title: Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro.
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 IC50value 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.
authors: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors: Madalena
authors: M
authors: Cipriano
authors: Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353, Berlin, Germany.
authors: Nora
authors: N
authors: Freyer
authors: Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353, Berlin, Germany.
authors: Fanny
authors: F
authors: Knöspel
authors: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors: Nuno G
authors: NG
authors: Oliveira
authors: ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451, Amadora, Portugal.
authors: Rita
authors: R
authors: Barcia
authors: ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451, Amadora, Portugal.
authors: Pedro E
authors: PE
authors: Cruz
authors: ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451, Amadora, Portugal.
authors: Helder
authors: H
authors: Cruz
authors: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
authors: Matilde
authors: M
authors: Castro
authors: ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451, Amadora, Portugal.
authors: Jorge M
authors: JM
authors: Santos
authors: Bioreactor Group, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, 13353, Berlin, Germany.
authors: Katrin
authors: K
authors: Zeilinger
authors: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. jmiranda@ff.ulisboa.pt.
authors: Joana P
authors: http://orcid.org/0000-0002-7804-4068
authors: JP
authors: Miranda
mesh_terms: D000818:Animals; D019149:Bioreactors; D016642:Bupropion / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004008:Diclofenac / Q000008:administration & dosage / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D000089762:Multidrug Resistance-Associated Protein 2; D051381:Rats; D017208:Rats, Wistar; D018874:Spheroids, Cellular / Q000378:metabolism*; D014116:Toxicology / Q000379:methods; D014508:Urea / Q000378:metabolism
chemical_list: C000715087:ABCC2 protein, human; D000089762:Multidrug Resistance-Associated Protein 2; D016642:Bupropion; D004008:Diclofenac; D014508:Urea; D019363:Cytochrome P-450 CYP1A1; D005947:Glucose
keywords: Hepatocyte-like cells; Hollow-fibre bioreactor; Human neonatal mesenchymal stem cells; In vitro toxicology; Spheroids