High Content Screening of Cellular Blebbing to Predict Sensitizing Potential of Contact Allergens

Abstract

Contact allergy is one of the most prevalent forms of immunotoxicity found in the Western world. Every day, we are exposed to a plenitude of chemicals. In order to replace animal testing which today is used for screening of skin sensitization, it would be great to develop a potential alternative in vitro tool. This thesis project was trying to develop an alternative, non-animal method to detect and predict contact allergens. It combined cellular cultivation with high content screening microscopy to monitor blebbing of keratinocytes (i.e. HEKn) exposed to chemicals in vitro. HEKn cells were seeded in 96-well plates and exposed to four sensitizing chemicals (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, 1-chloro-2, 4-dinitrobenzene,1, 2-Benzisothiazol-3(2H)-one and benzyl benzoate) at five different concentrations (i.e. 0.05 mM, 0.25 mM, 0.5mM, 1 mM and 2 mM) for 24 hours. This whole process was monitored using a high concent screening microscope. It was found that HEKn cells began bleb after exposure to chemicals with moderate or higher sensitizing potency. The strongest sensitizer caused the largest bleb-cell ratio. The order of sensitizing potency matched the order of bleb-cell ratio among these four chemicals. In addition, cell viability was investigated. It can be concluded that monitoring bleb formation from HEKn cells exposed to chemicals might be a potential alternative method to evaluate the sensitizing potency of chemicals.