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For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing. 14. Besides the cell model, an automated exposure system (AES) is used for the air-liquid exposure to aerosols 15,16. The AES has the advantage that it uses a continuous airflow to expose the cell model to aerosols. This is in contrast to other air-liquid exposure systems that usually use relatively high concentrations within a short period of time as a cloud (i.e., droplets containing particles that settle down rapidly) 17,18,19. These cloud systems do not reflect realistic long-term exposure to low concentrations of particles. By applying a continuous airflow using the AES, the cell model can be exposed to a low concentration of particles over a longer time period, reflecting realistic exposure conditions. Another advantage over cloud systems is that the AES has the option to connect particle characterization instruments, allowing measurement of particle

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