Name: the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections
Uploaded: 2017-05-08T14:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

作者希望承认Vertex Pharmaceuticals和囊性纤维化研究创新奖，为R.Quinn和NIH / NIAID提供资金赠款1 U01 AI124316-01，一种用于治疗多重耐药性病原体的系统生物学方法。我们还要感谢UCSD本科机械工程高级设计课程的机械和航空航天工程系，以便与这项工作的工程方面进行协作。

1.股票的研制人工痰媒体<ol><li>创建一个5％粘蛋白的溶液。添加1.0克脱水猪胃粘蛋白的〜20毫升去离子水。高压釜中加入所得溶液。 注:粘蛋白消毒会破坏它的内在结构;其他方法来灭菌粘蛋白在其干燥形式包括紫外灭菌和辐射。这些方法还没有被广泛用于WinCF系统不过。 </li><li> 2.2克氯化钾添加到50毫升的去离子水，并允许溶解。 5.0克的NaCl添加至50mL的去离子水，并允许溶解。高压灭菌这两种解决方案。 </li><li> 100毫克的鲑鱼精子DNA的添加至10mL无菌去离子水。在水浴中加热该溶液至约85℃下进行几个小时，以确保溶解。 </li><li> 5.0毫克铁蛋白添加到5.0毫升无菌去离子水。 </li></ol> 2.人工痰介质的制备<ol><li>结合以下组分:16mL粘蛋白储备溶液，2.0mL KCl储备溶液，2.0mL NaCl储备溶液，200μL蛋黄乳液，5.6mL DNA储备溶液，120μL铁蛋白储备溶液，5.78mL必需碱氨基酸溶液，5.78mL非必需氨基酸溶液和2.44mL无菌水。 <ol><li>如果出现少量沉淀物，请轻轻晃动混​​匀。 </li><li>将5.0mL培养基吸入八只无菌的15mL离心管中。 注意:每个管的化学条件可以根据需要进行操作。例如，可以将缓冲溶液和pH指​​示剂添加到每个管中，目的是比较不同pH值下的微生物行为。在代表性结果部分中显示了这一点，其具有8个不同的pH水平，从5.0到8.5，0.5个pH增量。 </li></ol></li><li>一旦介质的化学条件成功操作，冻结以供以后使用。媒体将保持稳定禅在-20℃下数月。在解冻时旋涡。 </li></ol> 3.毛细管的控制运行的制备<ol><li>在无菌biohood，填充的每一媒体250μL8个无菌微量离心管中。 </li><li>获得八个无菌的15个毫升离心管中，每管对应于在步骤3.1中提到一个微量离心管中。 <ol><li>消毒用70％乙醇溶液纸巾并晾干。一旦干燥，撕毛巾成关于每个4平方英寸件，并在一个15毫升的离心管的底部揉碎每一块。对于另外的管，喷雾和干燥的附加纸巾根据需要。 </li><li>在每个离心管的底部的纸张一个丛，稍微蘸用约1.0毫升的无菌水中每个纸丛创建一个潮湿的环境。 </li></ol></li><li>获得三个玻璃毛细管在步骤3.1制得的每个微量离心管中并毛细管油灰seala的方块NT。 </li><li>对于每个微量离心管中，填充介质3个的毛细管至约5mm从靠近管的顶部的蓝色标记程。 <ol><li>通过保持在微量离心管中的管的一端并且朝向水平方向倾斜，从而允许毛细作用于介质引导到管填充（参见图1）。 </li><li>轻轻放置带手套的手指在管的开口端停止填充，然后通过按下它分解成密封剂块密封管的另一端。 </li></ol></li><li></li></ol><img alt="图1" src="/files/ftp_upload/55532/55532fig1.jpg" /> 图1:实施例的pH梯度，填充毛细管人工痰平台。该介质是通过将管插入液体的一端，并且倾斜以促进毛细管作用加入。本例中的介质的着色是由于pH指示剂加入到帮助妖STRATE培养后在酸性电位的变化。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>将每个组三个毛细管放入盛有无菌水，腻子密封侧完全润湿向下纸巾15个毫升离心管中。盖上管和标签。这三个毛细管是用于每个控制条件复制。 </li><li>当所有15个毫升离心管中填充有其指定的毛细管，将管在保持架上。放置架，使得管将被水平地温育（捉气泡）（参见图2）。