Name: label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics
Uploaded: 2018-06-07T14:30:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

这项工作得到了 nih/NIAID (R21AI119042) 以及 nih U24 样本保留化验计划 (U24-AI118656) 的支持。

样本收集是由匹兹堡大学机构审查委员会 (IRB PRO16060443, PRO10110387) 批准的。所有实验均在安全柜内进行, 并配备适当的个人防护设备。
1. 器件制造和软光刻
注: 标准软光刻技术15、16用于创建烷微通道。
<ol><li>在10:1 的碱基和固化剂的配比中混合了该聚硅烷前驱体。</li>
	<li>在微机械加工的铝模上倒入30克的前体细胞混合物 (见图 1为尺寸) 和 20 g 的在 100 mm 培养皿上的前体混合物。 注: 培养皿用于制作支撑层的厚膜 (3 毫米)。在微流体的支撑层提供了均匀的物理表面特性。另外, 还可以使用玻璃滑梯上的一层薄薄的塑料薄膜。利用铝板上的微铣削设计制造了具有特定通道尺寸的主模。本研究采用的螺旋通道是一个8环的螺旋微通道, 其中一个入口和两个插座, 半径从8毫米增加到24毫米, 以有效的大小为基础的分离。</li>
	<li>将模具和培养皿放入真空干燥, 直到表面上没有气泡, 通常为 5-10 分钟. 使用干燥的房子真空。</li>
	<li>释放真空, 将模具和培养皿放在90摄氏度烤箱中1小时。 注: 可使用热板或其他加热工具代替烤箱。</li>
	<li>从烤箱中取出模具和培养皿, 让它在室温下冷却10分钟。</li>
	<li>小心地剪下轮廓, 用2毫米冲床在设备上打上流体通路孔。 注: 冲床的大小可以根据油管或流体导轨的大小而变化。</li>
	<li>将胶带粘附在设备的通道侧, 并将其厚膜涂上, 并用镊子小心剥离以除去灰尘。</li>
	<li>将该设备的通道侧和普通的与空气等离子和粘结的纯聚硅烷 (图 1A) 制成的支撑层。</li>
	<li>将芯片放置在50毫米 x 70 毫米玻璃上, 并将其放置在70摄氏度烤箱中, 以提高粘结强度。</li>
</ol>2. 机械通气病人的气管分泌物收集
注: 气管分泌物可以从机械通气的病人在正常例行气道护理, 使用从常规方法修改的协议, 以适应标准, 成人通气病人。样品被取消辨认并且立刻被送去处理。
<ol><li>如果表示气管吸入, 使用导管提取气道分泌物。</li>
	<li>将导管小心的中途插入气管导管, 并从预先填充的10毫升注射器中注入5毫升0.9% 无菌生理盐水。</li>
	<li>当导管完全推进, 或抵抗被满足, 吸入分泌物并收集成无菌痰收集容器。</li>
	<li>收集10毫升的气管分泌物在一个无菌的痰容器, 并把它放在冰上。立即发送样品进行处理。</li>
</ol>3. 气管分泌物样品制备
注: 所有实验必须在安全柜内进行, 并配备适当的个人防护设备。
<ol><li>使用塑料10毫升注射器 (用于10x 稀释), 分散1毫升的气道分泌物样品在9毫升磷酸盐缓冲盐水 (PBS)。用钝吸管融汇粘液样本。</li>
	<li>将稀释剂与40µm 尼龙细胞过滤器一起去除大块或血块, 这样可以阻断微流体进入孔或通道。加工后将样品放在冰上。</li>
	<li>使用100x 体积的 PBS 缓冲器分散每个样品的稀释剂, 导致1,000x 稀释悬浮。在整个离解操作过程中把样品放在冰上。</li>
</ol>4. 实验设置
注: 所有实验必须在安全柜内进行, 并配备适当的个人防护设备。
<ol><li>将该芯片与流体导轨组装, 将均匀流动应用于四螺旋微 (图 1B)。 注: 四通道通过并行化提高了吞吐量, 并优化了由此产生的悬浮液的体积和细胞密度。流体导向是设计和制造的光固化成形型3D 打印机与透明树脂。该流体导向器的入口和出口端使用女性鲁尔接头, 便于连接和在操作过程中保持稳定的密封。</li>
	<li>将1/16 英寸的男性鲁尔连接器连接到进气道、内壁 (爱德) 插座和流体导轨的外壁 (下) 出口端, 并将硅胶管连接到样品悬浮液中。</li>
	<li>向入口和插座的每一端插入钝尖, 到达样品库的底部。</li>
	<li>将蠕动泵与进油管连接。</li>
	<li>将进油管和爱德出口油管的末端放置在样品库中, 并将其出口油管的末端放置在废物库中 (图 1C, D)。</li>
	<li>将经过过滤的 PBS 的50毫升管放置在样品库中, 不含钙和镁。</li>
	<li>以低流速 (~ 1 毫升/分钟) 开始泵浦, 以使该装置成为质数。 注: 当气泡在通道中捕获时, 将通道顶部与镊子, 以稳定和消除气泡。当设备完全充满缓冲液时, 改变 PBS 管, 准备气道分泌样管。</li>
	<li>当样品放置在样品库中时, 在蠕动泵上切换, 并将流速设置为4毫升/分 (图 1C, D)。</li>
	<li>当试样体积达到指定体积 (~ 1 毫升) 时, 停止操作并断开硅胶管。 注: 该方法更有效地减少了大量稀释剂 (> 50 毫升) 成微离心管容积 (~ 1 毫升) (图 2)。</li>
</ol>5. 流式细胞仪分析
注: 比较分离方法 (微流体和裂解), 采用免疫荧光法进行流式细胞术。
<ol><li>添加异硫氰酸荧光素 (FITC)-共轭小鼠抗人 CD66b 单克隆抗体和复方阿司匹林 (APC)-共轭小鼠抗人 CD45 单克隆抗体的初始悬浮 (步骤 3.3) 和结果悬浮 (步骤 4.9) (200 µL每个抗体) 的比率为1:25 伏/v。</li>
	<li>在黑暗中孵化4摄氏度的样品30分钟。</li>
	<li>在流式细胞仪上分析两个样本以量化 PMNs。 注: FITC-APC 双阳性、CD66b 阳性和 CD45 阳性的人群被认为是中性粒细胞。恢复是由输入的总细胞数和由此产生的悬浮的比率来计算的。</li>
</ol>6. NE 释放分析
注: 用微流体和裂解法比较孤立中性粒细胞的功能, 采用 NE 测定试剂盒。
<ol><li>在检测试剂盒中添加一个 (在步骤4.9 中获得的) 到最后浓度为 50 nM 的。</li>
	<li>将样品孵化37摄氏度, 2 小时。</li>
	<li>离心机悬浮在 300 x g 为10分钟。</li>
	<li>将每个上清的10µL 转移到96井板上 (在化验试剂盒中提供)。 注:96-井微板块与平底和黑聚苯乙烯可以使用以及。</li>
	<li>在每个井中添加90µL 稀释化验缓冲器 (在化验试剂盒中提供)。</li>
	<li>添加10µL 弹性蛋白酶基底 (Z-阿拉巴马-阿拉巴马 2Rh110, 提供的化验试剂盒)。</li>
	<li>在37摄氏度孵育1.5 小时。</li>
	<li>阅读96井板使用一个荧光计在 485 nm 的激发波长和发射波长 525 nm。 注: 从流式细胞术分析中, NE 的水平除以中性粒度计数。</li>
</ol>

