这项工作是由台湾国科会grants- NSC 99-2320-B-007-005-MY2（CC）和NSC 101-2628-E-007-011-MY3（CMC），以及荣总部分支持医院和台湾联合研究计划大学系统（CC）。

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<ol><li>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。 </li><li>修改使用在下面的步骤28中所述的化学缀合方法纸装置。 <ol><li>处理纸的设备用氧等离子体（100毫瓦，1％的氧气，30秒）在等离子体室中。 注意:用3-巯丙基三甲氧基硅烷和 N-γ-马来酰亚胺琥珀酰亚胺工作时特别小心，必须采取酯（GMBS），因为它们都是湿气敏感和毒性。戴防护手套，避免吸入或与皮肤和眼睛接触。保持股市瓶子盖紧并允许它打开，以避免冷凝水之前平衡至RT。或者，打开股票瓶内充氮的手套袋中或框。分装成小药瓶，以避免频繁开股票瓶。 </li><li>立即处理的孵育在4％（体积/体积）的3-巯基丙基三甲氧基硅烷的乙醇（200标准强度）为30分钟，溶液纸装置。 </li><li>冲洗纸装置，用乙醇和孵化它们与0.01微摩尔/毫升GMBS在乙醇中15分钟。 </li><li>冲洗，用乙醇（200标准强度），并培育纸装置用10μg/ ml的抗生物素蛋白在磷酸盐缓冲盐水（PBS），在4℃的溶液1小时。如果需要储存在4℃下，并在4周内使用。 </li></ol></li><li>湿纸的每个测试区与10微升的PBS含有1％（重量/体积）牛血清白蛋白（BSA）为察觉10微升生物素化的捕获分子3×10分钟前，3×10分钟。使用抗CD63抗体（20微克/毫升的PBS含有1％（重量/体积）BSA和0.09％（重量/体积）叠氮化钠）或膜联蛋白V（1:20，体积/体积在膜联蛋白V结合缓冲液），为捕获分子。 </li><li>冲洗掉，用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，分别未结合的抗CD63抗体或膜联蛋白V的分子。 </li></ol> 2.血清和房水样品采集与处理<ol><li>血清集合。 <ol><li>在血清分离管中收集10毫升外周血通过静脉穿刺，轻轻颠倒试管5次。设置该管处于垂直位置，并等待30分钟。 </li><li>离心机在1200×g下15分钟。再次传输从顶层血清到一个干净的试管中，离心以3000×g的30分钟。 </li><li>通过0.8微米FIL通过上清器。保持样品在-80℃下直到使用。 </li></ol></li><li>房水的收集:收集房水样本的患者可直接通过侵入性操作和储存样品在-80°C，直到使用诊断眼科医生。 </li></ol> 3.隔离外囊泡<ol><li>点样到每个纸测试区以5微升/分钟的速率。 </li><li>冲洗掉未结合的电动汽车用10微升相应的PBS含有1％（重量/体积）BSA或膜联蛋白V结合缓冲液进行3×1分钟，用于分别官能用抗CD63抗体或膜联蛋白V的分子，造纸设备。执行以下下游试验。 </li></ol> 4.下游测定实施例1:扫描Electromicrographs <ol><li>修复捕获使用10微升0.5×Karnovsky固定液10分钟官能试纸测试区的电动车。 </li><li>冲洗了充足的PBS样品2×5分钟。 </li><li>脱水样品随后35％乙醇10分钟，50％乙醇为2×10分钟，70％乙醇中2×10分钟，95％乙醇为2×10分钟，100％乙醇为4×10分钟。 </li><li>临界干燥和溅射涂层与钯/金的样品中，并且检查使用在低电子加速电压操作的扫描型电子显微镜（〜5千伏）。 </li></ol> 5.下游分析实施例2:基于纸张的ELISA <ol><li>在PBS 1000稀释含电动车捕捉每一个测试区:添加含有抗CD9一抗5微升溶液中1。等待1分钟。 </li><li>冲洗样品用30ml PBS中振摇在100rpm下30秒。 </li><li>添加5微升含有溶液辣根过氧化物酶（HRP）为1 - 连接的二级抗体:1,000稀释在PBS中，并等待1分钟。 </li><li>冲洗样品用30ml PBS中振摇在100rpm下30秒。 </li><li>添加含有1 5微升比色底物:过氧化氢的1体积比ð3,3&#39;，5,5&#39;-四甲基联苯胺（TMB）和扫描使用台式扫描器。 </li></ol> 6.下游试验例3:RNA分离<ol><li>浸泡样品中35微升的聚乙烯吡咯烷酮为基础的RNA分离援助和265微升裂解/结合缓冲液裂解捕获电动车。涡大力。 </li><li>加在分离试剂盒的裂解物提供30微升匀浆。涡大力和雪藏了10分钟。 </li><li>使用在下面的步骤中描述的酸苯酚 - 氯仿分离提取的RNA。 <ol><li>取330微升酚 - 氯仿从瓶底层并添加到匀浆/溶胞产物。涡大力。 </li><li>离心以10,000×g离心5分钟。水性（上部）相转移到一个干净的试管，并注意除去的体积。 </li></ol></li><li>沉淀的总RNA使用本身是在前面的步骤和共除去水相的1.25倍的体积的体积的100％乙醇llect在预热至95℃的洗提溶液的核糖核酸。 </li><li>浓缩和进一步纯化根据生产商的方案使用RNA净化试剂盒的RNA。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

