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，但仍有大量信息不足，特别是在亚毒性和重复暴露情况的评估方面，以及在对颗粒超载造成的应激反应和对低剂量暴露的适应性反应的潜力方面的了解方面。目前尚不清楚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是否存在实质性差异。本设计的目的是确定纳米硫酸钡和二氧化钛(阳性对照)对人肺呼吸组织(L-MOC)以及人肺细胞和内皮细胞3D共培养的迁移率和细胞效应。为了避免颗粒超载，生物材料会暴露在亚毒量的纳米气溶胶(BaSO4、TiO2O5)中。将重复应用暴露方案，并将在几周内监测效果。该项目将专注于以下任务：1)细胞培养插入物(MatriGrids®)的3D配置与2D培养系统在纳米颗粒沉积方面有何不同？此外，细胞的反应是否相应地不同？2)当两种细胞类型保持在3D共培养条件下时，原代肺细胞与永久性细胞系A549对应激反应的敏感性是否相同。3)与3D共培养相比，L-MOC外周(呼吸)肺细胞的敏感性是否在生理形态上发生变化？纳米颗粒气雾剂将以单次或重复剂量的形式应用于暴露室内，并在原代肺组织上放置MatriGrids®培养插入物(Expertify TILUM)。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(活性、活性氧的标志物、谷胱甘肽水平、细胞因子的表达和释放)、上皮形态的标志物和细胞间的接触。特别的重点将放在低亚毒性暴露水平和几周内细胞反应的监测上。目的是确定哪些影响是直接与粒子相关的，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，以建立和控制气雾剂产生和应用于细胞培养的条件。曝光室和MatriGrids®3D插件是在TIULM建立的，并将根据项目的需要进行优化。这些结果将为得出BaSO4的NOAEL提供基础，以便能够对体内情况下的纳米颗粒进行风险评估，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.