Comparison of human primary lung and 3D co-culture with respect to adaptive response towards Bariumsulphate nanoparticle aerosol

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Despite all progress in toxicological risk assessment there are still substantial information deficits, in particular concerning assessment of subtoxic and repeated exposure scenarios as well as in the understanding in the stress response due to particle overload and the potential of adaptive response towards low dose exposure. It is still not understood whether the differences concerning sensitivity and variability between human tissue and other species or between tumor cell lines and primary cells are substantial. The designed project aims to determine the mobility and cellular effects of Bariumsulphate and Titandioxide nanoparticle (positive control) on respiratory tissue of native human lung (L-MOC) as well as on 3D co-cultures of human lung cells and endothelial cells. The biological material will be exposed towards subtoxic amount of nanoparticle aerosols (BaSO4, TiO2) in order to avoid particle overload. The exposure scenario will be applied repeatedly and effect will be monitored over several weeks. The project will focus on following tasks: 1) Is there any difference between 3D Configuration of cell culture inserts (MatriGrids®) and 2D culture systems with respect of deposition of nanoparticles? Furthermore does the cellular response differ accordingly?2) Is the sensitivity concerning stress reaction the same in primary lung cells compared to permanent cell line A549 when both cell types are maintained in 3D co-culture conditions. 3) Does the sensitivity of peripheral (respiratory) lung cells change between physiological morphology in L-MOC compared to 3D co-culture configuration?The nanoparticle aerosols will be applied as single or repeated dose in an exposure chamber with MatriGrids® culture inserts (expertise TILUM) on primary lung tissue (expertise MLU). Monitoring time of cellular responses will be extended up to four weeks. The parameters of interest are cytotoxic effects (viability, marker for reactive oxygen species, level of glutathione, expression and release of cytokines), markers for epithelial morphology and cell to cell contacts. The special focus will be laid on low subtoxic exposure levels and on monitoring of cellular reactions over several weeks. The aim is to determine which effects are direct particle associated and which are induced by mediators of inflammation. The project will combine the expertise from engineering science and toxicology in order to establish and control conditions of aerosol generation and application on cell culture. The exposure chambers and MatriGrids® 3D inserts are established at TIULM and will be optimized according to the needs of the projects. The results will provide the bases to derive NOAEL for BaSO4 to enable risk assessment for nanoparticles for in vivo situation neither to overestimate nor underestimate.