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附。相比于另一种常用的多孔材料，硝酸纤维素，纤维素纸具有低得多的非特异性蛋白质吸附34。我们还发现在这项研究电动车的非特异性很少捕获。另外，它的颗粒保留水平，11微米，比电动汽车35的尺寸大，从而允许在特定的捕获相反，电动车的物理诱捕。在这里，我们专注于一个更小尺寸的电动汽车，通过一个0.8微米的过滤器，其步骤可以被修改时较大的电动汽车是需要研究经过血清样品。 表面化学改性可一键实现捕获分子的稳定，高覆盖面的提高EV隔离效率的手段上。纤维素表面改性进行了审查36,37。有高数量的羟基（-OH）基团的纤维素的表面上，它们可以与硅烷反应被用氧等离子体或其他缩合方法活化后，得到稳定的化学的Si-OC键。在这项研究中所用的硅烷是（3-巯基丙基）三甲氧基硅烷，（的MeO）3 Si-所示（CH 2）3 -SH。硫醇（-SH）基团可与GMBS反应以提供短空臂（0.73毫微米）和交联基团，夫妻与伯胺。捕获分子与胺基团可缀合直接专线小巴。在这项研究中，但是，我们的NeutrAvidin缀合代替，以提供一个总方案偶联各种捕获分子是生物素标记。然而，在EV亚型特异性标志物的共识尚未达成17。的NeutrAvidin具有非常强的亲和力的生物素和一个近中性等电点（pH值6.3），最小化与带负电荷的对象的非特异性相互作用， 例如电动汽车的表面上。与此相反，抗生物素蛋白10.5的等电点携带正电荷，在中性pH值。抗CD63抗体的浓度较高时比在Chen 等人 20，主要是由于施加的小体积和纸装置的较大的表面与体积比。然而，其量和吸附到纸纤维蛋白的​​组合物还没有被表征，其可以是样品依赖性。因此，必须小心解释蛋白质分析的结果时，可采取。 收集的双向ological样品应轻轻处理和快速地处理，以防止artifactually增大的EV电平。因此建议以除去细胞和血小板，如果有的话，保存前在-80℃下。在EV研究的样品处理电流的标准化可以在本次审查17被发现。 正确的实验室卫生措施应被采用。一定要穿戴手套和变化频繁的手套，以尽量减少引进的尘埃粒子和核糖核酸的。从电动车RNA在细胞培养条件培养基分离可以作为相比，提取使用超速离心机方法（未出版），从电动车的产量集中使用的纸张的设备超过70倍的浓度。 本文所描述的协议使用与捕获分子的化学修饰来隔离电动车辆的不同亚组的纤维素纸的设备。该方法简便，高效，尤其是有价值的，当样本量是有限的。

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 routes1-9. 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 precipitation17-19, and microfluidic techniques20-22. 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 nonspecific14,23,24. 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 polymers25-27, 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.