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作者已经在这里描述的技术申请了专利申请。

在惯性微流体中, 粒子和细胞以5、18、19、20的大小在微通道中的某一侧向位置定位。由于在弯曲微通道中, 系主任阻力和惯性升力的联合作用, 大颗粒或中性粒细胞 (> 10 µm) 位于通道内, 小颗粒、粘液骨料和小于6µm 的碎片被定位在渠道外面在某些流速情况10,11</su...

由于细胞被封装在气道分泌物中的大量粘液中,体外测定对白细胞的功能评估受到了阻碍。Dithiothreitol () 是最常见的裂解缓冲液, 用于分离和融汇用于细胞学分析和检测的痰液, 同时为隔离细胞提供可耐受的生存能力1,2。然而, 德勤可能干扰气道中性粒细胞的表面束缚抗原, 导致中性粒细胞功能紊乱, 如弹性蛋白酶和髓 (过氧化物) 释放2,3。因此, 对人气道中性粒细胞功能的研究很少与外周血中性粒细胞有关, 这可能无法揭示4肺的生理特性。同时, 惯性微流体已取得进展, 隔离细胞从不同的病人 biomatrices5,6。惯性升力和迪恩阻力之间的平衡根据其大小对颗粒/细胞进行调整, 允许无标签粒子分离7。我们小组以前介绍了一个样品制备方法为循环肿瘤细胞8,9, 病原体在血液8, 细胞从悬浮培养10,11, 12, 和中性粒细胞 (PMNs) 从血液13,14。
在这里, 我们引入了一个协议, 用闭环惯性微流体来制备病人呼吸道分泌物的免疫细胞, 如中性粒细胞弹性蛋白酶 (NE) 测定。这种方法提供高浓度和恢复, 特别是当细胞/粒子的横向方向有明显的重叠, 细胞/粒子的利益将被删除, 这是通常观察到的临床样本。通过将内壁 (爱德) 聚焦的大颗粒或细胞回流到输入样品管, 粒子或细胞的兴趣集中在原储层, 而带有小粘着骨料的背景流体通过废物库。尽管临床样品的非均质流体特性, 该方法从气道分泌物样品中恢复到95% 以上的中性粒细胞, 稀释1000倍于干净的盐水溶液 (~ 1 毫升)。与此相反, 裂解方法根据样品条件提供了 PMNs 回收率的范围。拟议的议定书以无标签的方式捕获白细胞, 没有物理或化学干扰, 这就提供了从临床上具有挑战性的生物特征识别中获得微妙细胞的可能性, 并具有微创程序。

