Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

[1]  M. Yliperttula,et al.  Peptide nanofiber hydrogel induces formation of bile canaliculi structures in three-dimensional hepatic cell culture. , 2012, Tissue engineering. Part A.

[2]  Enrico Drioli,et al.  Human hepatocytes and endothelial cells in organotypic membrane systems. , 2011, Biomaterials.

[3]  O. Silvennoinen,et al.  Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines , 2013, Stem cells translational medicine.

[4]  G. Poda,et al.  Structure-Activity Relationships for Interaction with Multidrug Resistance Protein 2 (ABCC2/MRP2): The Role of Torsion Angle for a Series of Biphenyl-Substituted Heterocycles , 2007, Drug Metabolism and Disposition.

[5]  N. Feller,et al.  ATP‐dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion , 1995, FEBS letters.

[6]  Juha Salmela,et al.  Flocculation of microfibrillated cellulose in shear flow , 2012, Cellulose.

[7]  Shouren Ge,et al.  Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties. , 2007, Biomaterials.

[8]  Dieter Klemm,et al.  Nanocelluloses: A New Family of Nature-Based Materials , 2011 .

[9]  E. Hudson,et al.  The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). , 2000, Journal of cell science.

[10]  Olivier Fardel,et al.  Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. , 2006, European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences.

[11]  R. Linhardt,et al.  Influence of a three-dimensional, microarray environment on human cell culture in drug screening systems. , 2012, Biomaterials.

[12]  T. Andersson,et al.  Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor , 2011, Drug Metabolism and Disposition.

[13]  Olli Ikkala,et al.  Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture. , 2012, Journal of controlled release : official journal of the Controlled Release Society.

[14]  S. Knuutila,et al.  Distinct differentiation characteristics of individual human embryonic stem cell lines , 2006, BMC Developmental Biology.

[15]  Jörg C Gerlach,et al.  HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. , 2011, Journal of pharmacological and toxicological methods.

[16]  Valérie Fessard,et al.  Long-Term Functional Stability of Human HepaRG Hepatocytes and Use for Chronic Toxicity and Genotoxicity Studies , 2008, Drug Metabolism and Disposition.

[17]  Anthony Atala,et al.  Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function. , 2012, Biomaterials.

[18]  Adam S. Hayward,et al.  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 , 2013, Archives of Toxicology.

[19]  G. Prestwich,et al.  Disulfide-crosslinked hyaluronan-gelatin sponge: growth of fibrous tissue in vivo. , 2004, Journal of biomedical materials research. Part A.

[20]  E. Wang,et al.  Fluorescent Substrates of Sister-P-Glycoprotein (BSEP) Evaluated as Markers of Active Transport and Inhibition: Evidence for Contingent Unequal Binding Sites , 2003, Pharmaceutical Research.

[21]  G. Prestwich,et al.  Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering. , 2009, Macromolecular bioscience.

[22]  André Guillouzo,et al.  EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS , 2006, Drug Metabolism and Disposition.

[23]  Christian Trepo,et al.  Infection of a human hepatoma cell line by hepatitis B virus , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  B. Drénou,et al.  Transdifferentiation of hepatocyte‐like cells from the human hepatoma HepaRG cell line through bipotent progenitor , 2007, Hepatology.

[25]  Steven N. Hart,et al.  A Comparison of Whole Genome Gene Expression Profiles of HepaRG Cells and HepG2 Cells to Primary Human Hepatocytes and Human Liver Tissues , 2010, Drug Metabolism and Disposition.

[26]  M. Mcdougall,et al.  Proluciferin Acetals as Bioluminogenic Substrates for Cytochrome P450 Activity and Probes for CYP3A Inhibition , 2011, Drug Metabolism and Disposition.

[27]  Tommy B. Andersson,et al.  HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans , 2008, Drug Metabolism And Disposition.

[28]  Jian Ye,et al.  Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction , 2012, BMC Bioinformatics.

[29]  O. Ikkala,et al.  Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels. , 2007, Biomacromolecules.

[30]  Christel A. S. Bergström,et al.  Early Identification of Clinically Relevant Drug Interactions With the Human Bile Salt Export Pump (BSEP/ABCB11) , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[31]  U. Müller-Vieira,et al.  An integrated approach to improved toxicity prediction for the safety assessment during preclinical drug development using Hep G2 cells. , 2009, Toxicology and applied pharmacology.

[32]  Jan Schrooten,et al.  Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures. , 2012, Tissue engineering. Part C, Methods.

[33]  Jari Vartiainen,et al.  Health and environmental safety aspects of friction grinding and spray drying of microfibrillated cellulose , 2011 .

[34]  L. Schwarz,et al.  Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Elmar Heinzle,et al.  3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[36]  Kim L R Brouwer,et al.  Use of Sandwich-Cultured Human Hepatocytes to Predict Biliary Clearance of Angiotensin II Receptor Blockers and HMG-CoA Reductase Inhibitors , 2009, Drug Metabolism and Disposition.

[37]  Arto Urtti,et al.  The use of nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells. , 2014, Stem cells and development.

[38]  Glenn D Prestwich,et al.  3-D culture in synthetic extracellular matrices: new tissue models for drug toxicology and cancer drug discovery. , 2007, Advances in enzyme regulation.

[39]  A. Guillouzo,et al.  Running title page Running title : Functional stability of CYP P 450 enzymes in HepaRG cells , 2009 .

[40]  A. Guillouzo,et al.  Dose- and time-dependent effects of phenobarbital on gene expression profiling in human hepatoma HepaRG cells. , 2009, Toxicology and applied pharmacology.

[41]  D. Cassio,et al.  Efficient In Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF‐B and WIF‐B9 cells , 1998, Hepatology.