<table><tbody><tr><td>Chromatography Paper</td><td>GE Healthcare Life Sciences</td><td>3001-861</td><td>Whatman&amp;reg; Grade 1 cellulose paper</td></tr><tr><td>(3-Mercaptopropyl) trimethoxysilane</td><td>Sigma Aldrich</td><td>175617</td><td>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.</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818</td><td>Absolute, 200 Proof, molecular biology grade</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>BioShop Canada Inc.</td><td>ALB001</td><td>Often referred to as Cohn fraction V.</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-&amp;#947;-maleimidobutyryloxy succinimide ester (GMBS)</td><td>Pierce Biotechnology</td><td>22309</td><td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td></tr><tr><td>Avidin</td><td>Pierce Biotechnology</td><td>31000</td><td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td></tr><tr><td>Biotinylated mouse anti-human anti-CD63</td><td>Ancell</td><td>215-030</td><td>clone AHN16.1/46-4-5</td></tr><tr><td>biotinylated annexin V</td><td>BD Biosciences</td><td>556418</td><td>Annxin V has a high affinity for phosphatidylserine (PS)</td></tr><tr><td>Primary anti-CD9 and secondary antibody</td><td>System Biosciences</td><td>EXOAB-CD9A-1</td><td>The secondary antibody is horseradish peroxidise-conjugated</td></tr><tr><td>Serum separation tubes</td><td>BD Biosciences</td><td>367991</td><td>Clot activator and gel for serum separation</td></tr><tr><td>Annexin V binding buffer</td><td>BD Biosciences</td><td>556454</td><td>10x; dilute to 1x prior to use.</td></tr><tr><td>TMB substrate reagent set</td><td>BD Biosciences</td><td>555214</td><td>The set contains hydrogen peroxide and 3,3&amp;rsquo;,5,5&amp;rsquo;-tetramethylbenzidine (TMB)</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>RNA isolation kit</td><td>Life Technologies</td><td>AM1560</td><td>MirVana RNA isolation kit</td></tr><tr><td>Polyvinylpyrrolidone-based RNA isolation aid</td><td>Life Technologies</td><td>AM9690</td><td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td></tr><tr><td>RNA cleanup kit</td><td>Qiagen Inc.</td><td>74004</td><td>MinElute RNA cleanup kit is designed for purification of up to 45&amp;nbsp;&amp;mu;g RNA.</td></tr><tr><td>Plasma chamber</td><td>March Instruments</td><td>PX-250</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Hitachi Ltd.</td><td>S-4300</td><td></td></tr><tr><td>Desktop scanner</td><td>Hewlett-Packard Company</td><td>Photosmart B110</td><td>8-bit color images were captured. Cameras and smart phones may be also used.</td></tr><tr><td>Image-record system</td><td>J&amp;amp;H Technology Co</td><td>GeneSys G:BOX Chemi-XX8</td><td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td></tr></tbody></table>

