Fig. 2

Hepatocyte cell lines retain key hepatic properties. a Albumin and Glucose-6-phosphatase gene expression analysis of expandable hepatocytes cultured in standard 2D or 3D (9 days of cultivation as spheroids in spinner flasks) conditions and primary hepatocytes (pHep) either cultured in 3D conditions or freshly isolated (expression normalized to Gapdh, n = 3 independent experiments, except for pHep were n = 2 independent experiments, bars represent mean). b Immunofluorescence-based detection of Albumin and E-cadherin in e-mHepA expandable hepatocytes counterstained for nuclei with DAPI. Scale bar, 20 μm. c Glycogen storage in expandable hepatocytes and control fibroblasts as analyzed by Periodic Acid-Schiff staining. Representative bright field micrographs and quantification of multiple field of views are shown. Scale bar, 100 μm (mean, pooled data from n = 3 independent experiments, field of views analyzed in total 35 to 42). d Immunofluorescence micrograph of Albumin and Hnf4α in e-mHepB hepatocytes after 3D culture as spheroids for 9 days. Actin filaments and nuclei were counterstained with phalloidin and DAPI, respectively. Scale bar, 20 µm. e Induction of phase I metabolic enzyme expression in expandable hepatocytes cultured in 2D or 3D conditions and control fibroblasts in response to stimulation with 50 μM Dexamethasone or 2 μM 3-Methylcholanthrene for 72 h (expression normalized to Gapdh, n = 3 independent experiments, except for e-mHepB Cyp1a1 expression were n = 6 independent experiments, bars represent mean). f Luminescence-based detection of Phase I enzymatic activity in expandable hepatocytes and control fibroblasts after cells were treated as indicate above (activity normalized to 10⁴ cells, pooled data from n = 2 independent experiments with n = 3 technical replicates each). g Staining of livers of Fah^–/–/Rag2^–/–/Il2rg^–/– (FRG) mice for FAH and GFP, 90 days after transplantation of GFP-tagged expandable hepatocytes. Scale bar, 100 µm