Name: sample preparation and imaging of exosomes by transmission electron microscopy
Uploaded: 2018-01-04T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

这项研究得到了生物和 #38 的支持; 国家研究基金会 (NRF) 和 #38 的医疗技术发展方案; 由韩国政府资助 (MSIP 和 #38; 保健) (No. 2016M3A9B6904244)。我们还感谢医学 Bioconvergence 研究中心的成员外切纯化。

1. 外切块的制备、切片、染色和成像
<ol>
	<li>将 HCT116 细胞培养上清液中的体, 在 10万 x g 为 1.5 h23进行离心。去除培养上清液, 在1° c 的 0.1 M 钠甲溶液 (pH 7.0) 中, 用1毫升2.5% 戊二醛仔细修复纯化的外切颗粒, 4 小时。对0.1 米甲钠, 溶解4.28 克胂酸在160毫升蒸馏水 (DW)。将 pH 值调整到7.4 与0.1 米 HCl, 然后用蒸馏水构成200毫升。</li>
	<li>在室温下用1毫升的0.1 米甲缓冲液去除固定剂并冲洗颗粒。重复三次, 每次变化持续10分钟。</li>
	<li>在4° c 的情况下, 用1毫升2% 锇氧化在1小时后修复样品。</li>
	<li>取下定影液, 冲洗三次, 每10分钟用0.1 米甲缓冲。</li>
	<li>用分级丙酮系列 (50%、60%、70%、80%、90%、95%、100%、分别) 在振动筛上孵育10分钟。</li>
	<li>除去丙酮, 孵育3:1 丙酮的溶液: 低粘度嵌入混合物30分钟。外切颗粒在试管里。</li>
	<li>去除培养基, 加入1:1 丙酮: 低粘度的嵌入混合物培养基, 然后孵育30分钟。</li>
	<li>去除培养基, 加入1:3 丙酮: 低粘度的嵌入混合物培养基, 然后孵育30分钟。</li>
	<li>去除培养基, 加入100% 低粘度的嵌入混合物, 在室温下过夜。</li>
	<li>将样品嵌入纯低粘度的嵌入混合物中, 并在65° c 下烘烤24小时。</li>
	<li>通过 ultra-microtome 准备 60 nm 厚度的剖面。</li>
	<li>双色染色, 2% 铀醋酸20分钟, 柠檬酸铅10分钟。 对于雷诺铅溶液, 添加1.33 克硝酸铅和1.76 克柠檬酸钠, 总共50毫升蒸馏水。</li>
	<li>在 80 kV 的透射电镜下观察网格。</li>
	<li>单击 "获取", 然后在 80 kV 的电子显微镜下, 在 CCD 摄像系统中单击 "文件" 和 "另存为"。按照曝光时间的自动设置进行。</li>
</ol>
2. 切片和成像的免疫染色 (图 1)
<ol>
	<li>使用 ultra-microtome 切割60纳米超薄切片, 并在镍网格上收集。</li>
	<li>孵育网格在50µL 滴0.02 米甘氨酸为10分钟到淬火游离醛组。</li>
	<li>在100μ l 的 DW 中冲洗三次, 每次10分钟, 在室温下孵育 1 h 在含有 1% BSA 的 PBS 中。</li>
	<li>孵育网格在 50-100 µL 滴抗 KRS 抗体24 (1:100 在 PBS 中含有 0.1% BSA) 为 1 h (如有必要, 此步骤应在夜间在4° c 进行。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。</li>
	<li>传输网格到下落 2nd抗体为 1 h (兔 IgG 共轭到10毫微米金微粒 (1:100) 在 PBS 包含 0.1% BSA)。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 每 10 min。</li>
	<li>双染色2% 铀醋酸20分钟在黑暗条件下, 并与雷诺的铅柠檬酸10分钟, 分别。</li>
	<li>单击 "获取", 然后在 80 kV 的 TEM 下, 在 CCD 摄像系统中点击 "文件" 和 "另存为"。曝光时间遵循自动设置。</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56482/56482fig1.jpg"> 
图 1: 样品制备和免疫的过程.(A)双固定外切的脱水和渗透低粘度嵌入混合树脂。(B)树脂嵌入。(C)超薄截面带金刚石刀。(D)设置免疫金标签。与超薄部分的网格放在液滴上的膜, 这是放置在湿纸巾。每节都用 50-100 µL 抗体的飞沫孵育, 然后用缓冲液冲洗。(E)双染色, 醋酸铀和柠檬酸铅。盖子上覆盖着铝箔, 用于重金属染色。(F)染色液由滤纸移除。<a href="//ecsource.jove.com/files/ftp_upload/56482/56482fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 阴性染色
<ol>
	<li>辉光放电的薄 formvar/碳膜覆盖200网状铜 EM 栅1分钟的辉光料器。</li>
	<li>1毫升2% 甲醛 (粉煤灰) 固定纯化体5分钟。 警告: 甲醛的烟雾是有毒的。所有工作应在通风油烟罩。</li>
	<li>在网格上加载 5-7 µL 外切悬浮液, 孵育1分钟。如果外切浓度过高, 稀释浓度为 1/2-1/5。</li>
	<li>立即染色与〜20滴过滤1% 铀醋酸盐 (UA) 解决方案在表面的 EM 网格的注射器。</li>
	<li>通过与过滤纸接触网格边缘, 删除网格上多余的 UA 解决方案。</li>
	<li>用一滴水迅速冲洗网格。此步骤将删除多余的染色液。</li>
	<li>用镊子把网格放在桌上, 用一个培养皿把网格部分盖住, 在室温下晾干10分钟。</li>
	<li>将网格存储在 EM 网格框中, 以便在80伏的 TEM 上进行观察。</li>
</ol>
4. 全山免疫
<ol>
	<li>辉光放电薄 formvar/碳膜涂层200网状铜 EM 网三十年代。</li>
	<li>1毫升2% 甲醛 (粉煤灰) 固定纯化体5分钟。 警告: 甲醛的烟雾是有毒的。所有工作应在通风油烟罩。</li>
	<li>在网格上加载 5-7 µL 固定外切溶液, 孵育5分钟, 100 µL PBS 三次, 每10分钟冲洗一次。</li>
	<li>治疗50µL 0.05 米甘氨酸10分钟, 以淬火游离醛组的网格。</li>
	<li>将网格转移到一滴阻塞缓冲器 (PBS 含有 1% BSA), 用于30分钟。</li>
	<li>孵育网格与 50-100 µL 抗 PD-L1 抗体 (1:100 在 PBS 包含 0.1% BSA) 为 1 h (如果需要, 这步应该执行在4° c 隔夜)。</li>
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。</li>
	<li>将网格传输到 1 h 的 2nd抗体的下落。</li></ol>抗鼠 IgG 共轭 9-11 nm 的黄金粒子稀释在1:100 在 PBS 含有 0.1% BSA。
	<li>洗涤网格与五单独下落 (50 µL) PBS 包含 0.1% BSA 为10分钟每个。用两个单独滴 (50 µL) 的 DW 洗涤网格。用2% 铀醋酸酯进行阴性染色, 描述从3.4 到3.8。</li>
	<li>将网格存储在 EM 网格框中, 以便在80伏的 TEM 上进行观察。</li>