在37℃下孵育离心管（包含湿纸上团块）内的毛细管48小时。 </li></ol><img alt="图2" src="/files/ftp_upload/55532/55532fig2.jpg" /> 图2:实例pH梯度，毛细管准备孵化。一旦三个毛细管被填充和密封，它们被放置在离心管中，在底部带有湿纸巾。然后将该管封盖并放入架子中。在孵化期间，机架必须侧向定位，如图所示，以便一旦孵化完成就可观察到气体产生。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 孵化后控制毛细管成像<ol><li>从培养箱中取出离心管架，确保管子保持水平。仔细地将毛细管从离心管中滑出，保持每组三个与其他组分离。 </li><li>将毛细管彼此排列在灯箱上，均匀排列，使管的内容物相对可见并照亮。每隔三个管子分隔不同的化学条件。 </li><li>管子对齐，灯箱开启后，直接从上方拍照。 （ 见图3 ） </li></ol><img alt="图3" src="/files/ftp_upload/55532/55532fig3.jpg" /> 图3:实施例pH梯度，对照运行，预孵育，无痰添加。人造痰培养基被添加到三套毛细管中后，pH从左到右增加。添加到培养基中的指示剂的组合导致更多的酸性管出现更多的黄色，而较少的酸性管变得更加紫色。管子水平排列，从下方照射，从上方拍摄。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="4"><li>处理控制材料的生物危害性的废物。 </li></ol> 5.痰样品接种WinCF毛细管<ol><li>在无菌biohood，填充的每一媒体225μL8个无菌微量离心管中。 </li><li>通过抽出，并用3mL注射器（塑料注射器没有针头）反复喷射痰均化痰样品。这样做，直到痰是光滑的一致性。 </li><li>添加25μL匀浆痰的在步骤5.1中制备的各微量离心管（1/10稀释在ASM介质）。然后涡管30秒以充分混合。 </li><li>媒体添加到毛细管按照相同的程序与步骤3.2至3.5。 </li></ol> 6.成像样本毛细管温育后<ol><li> 48小时的温育时间之后，按照相同的程序除去和图像毛细管与步骤4.1至4.3。 </li><li>如果存在气泡在管中，使确保拍摄的照片清楚地描绘了管中气泡和介质之间的描绘。如果存在生物膜，请确保照片可以清楚地描述其存在。 </li></ol> 7.删除下游应用程序的媒体<ol><li>为了方便下游分析，成像后从毛细管中取​​出培养基。潜在的应用包括培养和DNA / RNA测序和代谢组学分析。 注意:填充有病原体的玻璃毛细管是一个重要的生物危害因素，因此，必须使用特定的设备非常仔细地进行这些步骤。如果毛细血管破裂，则放在适当的生物危害尖锐的容器中。 </li><li>使用25号长度为0.5英寸的平头针，以移除介质。将钝头针插入管的堵塞端，以破坏密封。 </li><li>打破密封后，将毛细管上下颠倒，介质将从顶部滴出。一世f介质不会轻易滴落，使用200μL吸头的移液器，并将其插入毛细管末端时，通过向下移动移液器柱塞，将介质从管中排出。将排出的管介质收集在合适的容器（1.5mL离心管）中。 注意:对于转录组学或其他RNA分析，培养基可以直接排出到RNA稳定缓冲液中。 </li></ol> WinCF FLUD系统注意:WinCF流体加载实用程序（FLUD）系统是一套可选的补充设备，旨在优化WinCF系统的吞吐量。 WinCF FLUD系统主要由3D可打印材料组成。 3D印刷制造允许快速和容易地更换材料，以确保研究人员的最短停机时间以及最低的制造要求。设计，stl文件，3D打印说明和WinCF FLUD手册可在线补充NT。 <ol><li> 对于毛细管装载准备媒体 <ol><li>在无菌biohood，填充与每个介质的900μL8个无菌2个毫升微量离心管中。 </li><li>通过抽出，并用3mL注射器（塑料注射器没有针头）反复喷射痰均化任何痰样品。这样做，直到痰是光滑的一致性。 </li><li>加入100μL匀浆痰的在步骤8.1.1制备的每个微量离心管（1/10稀释在培养基中）。然后涡管30秒以充分混合。 </li><li>时隙中的填充和开放的微量离心管到定向成使得管是垂直旋转体管保持架。 </li></ol></li><li> 毛细管的位置 <ol><li>检索用于每个微量离心管3个的毛细管在步骤8.1。 </li><li>槽三个管入橡胶支架，使得它们与在该装置的另一端的离心管对准。确保管的标记末端远离微量离心管。将托架底部的三个孔适当地安装在支架支架上的三个螺柱上。 </li><li>将放置的毛细管放置在设备的引导通道中，将橡胶夯实牢固地固定在其中心部分以防止移动。 （ 见图4 ） </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/55532/55532fig4.jpg" /> 图4:完全装有毛细管的FLUD系统由橡胶垫片固定在其中心部分。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="3"><li> 将介质装入毛细管 <ol><li>仔细使用一只手抓住毛细管的设备末端并用另一只手握住装有微量离心管的旋转架。 </li><li>精心旋转微量离心机架，使微量离心管几乎处于水平位置，继续将毛细管慢慢推向毛细管。 （ 见图5 ） </li><li>当毛细管的端部与微量离心管中的介质接触时，确保毛细作用立即开始填充毛细管。要调整填充率和填充量，请将设备整体轻轻旋转。在执行此操作时，请注意不要将介质从微量离心管中溢出。 （ 见图6 ） </li><li>当毛细管被填充到所需的水平时，将装置水平放置在表面上，并小心地将微量离心管的架子从毛细管的端部拉出以停止填充。微量离心管现在可以一直回到垂直位置并关闭。 </li><li>通过将密封剂块压在三份一套上密封毛细管的突出端，一次密封一套，直到所有组密封。为了降低污染的风险，请将密封块的不同部分按压到每一套三份装置上（ 见图7 ）。 </li></ol></li></ol><img alt="图5" src="/files/ftp_upload/55532/55532fig5.jpg" /> 图5:具有中等管的FLUD系统部署到水平方向，准备与毛细管接触。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55532/55532fig6.jpg" /> 图6:带有毛细管的毛细管的FLUD系统通过毛细作用加载媒质。 </str翁&gt; <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/55532/55532fig7.jpg" /> 图7:密封充上FLUD系统1个重复的同时使用密封胶座毛细管管。此密封剂块沿着被切断，以防止在密封期间与相邻一式三份组接触边缘有塑料。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li> 孵化 <ol><li>取下橡胶夯实在管midsections并提起橡胶托架关闭主设备。这应该采取一切毛细管它。现在设置的摇篮，被关押在成像架管。该RAck有三个短杆，适合摇篮，还有小导轨，可以将管子放入。 </li><li>将成像架整体设置在透明塑料保护盒中。在无菌水中浸泡少量无菌纸巾，并沿着箱子的两个短边放置，以在孵育期间提供湿度。 （ 见图8 ） </li><li>完全关闭盒子，并置于37°C的孵化器内，确保管子保持水平。孵育48小时。 </li></ol></li></ol><img alt="图8" src="/files/ftp_upload/55532/55532fig8.jpg" /> 图8:从FLUD系统转移到成像架上的橡胶支架中的毛细管，已经放置在清洁温室中，潮湿纸巾旁边提供湿气。 <a href="http://ecsource.jove.com/files/ftp_upload/55532/55532fig8large.jpg" target="_blank">请点击这里查看更大的版本</a>是数字<ol start="5"><li> 成像和提取 <ol><li>从保护箱中取出保持管的成像架，并将其放在灯箱上，从下方照亮。使用管已经在成像架上，一式三份的组将被适当地间隔开并准备立即成像。 </li><li>将相机聚焦在管上，使得所有在视场中可见，并且来自灯箱的光提供了管染料中的颜色的足够的对比度和可视化。照片从上方。 </li><li>要提取管的内容物，请从橡胶支架中一次一组取出毛细管。轻轻地将管子提起并从支架中取出，或将其滑出。 </li><li>对于每一组三次，请按照步骤7.1至7.3中详述的提取程序。 </li></ol></li></ol>