paper-based devices for isolation and characterization of extracellular vesicles

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。

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。纸张测试区，然后用PBS清洗以除去未结合的PE-生物素分子。与此相反，10微升20微克/毫升PE-生物素在PBS中含有1％（重量/体积）BSA和0.09％（重量/体积）叠氮化钠被直接点样在纸测试区为3×10分钟，随后的PBS冲洗用于进行PE-生物素染料分子的物理吸附。化学修改纸装置（"纸+交联剂+染料"）与未处理纸装置（"纸"），修改与相同的协议没有PE生物素分子（"纸+交联剂"）和纸设备与物理吸附的应用程序文件设备PE-生物素分子（"物理吸附染料"），使用图像记录系统设置为540-560毫微米的激发被扫描，575纳米长通发射，并且该数据被保存在16位文件的形式和分析使用ImageJ软件。 如图2A所示 ，制备由化学缀合方法测试区的荧光强度基本上更高，因为相比物理涂布纸测试区（p值&lt;0.0005，每组5，学生t检验）。电动车辆的捕获也具体例举在图2B，C，其中少数电动汽车被保留在纸装置上时，所述捕获分子通过PBS溶液更换。 电动车是异质的SIZES，具体内容，和生物合成的路线。结合它们，涂覆有两种不同的EV捕获分子，抗CD63抗体和膜联蛋白V纸装置，进行制备。 CD63的四旋家族的成员（包括CD9，CD63，CD81和CD82分子）富含于EV膜31，和膜联蛋白V对磷脂酰丝氨酸（PS）的32,33有很强的亲和力。据发现，电动汽车是在早期凋亡或胁迫条件下组成性释放，在这种过程中的正常细胞的磷脂不对称性丧失，导致的PS上的EV膜的外小叶增加曝光量。 SEM图像的分析表明，CD63 +电动汽车和PS +电动汽车的尺寸分布是不同的，具有80的平均直径和分别为43纳米，（ 图3）。双尾学生t检验用于确定统计p值（p值&lt;0.0001）。出人意料的是，CD63 +电动车出现圆，比PS大，平均+电动汽车，其中生物意义仍有待澄清。 从捕获的纸装置上电动汽车的RNA可以被提取。分析用生物分析仪显示用于从CD63 +和PS +电动汽车（ 图4A）中提取总RNA类似的总体轮廓。相比的RNA利用微流体和超离心技术提取的量，分别为约两次，每血清样品的单位体积39倍以上的RNA提取利用所述纸装置中，虽然使用的是更小的样品体积（ 见表1）。 P-ELISA可以进行对电动汽车的检测。抗CD9初级抗体和HRP缀合的第二抗体被用于本研究。 图4B显示由HRP用于施加10微升PBS中，50％的血清纸装置的酶反应产生的灰度值的变化（用PBS稀释），或100 ％血清，分别。灰度值线性增加血清样本进行测试的范围内。 例如，超速离心，都可以使用。从患者房水样品中分离与涂覆有能够在SEM图像可以看出抗CD63抗体纸装置如电动汽车表现出在图5中。 <img alt="图1" src="/files/ftp_upload/52722/52722fig1.jpg" /> 图1的纸基试验旨在分离和细胞外囊泡的特征的制造和操作步骤:（A）过滤纸被切成所希望的形状并层叠。 （B）的纸张的表面进行改性以氧等离子体的简短处理，并与3-巯基丙基三甲氧基硅烷，N- &lt;反应/ em&gt;的-γ-马来酰亚胺琥珀酰亚胺酯，和的NeutrAvidin，以该顺序。生物素化的捕获分子， 例如，抗体，然后可通过生物 ​​素和的NeutrAvidin分子之间的亲和力强固定。 （C）的生物样品可以是吸移管到官能纸装置。外囊泡可以分离。 （D）下游检测，如SEM，转录和ELISA（如图所示），可以执行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52722/52722fig2.jpg" /> 图2.外囊泡（EVS），可以捕获功能化纸设备具有良好的特异性和灵敏性。（A）荧光标记的生物素分子被高度固定在纸张上通过化学缀合方法（"纸+交联剂+染料"）在对比物理吸附（"物理吸附染料"）的设备。未经处理的纸（"纸"）和共轭分子（"纸+交联剂"）贡献不大的荧光强度。 （B）的代表性SEM图像示出几个电动汽车被捕获的文件在设备上的非特异性当捕获分子被替换的PBS含有1％（重量/体积）BSA中。 （C）许多电动车保留在涂有抗CD63抗体纸装置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3-巯基丙基" src="/files/ftp_upload/52722/52722fig3.jpg" /> 图3.群外囊泡（EVS），可以有不同的粒度分布。（A）和（B）是捕获在涂有分别的抗CD63抗体和膜联蛋白V的分子，纸装置电动车辆的代表性SEM图像。 （C），CD63 +电动车看起来比在这项研究中所使用的实验条件下，PS +电动车大（p值&lt;0.0001，N = 124和203，学生t检验）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52722/52722fig4.jpg" /> 图4.下游转录和ELISA测定法:（A）生物分析仪的数据表示的强度（y轴，以任意单位）和大小（x轴，在核苷酸（nt））荧光标记的总RNA从电动汽车中提取的分布捕获抗CD63抗体或膜联蛋白V-涂滤纸。在〜25的迁移高峰NT代表<html>内标。 （B）的辣根过氧化物酶（HRP）在施加用PBS的10微升样品，50％血清（用PBS稀释）和100％的血清样品为纸装置的P-ELISA测定酶反应生成的灰色值改变。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig4highres.jpg" target="_blank">请这里查看这个数字的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52722/52722fig5.jpg" /> 图5.细胞外囊泡（电动汽车）的房水样品中，可以捕获在官能滤纸没有样品池。分离CD63的房水样品中的SEM图像+电动车从患者（a）年龄相关性黄斑变性，（B）的糖尿病黄斑水肿，和（C）的息肉状脉络膜血管病变，分别。加载/ 52722 / 52722fig5highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 隔离法 </td><td> 捕获分子 </td><td> 血清第一卷。 （微升） </td><td> 提取一般RNA（NG） </td><td> 平均提取RNA /血清 （PG /微升） </td><td> 平均RNA /血清比值 </td><td> 参考 </td></tr><tr><td>微流体装置</td><td>抗人CD63 </td><td> 400 </td><td> 4.14 </td><td> 10.4 </td><td> 20 </td><td> 20 </td></tr><tr><td>造纸设备</td><td>抗人CD63 </td><td> 72 </td><td> 1.49±0.08 </td><td> 20.7 </td><td> 39 </td><td> 22 </td></tr><tr><td>造纸设备</td><td>膜联蛋白V </td><td> 72 </td><td> 0.99±0.26 </td><td> 13.8 </td><td> 26 </td><td> 22 </td></tr><tr><td>超速离心</td><td> - </td><td> 2000 </td><td> 1.06±0.64 </td><td> 0.53 </td><td> 1 </td><td></td></tr><tr><td>血清</td><td> - </td><td> 72 </td><td> 1.23±0.58 </td><td> 17 </td><td> 32 </td><td> 22 </td></tr></tbody></table> 从健康志愿者的血清样品中提取表1总RNA用生物分析仪（n = 4时对所有的条件）进行分析。

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

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。

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.3K 次观看.  National Tsing Hua University. 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。 

视频: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

科研

生物化学

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

微量和外显子的纸基预集中和分离

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

用于从人尿液中连续分离细胞外囊泡的微流控装置

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

作者聚焦：用于生物反应器集成的不对称场流分离

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

使用分叉 A4F 微流控装置从大体积样品中单步分离细胞外囊泡

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

生物工程

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

神经科学

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

生物学

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

基于纸张的设备进行隔离外囊泡与表征

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此视频中的章节

基于荧光的人体血液单细胞外囊快速表征

微量和外显子的纸基预集中和分离

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

使用分叉 A4F 微流控装置从大体积样品中单步分离细胞外囊泡

使用分叉 A4F 微流控装置从大体积样品中单步分离细胞外囊泡

使用分叉 A4F 微流控装置从大体积样品中单步分离细胞外囊泡

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

从外周血中分离和鉴定的微泡

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics