Advertisement

HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism

  • 1145 Accesses

  • 25 Citations

Abstract

The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

ALB:

Albumin

ALF:

Acute liver failure

DMSO:

Dimethyl sulfoxide

3D:

Three-dimensional

2D:

Two-dimensional

MRP2:

Multidrug resistance protein 2

CYP3A4:

Cytochrome P450 family 3, subfamily A, polypeptide 4

CYP1A2:

Cytochrome P450 family 1, subfamily A, polypeptide 2

BAL:

Bioartificial liver

PSC:

Pluripotent stem cells

BLC:

Biliary-like cells

HLC:

Hepatocyte-like cells

PV:

Perivenous

PP:

Periportal

Rif:

Rifampicin

BNF:

β-Naphthoflavone

VC:

Vehicle control

CDFDA:

5-(and-6)-Carboxy-2,7-dichlorofluorescein diacetate

GS:

Glutamine synthase

HNF4α:

Hepatocyte nuclear factor 4 alpha

HNF3β:

Hepatocyte nuclear factor 3 beta

HH:

Human hepatocytes

PXR:

Pregnane X receptor

CPS1:

Carbamoyl phosphate synthase 1

CYP2C9:

Cytochrome P450 family 2, subfamily C, polypeptide 9

ZO-1:

Zonnula occludens protein 1

G6PC:

Glucose-6-phosphatase

References

  1. Antherieu S, Chesne C, Li R, Camus S, Lahoz A, Picazo L, Turpeinen M, Tolonen A, Uusitalo J, Guguen-Guillouzo C, Guillouzo A (2010) Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos 38(3):516–525. doi:10.1124/dmd.109.030197

  2. Bhatt T, Rizvi A, Batta SP, Kataria S, Jamora C (2013) Signaling and mechanical roles of E-cadherin. Cell Commun Adhes. doi:10.3109/15419061.2013.854778

  3. Cai ZH, Shi ZQ, O’Shea GM, Sun AM (1988) Microencapsulated hepatocytes for bioartificial liver support. Artif Organs 12(5):388–393

  4. Capone SH, Dufresne M, Rechel M, Fleury MJ, Salsac AV, Paullier P, Daujat-Chavanieu M, Legallais C (2013) Impact of alginate composition: from bead mechanical properties to encapsulated HepG2/C3A cell activities for in vivo implantation. PLoS ONE 8(4):e62032. doi:10.1371/journal.pone.0062032

  5. Celli JP, Rizvi I, Blanden AR, Massodi I, Glidden MD, Pogue BW, Hasan T (2014) An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models. Sci Rep 4:3751

  6. Cerec V, Glaise D, Garnier D, Morosan S, Turlin B, Drenou B, Gripon P, Kremsdorf D, Guguen-Guillouzo C, Corlu A (2007) Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 45(4):957–967. doi:10.1002/hep.21536

  7. Cheng N, Wauthier E, Reid LM (2008) Mature human hepatocytes from ex vivo differentiation of alginate-encapsulated hepatoblasts. Tissue Eng Part A 14(1):1–7. doi:10.1089/ten.a.2007.0131

  8. Du Y, Han R, Wen F, Ng San San S, Xia L, Wohland T, Leo HL, Yu H (2008) Synthetic sandwich culture of 3D hepatocyte monolayer. Biomaterials 29(3):290–301. doi:10.1016/j.biomaterials.2007.09.016

  9. Elkayam T, Amitay-Shaprut S, Dvir-Ginzberg M, Harel T, Cohen S (2006) Enhancing the drug metabolism activities of C3A—a human hepatocyte cell line—by tissue engineering within alginate scaffolds. Tissue Eng 12(5):1357–1368. doi:10.1089/ten.2006.12.1357

  10. Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689. doi:10.1016/j.cell.2006.06.044

  11. Gerets HHJ, Tilmant K, Gerin B, Chanteux H, Depelchin BO, Dhalluin S, Atienzar FA (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 Biol Toxicol (28):69–87. doi:10.1007/s10565-011-9208-4

  12. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Bottger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gomez-Lechon MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Haussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhutter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, Lecluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stober R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. doi:10.1007/s00204-013-1078-5

  13. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, Guyomard C, Lucas J, Trepo C, Guguen-Guillouzo C (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99(24):15655–15660. doi:10.1073/pnas.232137699

  14. Gunness P, Mueller D, Shevchenko V, Heinzle E, Ingelman-Sundberg M, Noor F (2013) 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci 133(1):67–78. doi:10.1093/toxsci/kft021

  15. Hart SN, Li Y, Nakamoto K, Subileau EA, Steen D, Zhong XB (2010) A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues. Drug Metab Dispos 38(6):988–994. doi:10.1124/dmd.109.031831

