SaferWorldbyDesign SaferLiver Products HepG2 BAC-GFP Reporter Cell Line Panel

HepG2 BAC-GFP Reporter Cell Line Panel

We provide a panel of reporter cell lines in which we have targeted biomarkers of cell stress response signal transduction pathways with a fluorescent tag. This assay panel enables us to dynamically observe activation of the stress pathways (upon chemical exposure) over time. As we are using an automated image acquisition and process pipe-line, we are able to perform this in a high throughput and cost effective fashion. In addition, we utilise an automated analysis pipeline to process the data and provide data quantitatively on stress signal pathway activation. The goal is to provide detailed mechanistic information and, if requested by the customer, a toxicity liability score to rank the chemicals based on mal-adaptive pathway activation.

The HepG2 BAC-GFP chemical safety testing platform consists of a panel of >50 human reporter liver cell lines. In each of these stable cell lines, a specific biomarker of a stress pathway is tagged with a fluorophore. Using live cell confocal microcopy, we can visualize and accurately quantify the induction of (chemically induced) cell toxicity.

Below you will find an overview of the tagged biomarkers and the associated stress pathways, which are covered by HepG2 BAC-GFP platform:

HepG2 BAC-GFP platform
An overview of the tagged biomarkers and the associated stress pathways, which are covered by HepG2 BAC-GFP platform.

The HepG2 BAC-GFP reporters can be cultured as a 2D monolayer. The automated image strategy allows for an accurate quantification of stress pathway induction over time on a single cell resolution. Besides the 2D culture, the cell system can also be cultured in a 3D matrigel environment in which stable (>3 weeks) reporter spheroids are formed. Especially the downstream targets (see figure above) can be applied in this setting in which we can also answer questions related to effects bio activation (due to increased human liver P450 enzyme expression) and bioaccumulation (since repeated dosing is possible in these stable spheroids).

Benefits of Approach

The HepG2 BAC-GFP platform is a high throughput and high content screening platform that:

  1. Provides a better prediction in toxicity testing.
  2. Provides dynamic (time and spatial) information on chemical stress pathway induction.
  3. Provides mechanistic information on intracellular perturbations upon chemical exposure.
  4. Provides an in vitro alternative to animal chemical safety tests.
  5. Provides an alternative to low throughput and/or low content in vitro toxicity evaluation assays.

The basis of the HepG2 BAC-GFP technology is a panel of human reporter cell lines which have targeted biomarkers of cell stress response signal transduction pathways with a fluorescent tag. This methodology enables us to dynamically observe activation of these stress pathways (upon chemical exposure) over time. The unique aspect of the approach is to capture key events of toxicity pathways at a physiologically-relevant level in single cells. This is feasible through the integration of the reporter cell lines with automated image acquisition and image processing. The high throughput platform allows cost effective generation of concentration response information required for refined hazard and risk assessment. The automated analysis pipeline to process the data and provide quantitative information on stress signal pathway activation is as crucial step in this entire process.

The HepG2 BAC-GFP reporter approach can dynamically verify not only if a specific chemical is toxic but also the underlying mechanism. This insight in the physiological-relevant toxicity pathway activation is essential for qualification of hazard and risk. The latter is crucial for translation of in vitro data to the in vivo situation. We can provide dynamic quantitative and qualitative mechanistic information on intracellular key toxicity pathway activation at the individual cell level after chemical exposure in a high throughput fashion for a very competitive pricing per sample. Besides the application of the HepG2 BAC-GFP technology we have also established know how on the interpretation of the stress pathway activation data, which is critical for the chemical safety assessment.

Quality Management

To assure high quality data, several internal quality controls are incorporated in every screen:

  1. The quality of the raw data is automatically verified by checking the individual confocal images using NIS elements software. Images that show discrepancies (e.g. out of focus, unequal cell seeding or auto fluorescence) are flagged and excluded from the analysis.
  2. For each reporter assay several negative control conditions are taken along (e.g. 0.2% DMSO, 0.2% H2Od, medium). These controls are negative and do not affect reporter activity.
  3. For each reporter assay a positive control in a full concentration range are taken along. These positive controls specifically monitor active reporter activity in a concentration-dependent manner allowing comparison of different plates and experiments.

We have verified the robustness of the assay by comparing the results of positive control compounds from one screen to the other. Due to our normalization (plate-wise min/max normalization) the results from one screen to the other can be directly compared.

References
  1. Hiemstra S, Ramaiahgari SC, Wink S et al. B. High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability. Arch Toxicol. 2019 Oct;93(10):2895-2911. doi: 10.1007/s00204-019-02552-0. Epub 2019 Aug 27. PMID: 31552476.
  2. Schimming JP, Ter Braak B, Niemeijer M, et al. System Microscopy of Stress Response Pathways in Cholestasis Research. Methods Mol Biol. 2019;1981:187-202. doi: 10.1007/978-1-4939-9420-5_13. PMID: 31016656.
  3. Bischoff LJM, Kuijper IA, Schimming JP et al. A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure. Arch Toxicol. 2019 Feb;93(2):435-451. doi: 10.1007/s00204-018-2353-2. Epub 2018 Nov 20. PMID: 30456486.
  4. Niemeijer M, Hiemstra S, Wink S et al. Systems Microscopy Approaches in Unraveling and Predicting Drug-Induced Liver Injury (DILI). 2018 In: Chen M, Will Y (eds) Drug-Induced Liver Toxicity. Springer New York, New York, NY, pp 611-625. doi:10.1007/978-1-4939-7677-5_29
  5. Wink S, Hiemstra SW, Huppelschoten S et al. Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury. Arch Toxicol. 2018 May;92(5):1797-1814. doi: 10.1007/s00204-018-2178-z. Epub 2018 Mar 3. PMID: 29502165; PMCID: PMC5962642.
  6. Wink S, Hiemstra S, Herpers B, van de Water B. High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities. Arch Toxicol. 2017 Mar;91(3):1367-1383. doi: 10.1007/s00204-016-1781-0. Epub 2016 Jun 29. PMID: 27358234; PMCID: PMC5316409.
  7. Herpers B, Wink S, Fredriksson L et al. Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα- induced cytotoxicity. Arch Toxicol. 2016 May;90(5):1163-79. doi: 10.1007/s00204-015-1536-3. Epub 2015 May 31. PMID: 26026609; PMCID: PMC4830895.
  8. Fredriksson L, Wink S, Herpers B et al. Drug-induced endoplasmic reticulum and oxidative stress responses independently sensitize toward TNFα-mediated hepatotoxicity. Toxicol Sci. 2014 Jul;140(1):144-59. doi: 10.1093/toxsci/kfu072. Epub 2014 Apr 20. PMID: 24752500.
  9. Ramaiahgari SC, den Braver MW, Herpers B et al. A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol. 2014 May;88(5):1083-95. doi: 10.1007/s00204-014-1215-9. Epub 2014 Mar 6. PMID: 24599296.
  10. Wink S, Hiemstra S, Huppelschoten S et al. Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment. Chem Res Toxicol. 2014 Mar 17;27(3):338-55. doi: 10.1021/tx4004038. Epub 2014 Feb 5. PMID: 24450961.
  11. Di Z, Herpers B, Fredriksson L et al. Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening. PLoS One. 2012;7(12):e52337. doi: 10.1371/journal.pone.0052337. Epub 2012 Dec 27. PMID: 23300644; PMCID: PMC3531459.
  12. Fredriksson L, Herpers B, Benedetti G et al. Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis. Hepatology. 2011 Jun;53(6):2027-41. doi: 10.1002/hep.24314. PMID: 21433042.