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.

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

“…We employed a lentivirus vector system to express so-called "upcyte/EPCC proliferation factors". The same approach was already used successfully to obtain human proliferating primary-like hepatocytes [11][12][13]. Here we infected primary cardiomyocytes that were enriched from atrial and ventricular biopsies by six cycles of trypsin-collagenase tissue dissociation followed by differential adhesion onto cell culture surfaces.…”

“…They are also required as models to study infection routes of myocarditis-inducing viruses such coxsackie virus B3 [29,30], for validation of new cardiovascular drugs and for toxicity testing. We aimed to establish procedures for proliferation induction of primary human cardiomyoctes by using the upcyte/EPCC approach which already had been implemented for long-term cultures of primary-like human hepatocytes [11][12][13]. Furthermore, we wanted to establish fast screening protocols for cardiomyocyte expression profiling by transcriptomics based on multiplex PCR of cDNAs.…”

“…A lentivirus vector system (Invitrogen, Karlsruhe, Germany) was used for transduction of proliferation-inducing genes (Upcyte genes) into cardiomyocytes as described recently for hepatocytes [12]. Sub-culturing of cells was performed with the Detach Kit 30 (PromoCell GmbH, Heidelberg, Germany).…”

“…We have been using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-derived cell types as an alternative to stem cells. This has already been demonstrated for human hepatocytes to be a very powerful procedure [11][12][13][14].…”

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization

“…PHH CYP 3A4 activity levels, measured by testosterone 6β‐hydroxylation, are between 26.6 and 67.4 pmol/min/mg protein 96 hours post‐thawing (Roymans et al, ); one study noted 55.0 pmol/min/mg protein for freshly cultured cells (Lübberstedt et al, ). Herzog et al () noted that UGT activity levels in freshly isolated PHH from two donors were 104.6 and 251.8 pmol/min/mg protein, respectively, as measured by hydroxycoumarin glucuronidation 48 h post seeding (Herzog et al, ). Thus, the values recorded for MutaMouse primary hepatocytes (Fig.…”