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
1Research Laboratory of Electronics,Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science,Massachusetts Institute of Technology, 3Department of Medicine,University of Pittsburgh, 4Department of Biological Engineering,Massachusetts Institute of Technology, 5Vascular Medicine Institute,University of Pittsburgh
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 在10:1 的碱基和固化剂的配比中混合了该聚硅烷前驱体。 在微机械加工的铝模上倒入30克的前体?…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| PDMS precursor | Dow corning | 184 SIL ELAST KIT 3.9KG | 10:1 ratio of base and curing agent |
| VWR gravity convection oven | VWR | 414005-128 | PDMS precursor to be cured in 90 deg. |
| 100mm petri dish | VWR | 89000-324 | Fabrication of PDMS Supporting layer |
| Harris Uni-core puncher | Sigma-aldrich | WHAWB100076 | 2mm diameter or other depending on the tubing size |
| Air plasma machine | Femto Science | Cute | Surface plasma treatment for PDMS device to bottom base. |
| 2” x 3” glass slide | TED PELLA, INC. | 2195 | To support PDMS device |
| Masterflex spooled platinum-cured silicone tubing, L/S 14 | Cole-Parmer | EW-96410-14 | Tubing for microfluidics and peristlatic pump |
| 1/16 inch Luer connector, male | Harvard apparatus | PC2 72-1443 | Connector for fluid guide |
| 50mL Falcon tube | Corning | 21008-940 | sample collection & preparation |
| Phosphate-Buffered Saline, 1X Without Calcium and Magnesium | Corning | 45000-446 | Buffer solution to dilute sample |
| Halyard Closed suction Catheter, Elbow, 14F/ channel 4.67mm | HALYARD HEALTH | 22113 | Tracheal seceation suction catheter |
| 0.9% Sterile Normal saline, 10mL pre-filled syringe | BD PosiFlush | NHRIC: 8290-306547 | For tracheal seceation collection from the patients |
| SecurTainer™ III Specimen Containers, 20mL | Simport | 1176R36 | Sterile sputum (airway secretion) collection container |
| Syringe with Luer-Lok Tip, 10mL | BD | BD309604 | To pipette homogenize the mucus sample and reach the bottom of sample tube |
| BD Blunt Fill Needle, with BD Luer-Lok Tip | BD | To pipette homogenize the mucus sample and reach the bottom of sample tube | |
| 40µm nylon cell strainer | Falcon | 21008-949 | To remove large chunk or blood clots, which can block the microfluidics access hole or the channel. |
| Peristaltic pump (Masterflex L/S Digital Drive) | Cole-Parmer | HV-07522-30 | operation of microfluidics |
| BD LSR II flow cytometer | BD Bioscience | LSR II flow cytometer | Quantification of cell recovery ratio |
| Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD66b monoclonal antibody | BD Bioscience | 561927 | Immunostaining of neutrophils for Flow cytometer analysis |
| Allophycocyanin (APC)-conjugated mouse anti-human CD45 monoclonal antibody | BD Bioscience | 561864 | Immunostaining of neutrophils for Flow cytometer analysis |
| Plate reader | Thermo Fisher scientific | Varioskan | Plate reader for neutrophil elastase assay, ex485/em525 |
| Neutrophil elastase assay kit | Cayman Chemical | 600610 | Neutrophil functionality assessment |
| Fluoresbrite YG Microspheres 10.0µm | PolyScience, Inc. | 18140-2 | Fluorescent particles to express white blood cell trajectory in microfluidics |
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Cite This Article
Ryu, H., Choi, K., Qu, Y., Kwon, T., Lee, J. S., Han, J. Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics. J. Vis. Exp. (136), e57673, doi:10.3791/57673 (2018).