Archives of Toxicology

, Volume 91, Issue 4, pp 1815–1832 | Cite as

Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro

  • Madalena Cipriano
  • Nora Freyer
  • Fanny Knöspel
  • Nuno G. Oliveira
  • Rita Barcia
  • Pedro E. Cruz
  • Helder Cruz
  • Matilde Castro
  • Jorge M. Santos
  • Katrin Zeilinger
  • Joana P. Miranda
In vitro Systems


3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.


Spheroids Hollow-fibre bioreactor Hepatocyte-like cells Human neonatal mesenchymal stem cells In vitro toxicology 



Two dimensional


Three dimensional










CCAAT/enhancer-binding protein




Cytochrome P-450




Dimethyl sulphoxide


Extracellular matrix


7-Ethoxyresorufin-O-deethylase assay


Fibroblast growth factor


Glyceraldehyde-3-phosphate dehydrogenase


Hepatoblast-like cells


Human embryonic stem cells


Hepatocyte growth factor


Hepatocyte-like cells


Hepatocyte nuclear factor-4α


Human neonatal mesenchymal stem cells


Human primary hepatocytes


Induced pluripotent stem cells


Lactate dehydrogenase


Multidrug resistance protein 2


Mesenchymal stem cells


3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt


Organic anion-transporting polypeptide C


Oncostatin M


Stem cell


Periodic acid Schiff’s staining


Tyrosine aminotransferase


ECBio’s proprietary Umbilical Cord eXpanded hnMSCs isolated from the Wharton’s Jelly


Uridine 5′-diphosphate glucuronosyltransferase



We acknowledge Pharmacelsus GmbH (Dr. Ursula Muller-Vieira) for the MS quantifications and analyses. We also acknowledge the support of the COST action BM1305 (A FACTT: Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies). This work was supported by FCT (Fundação para a Ciência e a Tecnologia) [EXPL/DTP-FTO/0308/2013, PTDC/SAU-TOX/110457/2009, UID/DTP/04138/2013, SFRH/BPD/96719/2013 and Ciência2008 to JPM, SFRH/BD/87508/2012 to M. Cipriano]. The work herein presented was performed at iMed.ULisboa, ECBio S.A. and BCRT.

Compliance with ethical standards

Human and animal rights statement

All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

204_2016_1838_MOESM1_ESM.docx (292 kb)
Supplementary material 1 (DOCX 291 kb)


