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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

Abstract

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.

Keywords

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

Abbreviations

2D

Two dimensional

3D

Three dimensional

3-MC

Methylcholanthrene

5-AZA

5-Azacytidine

AFP

Alpha-fetoprotein

ALB

Albumin

c/EBPα

CCAAT/enhancer-binding protein

CK

Cytokeratin

CYP

Cytochrome P-450

DAPI

4′,6-Diamidino-2-phenylindole

DMSO

Dimethyl sulphoxide

ECM

Extracellular matrix

EROD

7-Ethoxyresorufin-O-deethylase assay

FGF

Fibroblast growth factor

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HbLCs

Hepatoblast-like cells

hESCs

Human embryonic stem cells

HGF

Hepatocyte growth factor

HLCs

Hepatocyte-like cells

HNF-4α

Hepatocyte nuclear factor-4α

hnMSCs

Human neonatal mesenchymal stem cells

hpHep

Human primary hepatocytes

iPSCs

Induced pluripotent stem cells

LDH

Lactate dehydrogenase

MRP-2

Multidrug resistance protein 2

MSCs

Mesenchymal stem cells

MTS

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

OATP-C

Organic anion-transporting polypeptide C

OSM

Oncostatin M

SC

Stem cell

PAS

Periodic acid Schiff’s staining

TAT

Tyrosine aminotransferase

UCX®

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

UGT

Uridine 5′-diphosphate glucuronosyltransferase

Notes

Acknowledgments

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)

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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

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