An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.
An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung.
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DOI：
10.1186/1743-8977-10-31
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发表时间：
2013-07-26
影响因子：
10
通讯作者：
Gutleb AC
中科院分区：
文献类型：
--
作者：
Klein SG;Serchi T;Hoffmann L;Blömeke B;Gutleb AC
在世界上许多工业化地区，接触细颗粒和超细颗粒仍然是一个令人关切的问题，纳米技术的强化使用可能进一步增加接触小颗粒的机会。复杂的体外共培养系统可能是研究颗粒诱导过程和推断颗粒对肺的影响的有价值的工具。一个由四种不同的人类细胞系组成的系统，模拟体外肺泡表面的细胞反应，用于研究天然气溶胶暴露（Vitrocell™室）。该系统由肺泡ii型细胞系（A549）、分化的巨噬细胞样细胞（THP-1）、肥大细胞（HMC-1）和内皮细胞（EA.hy 926）组成，以3d定向植入微孔膜上。共聚焦激光扫描显微镜（CLSM）分析了细胞在四培养中的空间分布，显示在transwell的两侧有内皮细胞和上皮细胞的融合层。上皮细胞上可见巨噬细胞样细胞和肥大细胞。细胞在淹没条件下形成菌落，在ALI时消失。采用二氯二氢荧光素双乙酸酯（DCFH-DA）法和2,2 ' -偶氮氮-2-甲基-丙氨酰胺二盐酸盐（AAPH）作为氧化应激诱导剂，评价其对氧化应激的响应。与EA.hy 926、THP-1和HMC-1单培养相比，经阳性对照处理的四种培养对活性氧（ROS）产生的诱导作用较弱。与ALI培养相比，浸泡培养显示ROS和IL-8水平升高。Vitrocell™气溶胶暴露系统对存活率无显著影响。使用该系统，将细胞暴露于PBS中50 nm sio2 -罗丹明NPs的气溶胶中。用CLSM法评价了暴露后NPs在栽培中的分布。内化颗粒的荧光仅在cd11b阳性THP-1细胞中检测到。该系统可与天然气溶胶暴露系统结合使用，并可能最终导致对未来新化合物和/或新纳米级材料危害的更现实的判断。ROS生成和IL-8分泌的结果表明，淹没暴露可能导致对观察到的效应的高估。
Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane. The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2’-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.
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影响因子：
3.5
作者：
Aufderheide, M;Knebel, JW;Ritter, D
通讯作者：
Ritter, D
影响因子：
3.2
作者：
del Pilar Ramos-Godinez, Maria;Eunice Gonzalez-Gomez, Blanca;Alfaro-Moreno, Ernesto
通讯作者：
Alfaro-Moreno, Ernesto
影响因子：
7.4
作者：
HATCHER, EL;CHEN, Y;KANG, YJ
通讯作者：
KANG, YJ
影响因子：
1.7
作者：
BALIS, JU;BUMGARNER, SD;SHELLEY, SA
通讯作者：
SHELLEY, SA
影响因子：
3.3
作者：
Agu, RU;Jorissen, M;Verbeke, N
通讯作者：
Verbeke, N