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


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.

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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

UCX® :

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


Uridine 5′-diphosphate glucuronosyltransferase


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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.

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Correspondence to Joana P. Miranda.

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Cipriano, M., Freyer, N., Knöspel, F. et al. Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro. Arch Toxicol 91, 1815–1832 (2017).

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