Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm. As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins. 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

中文翻译：

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用于纳米粒子诱导的细胞基质改变的新型人类3D肺显微组织模型

已显示多壁碳纳米管（MWCNT）引起炎症和促纤维化介质的释放，以及暴露动物肺部的组织病理学变化。当前测试MWCNT和其他纳米颗粒（NP）的标准依赖于低通量体内研究来评估急性和慢性毒性以及对人类的潜在危害。几种利用二维（2D）体外测定法筛选工程化NP的替代测试方法报告了体外测定法和体内测定法之间相互矛盾的结果。与传统的2D体外或体内动物模型系统相比，三维（3D）体外平台已被证明可以更紧密地概括人类的生理状况，从而提供相关的，在分层的纳米材料毒性测试范例中评估急性毒性和慢性预后的更有效策略。由于吸入是纳米材料暴露的重要途径，因此将人肺成纤维细胞和上皮细胞与巨噬细胞共培养以形成无支架的3D肺微组织。将微组织暴露于多壁碳纳米管，M120炭黑纳米颗粒或青石棉石棉纤维4或7天，然后收集其用于表征微组织的生存力，组织形态以及与炎症和细胞外基质重塑相关的基因和所选蛋白质的表达。我们的数据证明了3D微组织在预测暴露于MWCNT或石棉纤维后的慢性肺终点的效用。将这些测试纳米材料掺入3D人肺微组织中，如使用光学显微镜观察到的那样。使用PCR阵列检测与急性炎症和细胞外基质重塑有关的基因的差异表达，并通过qRT-PCR分析和所选基因和蛋白质的Luminex分析进行确认。3D肺微组织为评估纳米材料诱导的细胞基质改变和毒性途径的描述提供了另一种测试平台，从而朝着一种更具预测性和生理学意义的体外NP毒性测试的方向发展。