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The airway epithelium together with the mucus layer coating it forms a protective system that efficiently filters and removes potentially harmful particles contained in inhaled air. The same mechanism, however, serves to entrap particulate drug carriers, precluding their interaction with their target. The mucus barrier is often neglected in in vitro testing setups employed for the assessment of pulmonary drug delivery strategies. Therefore, our aim was to more accurately model the bronchial barrier, by developing an in vitro system comprising a tight epithelial cell layer which may be optionally supplemented with a layer of human tracheal mucus. To form the epithelium in vitro, we used the cystic fibrosis cell line CFBE41o-, which can be grown as monolayers on Transwell® supports, expressing tight junctions as well as relevant transport proteins. In contrast to the cell line Calu-3, however, CFBE41o- does not produce mucus. Therefore, native human mucus, obtained from tracheal tubes of patients undergoing elective surgery, was used as a supplement. The compatibility of CFBE41o- cells with the human mucus was addressed with the MTT assay, and confirmed by fluorescein diacetate/propidium iodide live/dead staining. Moreover, the CFBE41o- cells retained their epithelial barrier properties after being supplemented with mucus, as evidenced by the high trans-epithelial electrical resistance values (˜1000 Ω cm2) together with a continued low level of paracellular transport of sodium fluorescein. Fluorescently-labeled chitosan-coated PLGA nanoparticles (NP, ˜168 nm) were used as a model drug delivery system to evaluate the suitability of this in vitro model for studying mucus permeation and cell uptake. Comparing CFBE41o- cell monolayers with and without mucus, resp., showed that the NP uptake was dramatically reduced in the presence of mucus. This model may therefore be used as a tool to study potential mucus interactions of aerosolized drugs, and more specifically NP-based drug delivery systems designed to exert their effect in the bronchial region.