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作者没有什么可透露的。

本文提出了一个协议, 以观察详细的外切的结构和标签的具体蛋白质。阴性染色被认为是外切成像17的最佳方法。这种传统的技术已经显示体的杯状结构。然而, 这个杯子形状是一种形式的人造制品, 可能发生由于干燥过程。低温透射电镜结果表明, 体在水溶液中具有完美的球形结构25,26。低温透射电镜技术是检测天然结构的一种非常有?...

胞外囊泡 (EVs) 是由细胞分泌的脂质双层囊泡, 其大小范围介于30和 300 nm 之间。EVs 是第一次报告在 1978年, 与证据从囊性霍奇金病患者的水泡1。据报道, 这些小泡的大小是40到120纳米。据报道, 在 1980年, EVs 参与了凝血系统和血栓形成, 在癌症患者的血管内产生了血块2。30年后, EVs 被报道是促进肿瘤侵袭、免疫逃逸和血管生成的重要因素3。此外, 在炎症、免疫紊乱、神经系统疾病和癌症等领域的细胞相互作用中, EVs 的功能已被研究为调节因子4。由于 EVs 包含特定的生物分子, 如蛋白质, mRNA, 和 rna5, 它们在诊断和治疗方面的潜在应用已被分析6,7。EVs 是一个由子群组成的类别, 包括体、prostasomes、oncosomes、dexosomes、微粒、promininosomes、argosomes 和外切样泡, 这取决于它们的细胞来源和生物学功能3。此外, 根据它们的生物, 这些 EVs 可分为微 (棚微、100-1000 nm) 和体 (30-300 nm)8,9。其中, 外切被报告为免疫应答的细胞通信器10、癌症11、12和传染性疾病13。
对体作为早期诊断的生物标记物的兴趣正在迅速增加, 体的纯化和鉴定必须伴有分子成像技术。不同的大小和形态的体, 根据它们的起源和功能14, 可以区分高分辨率的显微镜技术, 如电子显微镜。大多数体是由负染色透射电镜 (TEM)15,16,17, 这些结果是通过 immunolabeling 的一个特定的蛋白在整个芒泡的确认18. 有几个研究小组通过扫描电子显微镜、原子力显微镜、19、20、低温-TEM21、22来报告该结构。然而, 虽然这些技术是有用的研究外切结构, 他们是不足够的观察位置的特定蛋白质位于外切。因此, 我们介绍了一个协议的成像体与特定的蛋白质的标签。我们采用了块制备, 超薄切片和免疫, 以确定蛋白质的详细位置在外切。这是与阴性染色和整个芒免疫, 这是传统用于表征的外切。