  16. Higuchi Y, Kawai K, Yamazaki H, Nakamura M, Bree F, Guguen-Guillouzo C, Suemizu H (2013) The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice. Xenobiotica. doi:10.3109/00498254.2013.836257

  17. Hoekstra R, Nibourg GA, van der Hoeven TV, Ackermans MT, Hakvoort TB, van Gulik TM, Lamers WH, Elferink RP, Chamuleau RA (2011) The HepaRG cell line is suitable for bioartificial liver application. Int J Biochem Cell Biol 43(10):1483–1489. doi:10.1016/j.biocel.2011.06.011

  18. Hoekstra R, Nibourg GA, van der Hoeven TV, Plomer G, Seppen J, Ackermans MT, Camus S, Kulik W, van Gulik TM, Elferink RP, Chamuleau RA (2013) Phase 1 and phase 2 drug metabolism and bile acid production of HepaRG cells in a bioartificial liver in absence of dimethyl sulfoxide. Drug Metab Dispos 41(3):562–567. doi:10.1124/dmd.112.049098

  19. Hofmann U, Maier K, Niebel A, Vacun G, Reuss M, Mauch K (2008) Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng 100(2):344–354

  20. Jungermann K (1995) Zonation of metabolism and gene expression in liver. Histochem Cell Biol 103(2):81–91

  21. Koizumi T, Aoki T, Kobayashi Y, Yasuda D, Izumida Y, Jin Z, Nishino N, Shimizu Y, Kato H, Murai N, Niiya T, Enami Y, Mitamura K, Yamamoto T, Kusano M (2007) Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes. Cell Transplant 16(1):67–73

  22. Lan S-F, Starly B (2011) Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities. Toxicol Appl Pharmacol 256(1):62–72

  23. Le Vee M, Jigorel E, Glaise D, Gripon P, Guguen-Guillouzo C, Fardel O (2006) Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur J Pharm Sci 28(1–2):109–117. doi:10.1016/j.ejps.2006.01.004

  24. Le Vee M, Noel G, Jouan E, Stieger B, Fardel O (2013) Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells. Toxicol In Vitro 27(6):1979–1986. doi:10.1016/j.tiv.2013.07.003

  25. LeCluyse EL, Witek RP, Andersen ME, Powers MJ (2012) Organotypic liver culture models: meeting current challenges in toxicity testing. Crit Rev Toxicol 42(6):501–548. doi:10.3109/10408444.2012.682115

  26. Leite SB, Wilk-Zasadna I, Zaldivar JM, Airola E, Reis-Fernandes MA, Mennecozzi M, Guguen-Guillouzo C, Chesne C, Guillou C, Alves PM, Coecke S (2012) Three-dimensional HepaRG model as an attractive tool for toxicity testing. Toxicol Sci 130(1):106–116. doi:10.1093/toxsci/kfs232

  27. Lengyel G, Veres Z, Tugyi R, Vereczkey L, Molnar T, Glavinas H, Krajcsi P, Jemnitz K (2008) Modulation of sinusoidal and canalicular elimination of bilirubin-glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes. Hepatol Res 38(3):300–309. doi:10.1111/j.1872-034X.2007.00255.x

  28. Lin N, Lin J, Bo L, Weidong P, Chen S, Xu R (2010) Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold. Cell Prolif 43(5):427–434. doi:10.1111/j.1365-2184.2010.00692.x

  29. Mavri-Damelin D, Damelin LH, Eaton S, Rees M, Selden C, Hodgson HJ (2008) Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia. Biotechnol Bioeng 99(3):644–651. doi:10.1002/bit.21599

  30. McClelland R, Wauthier E, Uronis J, Reid L (2008) Gradients in the liver’s extracellular matrix chemistry from periportal to pericentral zones: influence on human hepatic progenitors. Tissue Eng Part A 14(1):59–70. doi:10.1089/ten.a.2007.0058

  31. Mueller D, Kramer L, Hoffmann E, Klein S, Noor F (2013) 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicol In Vitro. doi:10.1016/j.tiv.2013.06.024

  32. Nibourg GA, Chamuleau RA, van der Hoeven TV, Maas MA, Ruiter AF, Lamers WH, Oude Elferink RP, van Gulik TM, Hoekstra R (2012) Liver progenitor cell line HepaRG differentiated in a bioartificial liver effectively supplies liver support to rats with acute liver failure. PLoS ONE 7(6):e38778. doi:10.1371/journal.pone.0038778

  33. Pal R, Mamidi MK, Das AK, Bhonde R (2012) Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells. Arch Toxicol 86(4):651–661. doi:10.1007/s00204-011-0782-2