<table><tbody><tr><td>PDMS precursor</td><td>Dow corning</td><td>184 SIL ELAST KIT 3.9KG</td><td>10:1 ratio of base and curing agent</td></tr><tr><td>VWR gravity convection oven</td><td>VWR</td><td>414005-128</td><td>PDMS precursor to be cured in 90 deg.</td></tr><tr><td>100mm petri dish</td><td>VWR</td><td>89000-324</td><td>Fabrication of PDMS Supporting layer</td></tr><tr><td>Harris Uni-core puncher</td><td>Sigma-aldrich</td><td>WHAWB100076</td><td>2mm diameter or other depending on the tubing size</td></tr><tr><td>Air plasma machine</td><td>Femto Science</td><td>Cute</td><td>Surface plasma treatment for PDMS device to bottom base.</td></tr><tr><td>2&amp;rdquo; x 3&amp;rdquo; glass slide</td><td>TED PELLA, INC.</td><td>2195</td><td>To support PDMS device</td></tr><tr><td>Masterflex spooled platinum-cured silicone tubing, L/S 14</td><td>Cole-Parmer</td><td>EW-96410-14</td><td>Tubing for microfluidics and peristlatic pump</td></tr><tr><td>1/16 inch Luer connector, male</td><td>Harvard apparatus</td><td>PC2 72-1443</td><td>Connector for fluid guide</td></tr><tr><td>50mL Falcon tube</td><td>Corning</td><td>21008-940</td><td>sample collection &amp;amp; preparation</td></tr><tr><td>Phosphate-Buffered Saline, 1X Without Calcium and Magnesium</td><td>Corning</td><td>45000-446&amp;nbsp;</td><td>Buffer solution to dilute sample</td></tr><tr><td>Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm</td><td>HALYARD HEALTH</td><td>22113</td><td>Tracheal seceation suction catheter</td></tr><tr><td>0.9% Sterile Normal saline, 10mL pre-filled syringe</td><td>BD PosiFlush</td><td>NHRIC: 8290-306547</td><td>For tracheal seceation collection from the patients</td></tr><tr><td>SecurTainer&amp;trade; III Specimen Containers, 20mL</td><td>Simport</td><td>1176R36</td><td>Sterile sputum (airway secretion) collection container</td></tr><tr><td>Syringe with Luer-Lok Tip, 10mL</td><td>BD</td><td>BD309604</td><td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td></tr><tr><td>BD&amp;nbsp; Blunt Fill Needle, with BD Luer-Lok&amp;nbsp; Tip</td><td>BD</td><td></td><td>To pipette homogenize the mucus sample and reach the bottom of sample tube</td></tr><tr><td>40&amp;micro;m nylon cell strainer&amp;nbsp;</td><td>Falcon</td><td>21008-949</td><td>To remove large chunk or blood clots, which can block the microfluidics access hole or the channel.</td></tr><tr><td>Peristaltic pump (Masterflex L/S Digital Drive)</td><td>Cole-Parmer</td><td>HV-07522-30</td><td>operation of microfluidics</td></tr><tr><td>BD LSR II flow cytometer</td><td>BD Bioscience</td><td>LSR II flow cytometer</td><td>Quantification of cell recovery ratio</td></tr><tr><td>Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody</td><td>BD Bioscience</td><td>561927</td><td>Immunostaining of neutrophils for Flow cytometer analysis</td></tr><tr><td>Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody</td><td>BD Bioscience</td><td>561864</td><td>Immunostaining of neutrophils for Flow cytometer analysis</td></tr><tr><td>Plate reader</td><td>Thermo Fisher scientific</td><td>Varioskan</td><td>Plate reader for neutrophil elastase assay, ex485/em525</td></tr><tr><td>Neutrophil elastase assay kit</td><td>Cayman Chemical</td><td>600610</td><td>Neutrophil functionality assessment</td></tr><tr><td>Fluoresbrite YG Microspheres 10.0&amp;micro;m</td><td>PolyScience, Inc.</td><td>18140-2</td><td>Fluorescent particles to express white blood cell trajectory in microfluidics</td></tr></tbody></table>

label-free neutrophil enrichment from patient-derived airway secretion using closed-loop inertial microfluidics

