Toxicity assessment of aggregated/agglomerated cerium oxide nanoparticles in an in vitro 3D airway model: The influence of mucociliary clearance

• Testing local defense efficiently predicts local toxicity of particulate matter.
• 3D airway models with mucociliary apparatus can test local mechanical defense.
• Exposure via immersion to cell lines overestimate toxicity of particulate matter.
• Nano CeO2 has low toxicity for ciliated epithelium, despite expected deposition.
• 3D airway models can help reduce the need for animal models in toxicity assessments.

We investigated the toxicity of aggregated nanoparticles of cerium oxide (CeO2) using an in vitro 3D human bronchial epithelial model that included a mucociliary apparatus (MucilAir™). CeO2 was dispersed in saline and applied to the apical surface of the model. CeO2 did not induce distinct effects in the model, whereas it did in BEAS-2B and A549 cell cultures. The absence of effects of CeO2 was not because of the model’s insensitivity. Nanoparticles of zinc oxide (ZnO) elicited positive responses in the toxicological assays. Respiratory mucus (0.1% and 1%) added to dispersions increased aggregation/agglomeration to such an extent that most CeO2 sedimented within a few minutes. Also, the mucociliary apparatus of the model removed CeO2 from the central part of the apical surface to the borders. This ‘clearance’ may have prevented the majority of CeO2 from reaching the epithelial cells. Chemical analysis of cerium in the basal tissue culture medium showed only minimal translocation of cerium across the 3D barrier. In conclusion, mucociliary defence appeared to prevent CeO2 reaching the respiratory epithelial cells in this 3D in vitro model. This model and approach can be used to study compounds of specific toxicological concern in airway defence mechanisms in vitro.