  34. Parent R, Marion MJ, Furio L, Trepo C, Petit MA (2004) Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126(4):1147–1156

  35. Santos NC, Figueira-Coelho J, Martins-Silva J, Saldanha C (2003) Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. Biochem Pharmacol 65(7):1035–1041

  36. Schulze A, Mills K, Weiss TS, Urban S (2012) Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 55(2):373–383. doi:10.1002/hep.24707

  37. Su T, Waxman DJ (2004) Impact of dimethyl sulfoxide on expression of nuclear receptors and drug-inducible cytochromes P450 in primary rat hepatocytes. Arch Biochem Biophys 424(2):226–234. doi:10.1016/j.abb.2004.02.008

  38. Sun AM, O’Shea GM, Goosen MF (1984) Injectable microencapsulated islet cells as a bioartificial pancreas. Appl Biochem Biotechnol 10:87–99

  39. Takayama K, Kawabata K, Nagamoto Y, Kishimoto K, Tashiro K, Sakurai F, Tachibana M, Kanda K, Hayakawa T, Furue MK, Mizuguchi H (2013) 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing. Biomaterials 34(7):1781–1789. doi:10.1016/j.biomaterials.2012.11.029

  40. Torre C, Perret C, Colnot S (2010) Molecular determinants of liver zonation. Prog Mol Biol Transl Sci 97:127–150. doi:10.1016/B978-0-12-385233-5.00005-2

  41. Tostoes RM, Leite SB, Miranda JP, Sousa M, Wang DI, Carrondo MJ, Alves PM (2011) Perfusion of 3D encapsulated hepatocytes—a synergistic effect enhancing long-term functionality in bioreactors. Biotechnol Bioeng 108(1):41–49. doi:10.1002/bit.22920

  42. Wenzel C, Riefke B, Grundemann S, Krebs A, Christian S, Prinz F, Osterland M, Golfier S, Rase S, Ansari N, Esner M, Bickle M, Pampaloni F, Mattheyer C, Stelzer EH, Parczyk K, Prechtl S, Steigemann P (2014) 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. Exp Cell Res 323(1):131–143

  43. Tostoes RM, Leite SB, Serra M, Jensen J, Bjorquist P, Carrondo MJ, Brito C, Alves PM (2012) Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology (Baltimore, Md) 55(4):1227–1236

  44. You J, Park SA, Shin DS, Patel D, Raghunathan VK, Kim M, Murphy CJ, Tae G, Revzin A (2013) Characterizing the effects of heparin gel stiffness on function of primary hepatocytes. Tissue Eng Part A. doi:10.1089/ten.TEA.2012.0681

  45. Zamek-Gliszczynski MJ, Xiong H, Patel NJ, Turncliff RZ, Pollack GM, Brouwer KL (2003) Pharmacokinetics of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein and its diacetate promoiety in the liver. J Pharmacol Exp Ther 304(2):801–809. doi:10.1124/jpet.102.044107

Download references

Acknowledgments

The authors acknowledge Tiago Duarte for discussion and support in GC–MS technique and Daniel Simão for support in microscopy. This work was supported by PhD fellowship to S.R., SFRH/BD/70264/2010 and by PTDC/EBB-BIO/112786/2009, funded by Fundação para a Ciência e Tecnologia.

Author information

Correspondence to Paula M. Alves.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material Video 1 3D projection of the bile canalicular network, visualized through the excretion of the fluorescent substrate CDFDA (AVI 8444 kb)

Supplementary material Fig. S1 Analysis of proliferation by immunolocalization of the nuclear marker Ki67 a 2D cultures at the proliferative stage (day 2) used as positive control for the Ki67 antibody (green) and DAPI nuclear staining (blue). Cryosection of 3D cultures labeled for DAPI (blue) and Ki67 (green). b 2D cultures at the proliferative stage (day 2) used as positive control for the HNF3β antibody (red) and DAPI nuclear staining (blue). Cryosection of 3D cultures labeled for DAPI (blue) and HNF3β (red). Scale bars represent 10 µm (TIFF 1713 kb)

Supplementary material Fig. S2 Accumulation of CDFDA within the spheroids by inhibition of the phase III transporter MRP2 with 500 µM of Indomethacin. Scale bar represents 10 µm (TIFF 413 kb)

Supplementary material Video 1 3D projection of the bile canalicular network, visualized through the excretion of the fluorescent substrate CDFDA (AVI 8444 kb)

Supplementary material 4 (DOCX 12 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rebelo, S.P., Costa, R., Estrada, M. et al. HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol 89, 1347–1358 (2015) doi:10.1007/s00204-014-1320-9

Download citation

Keywords

  • Hepatic
  • CYP450
  • 3D
  • Toxicology
  • Xenobiotic