The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.
Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.
Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items.
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15.
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.
这里,我们已经证明人类器官支气管和鼻腔组织模型的适用性评估重复CS曝光的影响。作为替代动物试验,一些曝光系统的气溶胶暴露在体外毒理学评估被开发( 例如 ,Vitrocell,Cultex,艾丽斯等)。这些曝光模块也可以用于空气中的污染物,空气中的颗粒,纳米颗粒等的毒理评估在这项研究中,我们使用了Vitrocell系统,可以容纳多达48个不同的样品同时,允许对较大规模的实验和下部之间的变异治疗。对于每一个气溶胶暴露在体外组织培养污染的风险仍然是一个重大的风险,其缓解需要一个小心处理的组织培养在整个实验。
电子期间微量天平测量颗粒沉积在实时xposure实验允许监视来自曝光系统中产生的CS剂量符合期望。以确保所测量的颗粒沉积的精度,设定安装在线测量正确的曝光之前是临界指数, 例如 ,规模设置为0。此外,由于微天平(厘米2)的区域之间的差的和组织培养插入物(0.33 平方厘米),我们调整最后计算在培养插入物的区域:只有33%在微量天平沉积的反映在培养插入物的实际沉积。
测量TEER的确定紧结屏障功能,并评估上皮层的破坏是一个相对容易的过程来实现,这是我们这里报告。然而,因为支气管和鼻腔组织模型包含黏液产生穿插杯状细胞,根尖洗涤需要TEER测定值之前必须执行urement。心尖洗涤是至关重要的,因为该粘液层的存在和厚度可偏压TEER的测量的可变性,在CS曝光的影响interferring。这个概念是在与什么是报告的Hilgendorf和同事,其中Caco-2细胞的渗透性受共培养与粘液产生杯状细胞系HT29 23的协议。粘液需要之前TEER测量被洗掉,因为在曝光前右心尖洗涤可以与组织反应的CS干扰。因此,进行测量曝光前三天,之前曝光不正确。
我们发现,CYP1A1 / CYP1B1活动可以从以下CS暴露的器官培养模型测量虽然活性仅略有增加CS。这个弱信号可由CYP1A1 / 1B1基板的较长的温育扩增( 即 ,萤光素-CEE),例如用于24小时(数据未显示)。一个在目前的工作中的CYP活性测量的局限性是缺乏标准化到CYP蛋白质水平或向细胞计数,其可以被考虑为将来的研究,以确保对酶活性的改变不影响由任一蛋白水平或细胞计数的改变。
我们报道了CS暴露抑制纤毛击败两个鼻腔和支气管组织模型。类似的观察结果在不同的哺乳动物和非哺乳动物模型12完成。为睫状跳动测量,从而确保组织被处理,并且处理过的相似的方式是非常关键的,例如,如果介质变更实施,应适用于所有的样品。许特特和他的同事报道,pH值影响哺乳动物的纤毛跳动频率24。因此,比较以不同朱侃,陈万平/混合物,pH值处理的细胞之间跳动频率时调整,应考虑到最小化睫状打浆测量的可变性。此外,在该测量进行的温度也是关键的,因为睫状跳动的频率下降随着温度的降低。为了最大限度地减少由于这些变化,一个阶段顶培养箱,配备有温度,CO 2,湿度控制在这项研究中,使用的可变性。尽管这样,我们观察到,在睫状肌的CS曝光后的支气管组织跳动频率分别为高度可变( 即 ,增加在一个插入件和减小另一个插入件),这表明睫状打浆是曝光后高度干扰的权利。与此相反,我们观察到了不存在可测量的睫状打浆频率中的CS曝光后的鼻组织,这表明鼻组织的反应更加敏感和一致。这是在协议与先前的出版物表明该鼻组织具有较低的容量来解毒作为与支气管25相比。
最后,我们发现,基因表达的器官支气管及鼻部组织模式受到CS分析可以证明CS对异生代谢产生影响。有趣的是,在异生素代谢所观察到的改变的器官支气管和鼻腔的体外模型暴露的CS表现得像是在体内的情况在吸烟者中更详细地在先前的出版物15中讨论。用于基因表达分析,使用机器人的仪器使高通量分析成为可能。此外,自动机器人装卸进一步增加的基因表达的结果的一致性和准确性。然而,快速采集的组织样品是至关重要的,以避免RNA提取过程中的RNA降解。的RNA加工和这里描述转录方法,也可以应用到体内的组织样本。
The authors have nothing to disclose.
笔者要感谢莫里斯·史密斯和玛丽亚Talikka对稿件的审查。
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
MucilAir-human fibroblasts-bronchia | Epithelix Sárl, Geneva, Switzerland | http://www.epithelix.com/content/view/122/19/lang,en/ | |
MucilAir Culture Medium | Epithelix Sárl, Geneva, Switzerland | http://www.epithelix.com/content/view/84/16/lang,en/ | |
VITROCELL | VITROCELL systems GmbH, Waldkirch, Germany | http://www.vitrocell.com/index.php?Nav_Nummer=2&R= | |
3R4F reference cigarette | University of Kentucky | http://www2.ca.uky.edu/refcig/ | |
30-port carousel smoking machine SM2000 | Philip Morris, Int. | ||
CiliaMetrix camera and software | La Haute École de Gestion (HESGE), Geneva, Switzlerland | ||
Leica DMIL microscope | Leica, Heerbrugg, Switzerland | ||
LED light source | Titan Tool Supplies, Buffalo, NY | ||
Chopstick Electrode STX-2 | World Precision Instruments | http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/ | |
EVOMXTM Epithelial Voltohmmeter | World Precision Instruments | http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/ | |
Luciferin Detection Reagent | |||
MagNA Lyser Instrument | Roche | http://www.roche.com/products/product-details.htm?region=us&type=product&id=66 | |
chloroform | Sigma-Aldrich | http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en®ion= | |
QIAcube | Qiagen | 9001882 | |
NanoDrop | Thermo Scientific | http://www.nanodrop.com/ | |
Agilent 2100 Bioanalyzer | Agilent | http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106 | |
Affymetrix GeneChip High throughput 3’IVT Express Kit | Affymetrix | http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit | |
Scanner 3000 7G | Affymetrix | http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G |