Biological effects of inhalable substances - development of a concept to standardize in vitro studies using aerosols

The state-of-the-art method for lung related research in vitro is the air-liquid-interface culture (ALI) technique involving biological models (cell lines, primary cells, etc.) on microporous membranes. Techniques to use such ALI- models in inhalation related in vitro approaches have improved fundamentally and at the same time led to varying exposure setups. Concerning investigations on pure gases the reproducibility and relevance of these strategies could recently be shown in a prevalidation study comprising round-robin testing of several chemical gases, resulting in a prediction model [1]. However, in case of aerosols the comparability of results of individual labs is still lacking and has to be clearly improved to create relevant results. Therefore, as a first step, defined model setups together with model aerosols are needed to characterize different exposure procedures in individual labs and thus generate comparable and relevant data. To establish a test system which can easily be applied in different exposure setups, an entire concept was developed involving aerosols generated by nebulization and drying of salt solutions including toxic and non-toxic substances of different particle sizes. The aerosols were applied in an ALI exposure setup (P.R.I.T. ® ALI ExpoCube) on A549 cells and toxic effects were investigated based on viability measurements (WST-1). A first group consisting of one positive and one negative substance (SDS and NaCl) including different particle sizes was investigated in the aerosol exposure and resulted in reproducible dose- and substance dependent toxic effects with a higher toxicity using positive control (SDS) than the negative aerosol (NaCl). Thus, the proposed concept was successfully tested as a first step to set up a panel of positive and negative substances which can be used to generate aerosols for the characterisation of aerosol inhalation investigation procedures in vitro. By including simple methods for aerosol generation and quantification, a relevant cell line and fast and easy read-out methods, this concept can be applied easily to different exposure applications in vitro. Therefore, it can clearly improve the relevance of data generated using such methods by a concept of standardisation and normalization in the future.