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:
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:
- Provides a better prediction in toxicity testing.
- Provides dynamic (time and spatial) information on chemical stress pathway induction.
- Provides mechanistic information on intracellular perturbations upon chemical exposure.
- Provides an in vitro alternative to animal chemical safety tests.
- 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.
To assure high quality data, several internal quality controls are incorporated in every screen:
- 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.
- 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.
- 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.
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