气道分泌物含有大量与免疫相关的细胞,例如, 白细胞、巨噬细胞和淋巴细胞, 可作为评估各种肺部疾病的主要资源, 用于研究和临床目的。然而, 由于患者粘液的异质性和粘性, 目前尚无可靠的离解方法, 不损害患者气道分泌物的宿主免疫细胞。在本研究中, 我们介绍了一种利用惯性微流体进行患者免疫评估的样品制备方法。无论临床样品的非均质流体特性如何, 该方法均能从气道分泌物样品中恢复95% 以上的中性粒细胞, 稀释1000倍于干净生理盐水的毫升。通过将浓缩输出流循环到初始样品库, 提供了高浓度、回收率和纯度的免疫细胞;再循环被认为是一个权衡的单一运行的基于注射器的惯性微流体操作。螺旋微流体的闭环操作提供无物理或化学干扰的白细胞, 佛波 12-酯 13-醋酸酯 (PMA) 诱导的弹性蛋白酶释放排列的中性粒细胞。

我们实现了透明的免疫细胞悬浮与 mucolysis 和微流体离解 (图 3A)。微流体离解收集了 4.40 x 105 PMNs 平均 (2.1 x 104到 5.60 x 105 PMNs, n = 6) 从气道分泌样品稀释了1000倍 (50 毫升总容量) 在1毫升清洁悬浮。与最初稀释剂相比, 94.0% PMNs (CD66b+/CD45+) 在一个小体积内被回收, 持续超过6个临床样本。然而, mucolysis 方法显示, 平?...

Watch this Scientific Journal Video about Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics at JoVE.com

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

本研究以螺旋惯性微流体闭环操作为例, 对临床气道分泌物无标签的中性粒细胞分离方法进行了论证。该方法将扩大临床对各种呼吸道疾病的体外测定。

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

label-free-neutrophil-enrichment-from-patient-derived-airway

Research

JoVE Journal

Immunology and Infection

6.3K 次观看.  Massachusetts Institute of Technology. 本研究以螺旋惯性微流体闭环操作为例, 对临床气道分泌物无标签的中性粒细胞分离方法进行了论证。该方法将扩大临床对各种呼吸道疾病的体外测定。

视频: Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

科研

生物工程

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

免疫与感染

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

医学

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

生物学

Label-Free Neutrophil Separation from Tracheal Secretion Using Spiral Microfluidics Channel

Source: Ryu, H., et al., <a href="https://app.jove.com/v/57673">Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics.</a>&#160;J. Vis. Exp.&#160;(2018).
This video demonstrates separating neutrophils from tracheal secretions using a microfluidic device with spiral channels. The device achieves efficient separation based on cell size by utilizing varying thicknesses and trapezoidal-shaped channels. The interplay between forces results in large neutrophils accumulating near the inner wall while erythrocytes and mucin aggregates remain near the outer wall, ensuring effective neutrophil separation.

使用螺旋微流体通道从气管分泌物中分离无标记的中性粒细胞

Source: Ryu, H., et al., Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

Remove the deionized water from the device, and add 100 microliters of Fibronectin solution from the outlet. Wait three minutes to ensure that all the channels are filled with Fibronectin solution. Then, incubate the device in a covered Petri dish for an hour at room temperature.
Next, remove the Fibronectin solution from the device, and add 100 microliters of migration medium from the outlet. Again, wait three minutes to ensure that all of the channels are filled with the migration medium. Incubate the device for another hour at room temperature, before using the device in the chemotaxis experiment. Place 10 microliters of whole blood into a 1.5-milliliter tube. Then, add 2 microliters of the antibody cocktail in two microliters of magnetic particles from a neutrophil isolation kit, and gently mix the tube in order to magnetically label the antibody-tagged cells. Incubate the label mixture for five minutes at room temperature.
Next, attach two small magnetic disks to the two sides of the cell loading port of the device. Aspirate the medium from all ports of the device. Then, slowly pipette two microliters of the labeled blood mixture into the microfluidic device from the cell-loading port. Place the microfluidic device on the temperature-controlled microscope stage at 37 degrees Celsius. Wait a few minutes until enough neutrophils are trapped at the cell-docking area.
Next, add 100 microliters of the chemoattractant solution in 100 microliters migration medium to their designated inlet reservoirs, using two pipettors. This will generate a chemoattractant gradient by continuous laminar flow-based chemical mixing. The chemoattractant can be recombinant proteins, such as FMLP, or clinical samples, such as the supernatant of sputum from patients with COPD.
Once flow has stabilized, acquire fluorescence images of the FITC dextran gradient in the channel. Then, incubate the device on the temperature-controlled microscope stage, or in a conventional cell culture incubator for 15 minutes. Following incubation, image the gradient channel, using a 10 times objective to record the cell&#39;s final positions for data analysis.