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Aline Chary
Environmental Research and Innovation (ERIN), Luxembourg Institute of Science and Technology (LIST)

Difficult-to-test” substances: comparison between digital dispensing and cloud exposure in  context of respiratory sensitization  

Exposure to chemicals in occupational scenarios can lead to the development of respiratory allergies.  No in vitro method is yet validated for the identification of chemical respiratory sensitizers. An in vitro model grown at the air liquid interface (ALI) which includes alveolar type II epithelial cells (A549),  endothelial cells (EA.hy926), macrophages like cells (PMA differentiated THP-1) and dendritic like cells  (DC) (non-differentiated THP-1) was recently developed to identify respiratory sensitizers. During the  exposure, the solubility of chemicals needs careful consideration as certain sensitizers are either water  insoluble or are quickly hydrolysed after being in contact with water as it is the case for acid anhydrides.  

The alveolar model was exposed in different ways at the ALI to phthalic anhydride (PA) and trimellitic  anhydride (TMA) solubilized in DMSO. First, the Vitrocell cloud system was used, where the alveolar  model was exposed to solutions of PA and TMA diluted in PBS to allow the nebulization of a cloud.  Second, PA and TMA were digitally dispensed on the apical side of the alveolar model using the Tecan  D300e. The viability was evaluated using the Alamar blue assay and some markers relevant for  respiratory sensitization were measured, including CD54, CD86 and TSLPr expression on THP-1 cells  and the release of cytokines in the medium from the basolateral side of the model.  

Overall, both ways of exposures allow to measure comparable responses in term of cell surface markers  expression and cytokines release. The D300e allows the dispensing of higher concentrations as  compared to the Vitrocell cloud system as it does not require the use of water or PBS. In addition, as  anhydrides quickly hydrolyse into acids in contact of water, the D300e represents a promising technical  solution to overcome this issue and may be potentially used for challenging compounds.