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D., L&#252;nsdorf, H., Lam, J. S., R&#246;mling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcolony+formation:+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung.">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54 (Pt 7), 667-676 (2005).</li><li>Quinn, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogeochemical+forces+shape+the+composition+and+physiology+of+polymicrobial+communities+in+the+cystic+fibrosis+lung.">Biogeochemical forces shape the composition and physiology of polymicrobial communities in the cystic fibrosis lung.</a> mBio. 5 (2), (2014).</li></ol>

作者没有什么可以披露的。

与CF肺的微生物构成中含有种类繁多的生物，但在肺中的条件可能对什么样的微生物可以生存和发展13，15显著的影响。具体机制，通过这些条件的变化和它们对肺微生物的确切影响是目前通常不清楚。在该实验方法中，我们提出在模拟肺细支气管基于操纵化学条件变化微生物分析。 有协议的一些关键步骤。考虑到被分析的环境的微...

本手册中描述的方法称为WinCF系统1 。 WinCF的总体目标是提供一种能够模拟粘液填充肺支气管环境的实验装置。这将允许易感系统用粘液分泌过多的表型研究肺部疾病的微生物病原体，包括囊性纤维化（CF），慢性阻塞性肺病（COPD），哮喘等。该方法专门用于研究CF，其特征在于引起肺分泌物变稠且难以清除的突变，最终填充细支气管和其他具有粘液的小通道2 。肺中的这种阻塞抑制气体交换，因为吸入的空气不再能够到达许多肺泡，并且也为细菌定居3提供了栖息地。无法预防微生物生长肺过度粘液最终导致气道复杂的慢性感染的发展。这些社区含有多种生物，包括病毒，真菌，和类似的铜绿假单胞菌的细菌，都彼此5，6，7，8相互作用。在CF肺微生物的活性被认为是参与的症状称为肺部症状加重1，9，10，11耀斑。 WinCF能够围绕这些病情加重的微生物群落行为的研究，目前正扩大到作为一个基础的实验系统研究肺微生物生态学。传统上，加重已通过从肺部取出的样品的直接分析研究。许多混杂因素使微生物B的直接分析利用WinCF系统对肺部的行为进行挑战，除去许多这些因素，更直接地研究肺微生物组织的行为，从而更好地分析粘液塞细菌中的细菌活性。 WinCF系统提供了一种以有效模拟肺部环境的方式生长和分析细菌的方法。传统的肺细菌生长方法常常涉及在传统琼脂平板上培养样品。这些方法使样品对大气氧气开放，忽略了在粘液12,13接种的肺细支气管中发现的缺氧和经常缺氧的病症。在有氧条件下在琼脂上培养不像CF肺的环境，并且可能误导临床医生和研究人员关于他们试图治疗的病原体的行为。此外，琼脂平板上的细菌可用的营养物质与实际痰中可用的不相似，这通过使用人造痰介质（ASM）在WinCF中解决。如Sriramulu 等人的假单胞菌培养物所示。 14 ，ASM包括一组模拟可用于痰微生物的资源的组件，并且还可以复制痰的物理一致性。因为病肺具有特定的微生物组织，所以这种微生物的研究也应理想地发生在肺的特定条件下。 WinCF系统能够快速分析和轻松操作实验条件，以观察微生物的变化，类似于它们在实际肺细支气管中的发生。这种技术允许接种无数种相关样品，包括痰液，唾液，其他身体分泌物和纯的或混合的细菌培养物。实验设置的性质允许立即视觉解读微生物群落行为，旨在实现许多微生物和流行程序的下游应用。这些研究是重要的，因为细菌群落组成根据其环境的生理化学条件而变化。使用WinCF可以操纵介质的化学条件来分析对细菌活性的影响。例如，可以在接种样品之前改变培养基的酸度。孵育后，可以直接比较这些条件中的每一种的细菌活性，并且可以得出关于这些痰样品中的细菌如何响应不同pH值的行为的结论。在这里，我们概述了应用WinCF系统的程序和如何操作媒体化学来研究对肺微生物的影响的例子。

