Daniel Mueller

3D Organotypic Cultures of Human HepaRG Cells: A Tool for In Vitro Toxicity Studies

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.

3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.

Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects.

Stable isotope-assisted metabolomics to detect metabolic flux changes in mammalian cell cultures.

The determination of metabolic fluxes provides detailed information of cellular physiology, and the assessment of metabolic flux changes upon a certain perturbation can help to improve biotechnological and pharmaceutical processes. Stable isotope-assisted metabolomics using tracer-labeled substrates is the method of choice to determine the fluxes. Though well-established for microbial cultures, the application to mammalian cells is generally complex and still limited. However, there have been great achievements in recent years and it is now emerging that stable isotope-assisted metabolic flux analysis in mammalian cell cultures will help improving biotechnological production and will also support drug development and discovery.

In‐depth physiological characterization of primary human hepatocytes in a 3D hollow‐fiber bioreactor

As the major research focus is shifting to three‐dimensional (3D) cultivation techniques, hollow‐fiber bioreactors, allowing the formation of tissue‐like structures, show immense potential as they permit controlled in vitro cultivation while supporting the in vivo environment. In this study we carried out a systematic and detailed physiological characterization of human liver cells in a 3D hollow‐fiber bioreactor system continuously run for > 2 weeks. Primary human hepatocytes were maintained viable and functional over the whole period of cultivation. Both general cellular functions, e.g. oxygen uptake, amino acid metabolism and substrate consumption, and liver‐specific functions, such as drug‐metabolizing capacities and the production of liver‐specific metabolites were found to be stable for > 2 weeks. As expected, donor‐to‐donor variability was observed in liver‐specific functions, namely urea and albumin production. Moreover, we show the maintenance of primary human hepatocytes in serum‐free conditions in this set‐up. The stable basal cytochrome P450 activity 3 weeks after isolation of the cells demonstrates the potential of such a system for pharmacological applications. Liver cells in the presented 3D bioreactor system could eventually be used not only for long‐term metabolic and toxicity studies but also for chronic repeated dose toxicity assessment. Copyright

Organotypic Cultures of Hepg2 Cells for In Vitro Toxicity Studies

Tissue engineering of human liver cells in a three dimensional cell culture system could improve pharmacological studies in terms of drug metabolism, drug toxicity or adverse drug effects by mimicking the in vivo situation. In this study, we produced 3D organotypic cultures of HepG2 cells using the hanging drop method. 250 – 8000 seeded cells formed organotypic cultures within 2–3 days which increased in size during the first week. Viability and metabolic parameters (glucose, lactate) were analyzed during almost three weeks of cultivation. Liver specific albumin production was higher in the organotypic cultures as compared to both monolayer and collagen-sandwich cultures. Amino acid quantification revealed high production of glutamate as well as uptake of glutamine, alanine and branched-chain amino acids. CYP1A induction capacity was significantly improved by organotypic cultivation. The acute toxicity (24 h) of tamoxifen, an anti-cancer drug, was lower in the 3D cultures as compared to monolayer and collagen-sandwich cultures. This could be explained by a higher drug efflux through membrane transporter (MRP-2). We conclude that the engineered HepG2 cultures could be used for the investigation of CYP450 induction, anti-cancer drug effects and for the study of chemotherapy resistance. Applied to other cell types such as the human primary cells these 3D organotypic cultures may have potential in long term toxicity screening of compounds.