  1. Boelsterli UA (2003) Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity. Toxicol Appl Pharmacol 192(3):307–322. doi: 10.1016/s0041-008x(03)00368-5 CrossRefPubMedGoogle Scholar
  2. Bort R, Ponsoda X, Jover R, Gómez-Lechón MJ, Castell JV (1999) Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. The J Pharmacol Exp Ther 288(1):65–72PubMedGoogle Scholar
  3. Campard D, Lysy PA, Najimi M, Sokal EM (2008) Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology 134(3):833–848. doi: 10.1053/j.gastro.2007.12.024 CrossRefPubMedGoogle Scholar
  4. Cipriano M, Medeiros A, Filipe E et al (2014) 2014) UCX (R) cells: a primordial stem cell source for in vitro differentiation into hepatocyte-like cells (HLCs. Toxicol Lett 229:S140–S140. doi: 10.1016/J.Toxlet.2014.06.492 Google Scholar
  5. Fausto N (2004) Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39(6):1477–1487. doi: 10.1002/hep.20214 CrossRefPubMedGoogle Scholar
  6. Gerets HH, Tilmant K, Gerin B et al (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(2):69–87. doi: 10.1007/s10565-011-9208-4 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Gieseck RL 3rd, Hannan NR, Bort R et al (2014) Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One 9(1):e86372. doi: 10.1371/journal.pone.0086372 CrossRefPubMedGoogle Scholar
  8. Godoy P, Hewitt NJ, Albrecht U et al (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 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Guguen-Guillouzo C, Corlu A, Guillouzo A (2010) Stem cell-derived hepatocytes and their use in toxicology. Toxicology 270(1):3–9. doi: 10.1016/j.tox.2009.09.019 CrossRefPubMedGoogle Scholar
  10. Hamilton GA, Jolley SL, Gilbert D, Coon DJ, Barros S, LeCluyse EL (2001) Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions. Cell Tissue Res 306(1):85–99. doi: 10.1007/s004410100429 CrossRefPubMedGoogle Scholar
  11. Haruna Y, Saito K, Spaulding S, Nalesnik MA, Gerber MA (1996) Identification of bipotential progenitor cells in human liver development. Hepatology 23(3):476–481. doi: 10.1002/hep.510230312 CrossRefPubMedGoogle Scholar
  12. Hoffmann SA, Müller-Vieira U, Biemel K et al (2012) Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies. Biotechnol Bioeng 109(12):3172–3181. doi: 10.1002/bit.24573 CrossRefPubMedGoogle Scholar
  13. Kazemnejad S, Allameh A, Soleimani M et al (2009) Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold. J Gastroenterol Hepatol 24(2):278–287. doi: 10.1111/j.1440-1746.2008.05530.x CrossRefPubMedGoogle Scholar
  14. Knöspel F, Jacobs F, Freyer N et al (2016) In vitro model for hepatotoxicity studies based on primary human hepatocyte cultivation in a perfused 3D bioreactor system. Int J Mol Sci. doi: 10.3390/ijms17040584 PubMedPubMedCentralGoogle Scholar
  15. Komashko VM, Farnham PJ (2010) 5-azacytidine treatment reorganizes genomic histone modification patterns. Epigenet Off J DNA Methylation Soc 5(3):229–240CrossRefGoogle Scholar
  16. Leite SB, Teixeira AP, Miranda JP et al (2011) Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: improved in vitro models for toxicological applications. Toxicol In Vitro 25(4):825–832. doi: 10.1016/j.tiv.2011.02.002 CrossRefPubMedGoogle Scholar
  17. Lin J, Schyschka L, Muhl-Benninghaus R et al (2012) Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism. Arch Toxicol 86(1):87–95. doi: 10.1007/s00204-011-0733-y CrossRefPubMedGoogle Scholar
  18. Lübberstedt M, Müller-Vieira U, Biemel KM et al (2012) Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies. J Tissue Eng Regen Med. doi: 10.1002/term.1652 PubMedGoogle Scholar
  19. Miranda JP, Leite SB, Müller-Vieira U, Rodrigues A, Carrondo MJ, Alves PM (2009) Towards an extended functional hepatocyte in vitro culture. Tissue Eng Part C, Methods 15(2):157–167. doi: 10.1089/ten.tec.2008.0352 CrossRefGoogle Scholar
  20. Miranda JP, Rodrigues A, Tostoes RM et al (2010) Extending hepatocyte functionality for drug-testing applications using high-viscosity alginate-encapsulated three-dimensional cultures in bioreactors. Tissue Eng Part C, Methods 16(6):1223–1232. doi: 10.1089/ten.TEC.2009.0784 CrossRefGoogle Scholar
  21. Miranda JP, Filipe E, Fernandes AS et al (2015) The human umbilical cord tissue-derived MSC population UCX(R) promotes early motogenic effects on keratinocytes and fibroblasts and G-CSF-mediated mobilization of BM-MSCs when transplanted in vivo. Cell Transpl 24(5):865–877. doi: 10.3727/096368913X676231 CrossRefGoogle Scholar
  22. Müeller D, Tascher G, Müller-Vieira U et al (2011) In-depth physiological characterization of primary human hepatocytes in a 3D hollow-fiber bioreactor. J Tissue Eng Regen Med 5(8):e207–e218. doi: 10.1002/term.418 CrossRefPubMedGoogle Scholar
  23. Okura H, Komoda H, Saga A et al (2010) Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells. Tissue Eng Part C, Methods 16(4):761–770. doi: 10.1089/ten.TEC.2009.0208 CrossRefGoogle Scholar
  24. Ong SY, Dai H, Leong KW (2006) Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture. Biomaterials 27(22):4087–4097. doi: 10.1016/j.biomaterials.2006.03.022 CrossRefPubMedGoogle Scholar
  25. Paganelli M, Dallmeier K, Nyabi O et al (2013) Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection. Hepatology 57(1):59–69. doi: 10.1002/hep.26006 CrossRefPubMedGoogle Scholar
  26. Pinheiro PF, Pereira SA, Harjivan SG et al (2016) Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study. Arch Toxicol. doi: 10.1007/s00204-016-1792-x [Epub ahead of print] PubMedGoogle Scholar
  27. Rajan N, Habermehl J, Cote MF, Doillon CJ, Mantovani D (2006) Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc 1(6):2753–2758. doi: 10.1038/nprot.2006.430 CrossRefPubMedGoogle Scholar
  28. Ramasamy TS, Yu JS, Selden C, Hodgson H, Cui W (2013) Application of three-dimensional culture conditions to human embryonic stem cell-derived definitive endoderm cells enhances hepatocyte differentiation and functionality. Tissue Eng Part A 19(3–4):360–367. doi: 10.1089/ten.tea.2012.0190 CrossRefPubMedGoogle Scholar
  29. Santos JM, Camões SP, Filipe E et al (2015) Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing. Stem cell Res Ther 6:90. doi: 10.1186/s13287-015-0082-5 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Schwartz RE, Fleming HE, Khetani SR, Bhatia SN (2014) Pluripotent stem cell-derived hepatocyte-like cells. Biotechnol Adv 32(2):504–513. doi: 10.1016/j.biotechadv.2014.01.003 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Seeliger C, Culmes M, Schyschka L et al (2013) Decrease of global methylation improves significantly hepatic differentiation of Ad-MSCs: possible future application for urea detoxification. Cell Transpl 22(1):119–131. doi: 10.3727/096368912X638946 CrossRefGoogle Scholar
  32. Sengupta S, Johnson BP, Swanson SA, Stewart R, Bradfield CA, Thomson JA (2014) Aggregate culture of human embryonic stem cell-derived hepatocytes in suspension are an improved in vitro model for drug metabolism and toxicity testing. Toxicol Sci Off J Soc Toxicol 140(1):236–245. doi: 10.1093/toxsci/kfu069 CrossRefGoogle Scholar
  33. Sivertsson L, Synnergren J, Jensen J, Bjorquist P, Ingelman-Sundberg M (2013) Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused three-dimensional bioreactor. Stem Cells Dev 22(4):581–594. doi: 10.1089/scd.2012.0202 CrossRefPubMedGoogle Scholar
  34. Strassburg CP, Strassburg A, Kneip S et al (2002) Developmental aspects of human hepatic drug glucuronidation in young children and adults. Gut 50(2):259–265. doi: 10.1136/gut.50.2.259 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Subramanian K, Owens DJ, Raju R et al (2014) Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells. Stem cells and development 23(2):124–131. doi: 10.1089/scd.2013.0097 CrossRefPubMedGoogle Scholar
  36. Takayama K, Kawabata K, Nagamoto Y et al (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 CrossRefPubMedGoogle Scholar
  37. Talaei-Khozani T, Khodabandeh Z, Jaberipour M, Hosseini A, Bahmanpour S, Vojdani Z (2015) Comparison of hepatic nuclear factor-4 expression in two- and three-dimensional culture of Wharton’s jelly-derived cells exposed to hepatogenic medium. Rom J Morphol Embryol Rev Roum Morphol Embryol 56(4):1365–1370Google Scholar
  38. Tanimizu N, Mitaka T (2014) Re-evaluation of liver stem/progenitor cells. Organogenesis 10(2):208–215. doi: 10.4161/org.27591 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Tasnim F, Phan D, Toh YC, Yu H (2015) Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Biomaterials 70:115–125. doi: 10.1016/j.biomaterials.2015.08.002 CrossRefPubMedGoogle Scholar
  40. Tostões RM, Leite SB, Miranda JP et al (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 CrossRefPubMedGoogle Scholar
  41. Woo DH, Kim SK, Lim HJ et al (2012) Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice. Gastroenterology 142(3):602–611. doi: 10.1053/j.gastro.2011.11.030 CrossRefPubMedGoogle Scholar
  42. Xu J, Ma M, Purcell WM (2003) Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies. II. Spheroid cell spreading inhibition as a new cytotoxic marker. Toxicol Appl Pharmacol 189(2):112–119. doi: 10.1016/S0041-008X(03)00090-5 CrossRefPubMedGoogle Scholar
  43. Yoon HH, Jung BY, Seo YK, Song KY (2010) Park JK (2010) In vitro hepatic differentiation of umbilical cord-derived mesenchymal stem cell. Process Biochem 45(12):1857–1864. doi: 10.1016/J.Procbio.2010.06.009 CrossRefGoogle Scholar
  44. Yoshida Y, Shimomura T, Sakabe T et al (2007) A role of Wnt/beta-catenin signals in hepatic fate specification of human umbilical cord blood-derived mesenchymal stem cells. Am J Physiol Gastrointest Liver Physiol 293(5):G1089–G1098. doi: 10.1152/ajpgi.00187.2007 CrossRefPubMedGoogle Scholar
  45. Zeilinger K, Schreiter T, Darnell M et al (2011) Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies. Tissue Eng Part C, Methods 17(5):549–556. doi: 10.1089/ten.TEC.2010.0580 CrossRefGoogle Scholar
  46. Zhang YN, Lie PC, Wei X (2009) Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton’s jelly into hepatocyte-like cells. Cytotherapy 11(5):548–558. doi: 10.1080/14653240903051533 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Madalena Cipriano
    • 1
  • Nora Freyer
    • 2
  • Fanny Knöspel
    • 2
  • Nuno G. Oliveira
    • 1
  • Rita Barcia
    • 3
  • Pedro E. Cruz
    • 3
  • Helder Cruz
    • 3
  • Matilde Castro
    • 1
  • Jorge M. Santos
    • 3
  • Katrin Zeilinger
    • 2
  • Joana P. Miranda
    • 1
  1. 1.Research Institute for Medicines (iMed.ULisboa), Faculty of PharmacyUniversidade de LisboaLisbonPortugal
  2. 2.Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT)Charité - Universitätsmedizin BerlinBerlinGermany
  3. 3.ECBio S.A.AmadoraPortugal

Personalised recommendations