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

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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.

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Acute liver failure


Dimethyl sulfoxide






Multidrug resistance protein 2


Cytochrome P450 family 3, subfamily A, polypeptide 4


Cytochrome P450 family 1, subfamily A, polypeptide 2


Bioartificial liver


Pluripotent stem cells


Biliary-like cells


Hepatocyte-like cells










Vehicle control


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


Glutamine synthase


Hepatocyte nuclear factor 4 alpha


Hepatocyte nuclear factor 3 beta


Human hepatocytes


Pregnane X receptor


Carbamoyl phosphate synthase 1


Cytochrome P450 family 2, subfamily C, polypeptide 9


Zonnula occludens protein 1




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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.

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Correspondence to Paula M. Alves.

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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)

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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

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  • Hepatic
  • CYP450
  • 3D
  • Toxicology
  • Xenobiotic