LuSens test - an in vitro cell culture method
The video describes the experimental conduct of the cell-based LuSens assay.
The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using genetically modified keratinocytes (LuSens, Bauch et al. 2012 and Ramirez et al. 2014, and 2016). It uses a luciferase reporter gene under the control of an antioxidant response element (ARE) which is activated via the Keap1-Nrf2 pathway. The assay measures the up-regulation of the luciferase activity after 48 hours incubation with test substances. This up-regulation indicates cellular stress within the keratinocytes which can lead to skin sensitization.
The LuSens assay is performed in 96-well plates. Several test substance concentrations, as well as concurrent controls are tested in parallel. The luciferase activity is determined by the light emitted upon the oxidation of luciferin substrate (bio luminescence). In parallel a cytotoxicity test is performed in order to determine the relative cell viability. The cytotoxicity assay detects the decrease in viable cells which metabolize the diagnostic 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) substrate to a purple formazane, which is measured photometrically.
Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this adverse outcome. Therefore, a combination of several methods, addressing key events of the sensitization process are combined in an integrated testing strategy (Bauch et al. 2012, Urbisch et al. 2015). The key events leading to skin sensitization are modification of skin proteins (e.g. by covalent binding of the test substance), inflammatory reactions of keratinocytes (e.g. due to intracellular stress as detected by LuSens) and activation of dendritic cells (Bauch et al. 2012, Urbisch et al. 2015).
Protocols and guidelines
Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials
Caroline Bauch et al., Regulatory Toxicology and Pharmacology 63 (2012) 489–504
LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification
Tzutzuy Ramirez et al., Toxicology in Vitro 28 (2014) 1482–1497
Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers
Tzutzuy Ramirez et al., Toxicology in Vitro 32 (2016) 278–286
Assessing skin sensitization hazard in mice and men using non-animal test methods
Daniel Urbisch et al., Regulatory Toxicology and Pharmacology 71 (2015) 337–351