In vitro air-liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

BackgroundRecently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO2 and one nano-CeO2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed.ResultsDeposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used.ConclusionsWe showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0171-3) contains supplementary material, which is available to authorized users.