title: Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures.
abstract: Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Herzog
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Max
authors.initials: M
authors.lastname: Hansen
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Miethbauer
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Kai-Uwe
authors.initials: KU
authors.lastname: Schmidtke
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Anderer
authors.affiliation: Institute of Pharmacology and Toxicology, Jena University Hospital, Jena, Germany.
authors.forename: Amelie
authors.initials: A
authors.lastname: Lupp
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Charité University Medicine, Berlin, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Sperling
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Charité University Medicine, Berlin, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Seehofer
authors.affiliation: Department of General, Visceral and Transplantation Surgery, Charité University Medicine, Berlin, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Damm
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Scheibner
authors.affiliation: Faculty of Science, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
authors.forename: Jan-Heiner
authors.initials: JH
authors.lastname: Küpper
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D053533:Keratin-8 / Q000235:genetics / Q000378:metabolism; D027381:Liver-Specific Organic Anion Transporter 1 / Q000235:genetics / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism; D027702:Organic Cation Transporter 1 / Q000235:genetics / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism*; D014508:Urea / Q000378:metabolism
chemical_list: D051544:Cytochrome P-450 CYP3A; D006003:Glycogen; D053533:Keratin-8; D027381:Liver-Specific Organic Anion Transporter 1; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D027702:Organic Cation Transporter 1; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D014508:Urea; C000715087:ABCC2 protein, human; C503999:SLCO1B1 protein, human
keywords: CYP450; biotransformation; hepatocytes; in vitro drug testing; spheroids; xenobiotics