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

尽管在毒理学风险评估方面取得了所有进展，但仍然存在大量的信息缺陷，特别是关于亚毒性和重复暴露情景的评估，以及对颗粒过载引起的应激反应和对低剂量暴露的适应性反应的了解。人类组织与其他物种之间或肿瘤细胞系与原代细胞之间的敏感性和可变性差异是否存在实质性差异尚不清楚。设计的项目旨在确定硫酸钡和二氧化钛纳米颗粒（阳性对照）对天然人肺呼吸组织（L-MOC）的移动性和细胞效应，以及对人肺细胞和内皮细胞三维共培养的影响。生物材料将暴露在亚毒性的纳米颗粒气溶胶（BaSO4, TiO2）中，以避免颗粒过载。暴露情景将反复应用，效果将在数周内监测。该项目将重点关注以下任务：1)细胞培养插入（MatriGrids®）的3D配置与2D培养系统在纳米颗粒沉积方面有什么不同？此外，细胞反应是否相应不同？2)在3D共培养条件下，原代肺细胞与永久肺细胞系A549对应激反应的敏感性是否相同？3)与3D共培养相比，外周（呼吸）肺细胞的敏感性在L-MOC的生理形态中是否发生了变化？纳米颗粒气溶胶将在初级肺组织（专业知识MLU）上与MatriGrids®培养插入物（专业知识TILUM）的暴露室中作为单次或重复剂量应用。细胞反应的监测时间将延长至四周。感兴趣的参数是细胞毒性效应（活力、活性氧标记物、谷胱甘肽水平、细胞因子的表达和释放）、上皮形态标记物和细胞间接触。特别的重点将放在低亚毒性接触水平和监测细胞反应数周。目的是确定哪些影响是直接颗粒相关的，哪些是由炎症介质诱导的。该项目将结合工程科学和毒理学的专业知识，以建立和控制气溶胶的产生和细胞培养应用的条件。在TIULM建立了曝光室和MatriGrids®3D插入，并将根据项目的需要进行优化。研究结果将为获得BaSO4的NOAEL提供基础，使纳米颗粒在体内的风险评估既不高估也不低估。

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.