Optimization of upcyte® human hepatocytes for the in vitro micronucleus assay

Nörenberg, Astrid; Heinz, Stefan; Scheller, Katharina; Hewitt, Nicola J.; Braspenning, Joris; Ott, Michael

doi:10.1016/j.mrgentox.2013.09.008

Cited by 19 publications

(9 citation statements)

References 22 publications

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“…The authors could demonstrate significant expression levels of relevant phase I and phase II enzymes, their inducibility and biotransformation activity. In addition, Upcyte hepatocytes, which represent non-transformed cells genetically stable over many passages, should be an interesting tool to study hepatotoxicity and clearance of xenobiotics [68][69][70]. More specifically, a panel of 11 chemicals known to induce or non-induce CYP3A4 and CYP2B6 were run on second generation Upcyte hepatocytes which proved to be a good prediction model for those CYP enzymes [71].…”

Section: Upcyte / Hepafh Cellsmentioning

confidence: 99%

Human hepatocyte systems for in vitro toxicology analysis

Kammerer

Küpper

2018

JCB

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Drug induced liver injury (DILI) is still the leading single cause of drug failure during clinical phases and after market approval. Currently, many laboratories aim to develop appropriate in vitro systems to predict drug hepatotoxicity. Primary human hepatocytes are still the gold standard, but they have substantial disadvantages such as rapid dedifferentiation in vitro and lack of cell proliferation. In addition to primary human hepatocytes, liver cancer-derived cell lines such as HepG2, cytochrome P450 (CYP450) overexpressing HepG2 cell clones and HepaRG were studied intensively. In contrast to HepG2, HepaRG show promising characteristics of differentiated primary human hepatocytes, but they represent only one donor. There is some hope that this lack of donor variability can be solved by the use of iPS-derived hepatocytes. However, iPS technology still seems to need some improvement to produce physiologically relevant hepatocytes. Upcyte hepatocytes represent the most recent technical advancement combining some features of primary human hepatocytes such as physiological activity and donor variability with the ability of cell lines for extended proliferation. Altogether, more work is needed to develop and validate appropriate in vitro systems for precise prediction of DILI risk.

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Section: Upcyte / Hepafh Cellsmentioning

confidence: 99%

Human hepatocyte systems for in vitro toxicology analysis

Kammerer

Küpper

2018

JCB

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“…They show some improved liver functions compared to cell lines such as HepG2 or HepaRG, but perform still less than freshly isolated pHHs. Data on the expression and inducibility of CYP enzymes show that essential components of the phase I and phase II reactions are present and active [29][30][31][32][33].…”

Section: Introductionmentioning

confidence: 99%

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

Schulz

Kammerer

Küpper

2019

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BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte ® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte ® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte ® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte ® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

George

Rödiger

Schröder

et al. 2016

JCB

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Abstract.Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2a, MLC-2v, CHRM2, ADRB1, DES, EDRNB, Cx40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD, GATA-4, Cx43, SERCA2, BNP, Tbx5, EDNRA and ACTB. Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time.In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures.Keywords: Atrial appendages, ACTA1, ACTB ACTC1, ADRB1, ADRB2, ATP2A2, atrial fibrillation, BMP2, CHRM2, cluster analysis, DES, EDNRA, EDNRB, enhanced primary cell cultures (EPCC), GAPDH, GATA4, gene expression, GJA1, GJA5, HPRT1, KCNA5, KCNH2, multiplex PCR, MYL2, MYL7, MYOCD, NFATC4, NKX2-5, NPPA, NPPA, PLN, qPCR, reference genes, RPLP0, SCN5A, TBX5, TNNI3, TNNT2, upcyte genes, VIM Abbreviations EPCC enhanced primary cell cultures HE hematoxylin -eosin qPCR quantitative PCR 1 These authors contributed equally.

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Optimization of upcyte® human hepatocytes for the in vitro micronucleus assay

Cited by 19 publications

References 22 publications

Human hepatocyte systems for in vitro toxicology analysis

Human hepatocyte systems for in vitro toxicology analysis

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

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

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