Long‐term maintenance of HepaRG cells in serum‐free conditions and application in a repeated dose study

Chronic repeated‐dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species‐specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in ’omics‘‐based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum‐supplemented and serum‐free conditions. Viability and Cytochrome P450 (CYP) activity during long‐term cultivation were assessed. Liver‐specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum‐free and serum‐supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum‐free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long‐term repeated‐dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum‐free conditions opens up the opportunity for the use of these cells in human long‐term repeated‐dose hepatotoxicity studies and for application in systems toxicology. Copyright

Biotransformation of diclofenac and effects on the metabolome of primary human hepatocytes upon repeated dose exposure

In vitro repeated dose testing for the assessment of chronic drug-induced effects is a huge challenge in preclinical pharmaceutical drug development. Chronic toxicity results in discontinuation of therapy or post-marketing withdrawal of drugs despite in vivo preclinical screening. In case of hepatotoxicity, due to limited long term viability and functionality of primary hepatocytes, chronic hepatic effects are difficult to detect. In this study, we maintained primary human hepatocytes in a serum-free cultivation medium for more than 3 weeks and analyzed physiology, viability and drug metabolizing capacities of the hepatocytes. Moreover, we assessed acute (24 h) diclofenac toxicity in a range of (10-1000 μM) concentrations. The chronic (9 repeated doses) toxicity at one clinically relevant and another higher concentration (6.4 and 100 μM) was also tested. We investigated phase I and II metabolism of diclofenac upon repeated dose exposure and analyzed effects on the cellular exometabolome. Acute 24 h assessment revealed toxicity only for the highest tested concentration (1 mM). Upon repeated dose exposure, toxic effects were observed even at a low, clinically relevant concentration (6.4 μM). Biotransformation pathways were active for 3 weeks and diclofenac-acylglucuronide was detected as the predominant metabolite. Dose dependent diclofenac-induced effects on exometabolome, such as on the production of lactate and 3-hydroxybutyric acid as well as glucose and galactose metabolism, were observed upon nine repeated doses. Summarizing, we show that repeated dose testing on long-term functional cultures of primary human hepatocytes may be included for the assessment of long term toxic effects in preclinical screening and can potentially help replace/reduce in vivo animal testing.

Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay

Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors.

A Learner-Centered Design, Implementation, and Evaluation Approach of Learning Environments to Foster Acceptance

Most organizations donâ??t focus on what learners really do with their learning environments. Changing the perspective to the learner shows that they enlarge the organizational ones with their own technology-enhanced learning world, using a broad spectrum of technologies: company- and/or higher education-owned Learning Management Systems, social network platforms such as Facebook, mySpace, or Ning, search engines, open web services in the internet like blogs or wikis, and a lot more other applications. In this context, one of the challenges for todayâ??s organizations is how to create well-accepted and used learning environments that drive learnersâ?? success. As errors in requirements specifications have been identified as a major contributor to costly software project failures. Hence it may be highly beneficial if the designers and/or developers of learning environments could verify requirements by predicting learnersâ?? acceptance and usage based on evaluations during the earliest stages of the learning environment development phase. Previous findings of such an approach in the field of CRM software implementation showed that pre-prototype user acceptance tests did have almost equal informational value than their prototype counterparts that ensure hands-on experience with the system. More concrete, we focus on interventions of specific design features during the pre-implementation phase of learning environments. They may help minimizing learnersâ?? initial resistance by providing a realistic preview of the system to enable potential learners to develop accurate perceptions and find out how the system may help them to improve their learning process. The paper proposes a solution by examining the influence of specific design features of learning environments on user acceptance, particularly on the antecedents of perceived usefulness and perceived ease of use. We want to extrapolate the influence of specific design features on specific aspects of perceived usefulness and perceived ease of use in order to enhance designersâ?? and/or developers ability to identify and improve design features during learning environment implementation to enhance critical determinants. The goal of the current work is the improvement of design science in the topic of technology-enhanced learning. To ensure cross-organizational applicability, the approach suggests an application within higher education institutions as well as corporate organizations.

In Silico Modeling for the Prediction of Dose and Pathway-Related Adverse Effects in Humans From In Vitro Repeated-Dose Studies

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects is possible by reverse dosimetry using pharmacokinetic modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28-day exposure, the EC50 values for bosentan and VPA decreased by 21- and 33-fold, respectively. Using EC(10) as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified pharmacokinetic model for the extrapolation of in vitro toxicity to in vivo effect. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4% and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure, respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28-day EC(10) and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. We show that the combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights.