, M. José; Jiménez, Nuria; Hervás, David; Jover, Ramiro; Donato, M. Teresa (July 2016). "Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis". Toxicology and Applied Pharmacology. 302: 1–9. doi:10.1016/j.taap.2016.04.007. PMID 27089845.
  32. ^ Jossé, Rozenn; Aninat, Caroline; Glaise, Denise; Dumont, Julie; Fessard, Valérie; Morel, Fabrice; Poul, Jean-Michel; Guguen-Guillouzo, Christiane; Guillouzo, André (June 2008). "Long-Term Functional Stability of Human HepaRG Hepatocytes and Use for Chronic Toxicity and Genotoxicity Studies". Drug Metabolism and Disposition. 36 (6): 1111–1118. doi:10.1124/dmd.107.019901. PMID 18347083. S2CID 29337628.
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