<table><tbody><tr><td>Color-Coded Capillary Tubes</td><td>Fisher Scientific</td><td>22-260943</td><td></td></tr><tr><td>Cha-seal Tube Sealing Compound</td><td>Kimble-Chase</td><td>43510</td><td></td></tr><tr><td>Mucin from porcine stomach</td><td>Sigma</td><td>M1778</td><td></td></tr><tr><td>Ferritin, cationized from horse spleen</td><td>Sigma</td><td>F7879</td><td></td></tr><tr><td>Salmon sperm DNA Sodium salt (sonified)</td><td>AppliChem Panreac</td><td>A2159</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>MEM Amino Acids</td><td>Cellgro</td><td>25-030-CI</td><td></td></tr><tr><td>Egg Yolk Emulsion, 50%</td><td>Dalynn Biologicals</td><td>VE30-100</td><td></td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>P2157500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271500</td><td></td></tr><tr><td>15 mL centriguge tubes with Printed Graduations and Flat Caps</td><td>VWR</td><td>89039-666</td><td></td></tr><tr><td>50 mL centrifuge tubes with Printed Graduations and Flat Caps</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Corning</td><td>MCT-150-R</td><td></td></tr><tr><td>2.0 mL microcentrifuge tubes</td><td>Corning</td><td>MCT-200-C</td><td></td></tr></tbody></table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

许多慢性气道疾病导致气道粘液堵塞。具有囊性纤维化的个体的肺是其粘液性细支气管产生微生物定植的有利栖息地的示例性情况。各种病原体在这种环境中相互茁壮成长，并驱使许多与CF疾病相关的症状。像任何微生物群落一样，其栖息地的化学条件对社区结构和动力学有重大影响。例如，不同的微生物在不同水平的氧气或其他溶质浓度下繁殖。在CF肺中也是如此，其中氧浓度被认为是驱动社区生理和结构。这里描述的方法被设计为模仿肺环境并以与它们引起疾病的方式更相似的方式生长病原体。然后使用这些微生物的化学环境的操作来研究化学物质肺部感染的疾病控制其微生物生态学。称为WinCF系统的方法是基于人造痰中和细毛细血管，旨在提供与存在于粘液细支气管中的氧梯度相似的氧梯度。操作化学条件，例如痰液的介质pH或抗生素压力，允许使用有色指示剂观察这些样品的微生物差异，观察气体或生物膜产生，或提取和测序每个样品的核酸含量。

在整个样本中引起的各种化学条件下微生物生长显着在某些情况下，更巧妙地在别人改变。在活动许多变化是视觉性的，只要潜伏期结束是显而易见的。在pH值操作的例子中，在整个pH值范围的试样，如图由温育后变得很明显多种因素变化很大。当无痰样品加入到培养基，温育48个小时后在整个pH值谱所表现出的唯一的变化是在培养基体积轻微蒸发（ 图9）产生?...

Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

粘液堵塞囊性纤维化的气道（CF）患者是微生物病原体茁壮成长的理想环境。手稿描述了用于研究CF肺微生物的环境中模仿其中它们引起的化学条件和疾病如何改变可驱动微生物动力学的新方法。

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

the-wincf-model-an-inexpensive-tractable-microcosm-mucus-plugged

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Immunology and Infection

Unit 1

Lower Respiratory Disorders

SCIENTISTS IN ACTION

9.1K 次观看.  University of California, San Diego. 粘液堵塞囊性纤维化的气道（CF）患者是微生物病原体茁壮成长的理想环境。手稿描述了用于研究CF肺微生物的环境中模仿其中它们引起的化学条件和疾病如何改变可驱动微生物动力学的新方法。 

视频: The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

科研

免疫与感染

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

仿生的微流体模型肺呼吸腺泡航空公司

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

生物工程

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

建立用于呼吸道感染研究的 Lung-on-chip 模型

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

作者聚焦：开发微流体 Lung-on-Chip 模型，用于深入研究人类免疫反应和感染机制

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

在体内评估小鼠的粘液清除

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

摘要

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此视频中的章节

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

仿生的微流体模型肺呼吸腺泡航空公司

研究微生物群落

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

在体内评估小鼠的粘液清除

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

摘要

探索更多视频

此视频中的章节

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

仿生的微流体模型肺呼吸腺泡航空公司

研究微生物群落

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

在体内评估小鼠的粘液清除

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估