基于纸张的设备进行隔离外囊泡与表征

Summary

该协议细节的方法分离细胞外囊泡（电动汽车），从细胞中释放的小的膜的颗粒，从少至10微升血清样品。这种方法绕过需要超速离心，需要测定时间仅几分钟，并且使电动车从有限体积的样品的分离。

Abstract

胞外囊泡（电动汽车），从不同类型的细胞释放的膜的颗粒，持有用于临床应用的巨大潜力。它们含有核酸和蛋白质的货物和正日益被视为间通讯双方真核生物和原核生物细胞利用的手段。然而，由于它们的尺寸小，对电动汽车的隔离电流协议往往费时，繁琐，并且需要较大的样品体积和昂贵的设备，如超速离心。为了解决这些限制，我们开发了一种基于纸张免疫平台，用于分离电动汽车的亚组是容易的，高效的，并且需要的样品体积低至10微升。生物样品可以直接吸移到已经被化学修饰以捕获分子具有高亲和力的特定的EV表面标记纸测试区。我们验证该测定通过使用扫描电子显微镜（SEM），纸基酶联immunosorben吨测定（P-ELISA），和转录组分析。这些文件为基础的设备将使临床和研究设置电动车的研究，以帮助促进我们的健康和疾病EV功能的理解。

Introduction

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes^1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication¹⁰, immunity modulation¹¹, angiogenesis¹², metastasis¹², chemoresistance¹³, and the development of eye diseases⁹, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.

EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation¹⁴, ultrafiltration¹⁵, magnetic beads¹⁶, polymeric precipitation^17-19, and microfluidic techniques^20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific^14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met.

We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs²². Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers^25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

Protocol

操作步骤的总体框图如图1中提供。使用道德实践，我们收集了来自健康受试者的血液样本，并通过台中荣民总医院（TCVGH），台中，台湾在IRB批准协议获得房水样本的患者（ IRB TCVGH号CF11213-1）。 1.制造纸张的设备切色谱纸放入5mm的圆的直径，以提供相同的布局为一个96孔微量滴定板。夹着这些纸片具有两个聚苯乙烯片材与注册的通孔，和层压体。 修改使用在下面?…

Representative Results

造纸设备的隔离电动汽车的亚组的能力有效地依靠其敏感性和特异性识别的EV表面标志。纸纤维与捕获分子的稳定修饰是通过使用抗生物素蛋白-生物素化学如别处28-30所描述实现的。化学缀合的物理吸附方法的有效性，并且在使用基于荧光的读数评估。纸张测试区被以下置换为20微克/毫升的荧光团R-藻红蛋白缀合的生物素分子（PE-生物素），在步骤1.3协议步骤1），所不同的捕获分子制备。…

Discussion

为胞外囊泡的子组的成功分离的最重要的步骤是：1）一个良好的纸张选择; 2）稳定和高的纸纤维的表面上的捕获分子的覆盖范围; 3）样品的妥善处理; 4）一般实验室卫生习惯。

多孔材料已经被用于许多廉价且设备无测定。它们可以具有可调孔径，多用途功能，成本低和高的表面与体积之比，允许液体的被动的芯吸。我们选择色谱纤维素纸1级主要针对其适当的孔径和低蛋白吸…

Materials

Chromatography Paper	GE Healthcare Life Sciences	3001-861	Whatman® Grade 1 cellulose paper
(3-Mercaptopropyl) trimethoxysilane	Sigma Aldrich	175617	This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.
Ethanol	Fisher Scientific	BP2818	Absolute, 200 Proof, molecular biology grade
Bovine serum albumin (BSA)	BioShop Canada Inc.	ALB001	Often referred to as Cohn fraction V.
N-g-maleimidobutyryloxy succinimide ester (GMBS)	Pierce Biotechnology	22309	GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.
Avidin	Pierce Biotechnology	31000	NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin
Biotinylated mouse anti-human anti-CD63	Ancell	215-030	clone AHN16.1/46-4-5
biotinylated annexin V	BD Biosciences	556418	Annxin V has a high affinity for phosphotidylserine (PS)
Primary anti-CD9 and secondary antibody	System Biosciences	EXOAB-CD9A-1	The secondary antibody is horseradish peroxidise-conjugated
Serum separation tubes	BD Biosciences	367991	Clot activator and gel for serum separation
Annexin V binding buffer	BD Biosciences	556454	10X; dilute to 1X prior to use.
TMB substrate reagent set	BD Biosciences	555214	The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB)
RNA isolation kit	Life Technologies	AM1560	MirVana RNA isolation kit
Polyvinylpyrrolidone-based RNA isolation aid	Life Technologies	AM9690	Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.
RNA cleanup kit	Qiagen Inc.	74004	MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA.
Plasma chamber	March Instruments	PX-250
Scanning electron microscope	Hitachi Ltd.	S-4300
Desktop scanner	Hewlett-Packard Company	Photosmart B110	8-bit color images were captured. Cameras and smart phones may be also used.
Image-record system	J&H Technology Co	GeneSys G:BOX Chemi-XX8	16-bit fluroscence images were captured. Fluroscence microscopes may be also used.