<table><tbody><tr><td>Glutaraldehyde</td><td>EMS</td><td>16200</td><td></td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>EMS</td><td>12300</td><td></td></tr><tr><td>Osmium tetroxide 1 g crystal</td><td>EMS</td><td>19100</td><td>Use only in fume hood</td></tr><tr><td>acetone</td><td>JUNSEI</td><td>1B2031</td><td></td></tr><tr><td>Spurr medium</td><td>TED PELLA</td><td>18108</td><td></td></tr><tr><td>Ultra-microtome</td><td>Leica</td><td>UCT</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Lead citrate</td><td>EMS</td><td>17900</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>H7600</td><td></td></tr><tr><td>nickel grid</td><td>EMS</td><td>G200-Ni</td><td></td></tr><tr><td>Copper grid</td><td>EMS</td><td>G200-Cu</td><td></td></tr><tr><td>glycine</td><td>SIGMA</td><td>022K5404</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>SIGMA</td><td>P4417</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Aurion</td><td>25557</td><td></td></tr><tr><td>1st Antibody</td><td></td><td></td><td>purification in manually&amp;nbsp;</td></tr><tr><td>Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td><td>BioLegend</td><td>329710</td><td>Whole mount Immumogold</td></tr><tr><td>Purified Mouse IgG2b, &amp;kappa; Isotype Ctrl</td><td>BioLegend</td><td>401202</td><td>Whole mount Immumogold (Control)</td></tr><tr><td>2nd Anti-mouse igG conjugated 9-11 nm gold particle</td><td>sigma</td><td>G7652</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr><tr><td>Glow discharger</td><td>JEOL</td><td>JFC1100E</td><td></td></tr><tr><td>Carbon grid</td><td>EMS</td><td>121119</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19210</td><td></td></tr><tr><td>Formvar carbon coated Copper Grid (200 meh)</td><td>EMS</td><td>FCF200-CU</td><td></td></tr><tr><td>Formvar carbon coated&amp;nbsp; Nickel Grid (200 mesh)</td><td>EMS</td><td>FCF200-NI</td><td></td></tr></tbody></table>

sample preparation and imaging of exosomes by transmission electron microscopy

体是一种由体液分泌的纳米细胞胞外囊, 它能代表分泌它们的细胞的特征。分泌囊泡的含量和形态反映细胞的行为或生理状态, 例如细胞生长、迁移、分裂和死亡。体的角色可能高度依赖于大小, 体的大小从30到 300 nm 不等。最广泛使用的方法外切成像是阴性染色, 而其他结果是基于低温透射电镜, 扫描电镜, 和原子力显微镜。典型的外切的形态通过阴性染色评估是一个杯子形状, 但进一步细节还不清楚。通过结构研究的外切良好在医学和药学领域是必要的。因此, 功能相关的形态学应该通过电子显微镜技术来验证, 例如在外切的详细结构中标注特定的蛋白质。对体的结构、超薄切片图像和负染色图像进行了对比观察。在本协议中, 我们建议传输电子显微镜的成像体包括阴性染色, 全贴剂免疫染色, 块准备, 薄切片, 免疫金标签。

目前, 体被分为大小和形状类别的透射电子显微镜。图 2显示了负染色外切和免疫标签外切在整个安装状态。图 3显示了切片后的分段外切和免疫标记体。免疫金染色使用特异蛋白抗体, 用于积极识别外切和分类的蛋白质类型的外切。本协议采用全贴免疫和免疫塑料分段外切。
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Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

本协议描述了传输电子显微镜所需的各种技术, 包括阴性染色, 超薄切片的详细结构, 和免疫金标签, 以确定特定的蛋白质在体的位置。

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

sample-preparation-imaging-exosomes-transmission-electron

Research

JoVE Journal

Biology

38.8K 次观看.  Asan Medical Center. 本协议描述了传输电子显微镜所需的各种技术, 包括阴性染色, 超薄切片的详细结构, 和免疫金标签, 以确定特定的蛋白质在体的位置。

视频: Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

科研

生物化学

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

生物学

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

纳米等离子体增强散射和低放大显微镜成像对肿瘤衍生外质体的定量

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

生物工程

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

医学

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

用于生物医学应用中外泌体持续递送的微针贴片

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.

作者聚焦：用于增强外泌体递送和稳定性的创新微针策略

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.

用于加载和递送外泌体的微针贴片的制造和表征

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

透射电镜下体的样品制备与成像

摘要

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此视频中的章节

原子力显微镜对细胞外囊泡的成像

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

纳米等离子体增强散射和低放大显微镜成像对肿瘤衍生外质体的定量

分离和剖析人力胆汁含外来体的MicroRNA-

RNA含外来体的分离和鉴定

对于外来体定量和尺寸测量新方法研究

用于加载和递送外泌体的微针贴片的制造和表征

用于加载和递送外泌体的微针贴片的制造和表征

用于加载和递送外泌体的微针贴片的制造和表征

3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体