Animal experiments are still the gold standard to assess inhalation toxicology of nanomaterials (NMs). Due to financial and ethical reasons, it is of interest to create alternative methods based on human pulmonary in vitro models. There are accepted regulatory methods to investigate for example genotoxicity or sensitization like the Ames test, the micronucleus test, the Comet assay or the local lymph node assay. However, the toxicity of NMs also depends on the type of exposure. In contrast to standard submerse cell culture; air liquid interface (ALI) systems closer represent the in vivo situation as they allow the exposure of an aerosol containing the substance of interest, which is considered a promising possibility as alternative method. Therefore, ALI exposure should be considered when assessing nanomaterial toxicity. Until today, there is no in vitro method based on ALI systems which is regulatory accepted. This is mainly due to the fact, that there are currently no standardized protocols for testing and evaluating nanomaterials in ALI application. This thesis deals with the characterization of an ALI system to enhance data quality and reproducibility to further standardize ALI systems. Using a cause-and-effect (C&E) approach, several parameters like relative humidity, aerosol air temperature, flow rate and CO2 concentration in the aerosol were identified to be critical for the viability of the used cells. In addition to the type of exposure, the applied dose is also important for assessing toxicity. Since there is hardly any data on the concentration of CeO2 NPs (nanoparticles) in air, previous investigations are do not include realistic concentrations as they occur in the environment. For the first time, an intracellular delivery of CeO2 NPs similar to in vivo conditions has been verified by using the characterized and optimized ALI system. The production of equal intracellular concentrations is a necessary starting point to compare in vitro and in vivo data, representing an important step in the development of an alternative testing method. It was demonstrated that the application of environmentally relevant and realistic CeO2 NP concentrations can influence the composition of the cell membrane of the alveolar epithelium on a molecular level as a decrease in both phosphatidylcholines and lysophosphatidylcholines was detected. Since cell membrane lipids play an important role in the signaling cascade of proliferation and apoptosis, harmful effects like cancer development as a consequence of NP exposure cannot be excluded completely, even at such low NP concentrations. However, this needs further investigation. It is therefore important to conduct studies with very low doses in the future and include molecular level assessments. Furthermore, the newly developed hAELVi and huAEC cell lines as well as the 3D alveolar cell model EpiAlveolar were investigated in detail. All cell systems showed a clear cell-cell contact formation and a barrier function comparable to in vivo. Additionally, both cell lines showed similar biological responses to CeO2 NPs, comparable to the established but intact barrier function lacking cell model A549. In conclusion, the goal of this submitted doctoral thesis of creating a reliable in vitro platform to assess and characterize the toxicity of CeO2 NPs under realistic conditions with a commercially available ALI system was successful.
