This research was supported by the Bio &#38; Medical Technology Development Program of the National Research Foundation (NRF) &#38; funded by the Korean government (MSIP&#38;MOHW) (No. 2016M3A9B6904244). We also thank members of the Medicinal Bioconvergence Research Center for purification of exosome.

1. Block Preparation, Sectioning, Staining and Imaging of the Exosome
<ol>
	<li>Pellet the exosomes from culture supernatant of HCT116 cells by centrifugation at 100,000 x g for 1.5 h23. Remove the culture supernatant and carefully fix the purified exosome pellet with 1 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate solution (pH 7.0) for 1 h at 4 °C. For 0.1 M sodium cacodylate, dissolve 4.28 g cacodylic acid in 160 mL of distilled water (DW). Adjust pH to 7.4 with 0.1 M HCl then make up to 200 mL with distilled water.</li>
	<li>Remove the fixative and rinse the pellets with 1 mL of 0.1 M sodium cacodylate buffer at room temperature. Repeat three times with each change lasting 10 min.</li>
	<li>Post-fix the samples with 1 mL of 2% Osmium tetroxide for 1 h at 4 °C.</li>
	<li>Remove the fixative and rinse three times with 0.1 M sodium cacodylate buffer every 10 min.</li>
	<li>Incubate for 10 min with a graded acetone series (50%, 60%, 70%, 80%, 90%, 95%, 100%, respectively) on the shaker.</li>
	<li>Remove the acetone and incubate the solution of 3:1 acetone:low viscosity embedding mixture for 30 min. The exosome pellet is in the tube.</li>
	<li>Remove the medium and add 1:1 acetone:low viscosity embedding mixture medium, then incubate for 30 min.</li>
	<li>Remove the medium and add 1:3 acetone:low viscosity embedding mixture medium, then incubate for 30 min.</li>
	<li>Remove the medium and add 100% low viscosity embedding mixture and incubate overnight at room temperature.</li>
	<li>Embed the sample in pure low viscosity embedding mixture using the embedding mold and bake for 24 h at 65 °C.</li>
	<li>Prepare sections with 60 nm thickness through an ultra-microtome.</li>
	<li>Double-stain with 2% uranyl acetate for 20 min and lead citrate for 10 min. 
	For Reynolds lead solution, add 1.33 g of lead nitrate and 1.76 g of sodium citrate to a total of 50 mL distilled water.</li>
	<li>Observe the grid under transmission electron microscopy at 80 kV.</li>
	<li>Click "Acquire" and then click "File" and "Save as" in CCD camera system under the electron microscope at 80 kV. Follow automatic settings for the exposure time.</li>
</ol>
2. Immuno-staining of Sections and Imaging (Figure 1)
<ol>
	<li>Cut 60 nm ultrathin sections using an ultra-microtome, and collect on the nickel grid.</li>
	<li>Incubate grids in 50 µL drops of 0.02 M glycine for 10 min to quench free aldehyde groups.</li>
	<li>Rinse in 100 μL of DW three times each for 10 min. Incubate at room temperature for 1 h in PBS containing 1% BSA.</li>
	<li>Incubate grids in 50 - 100 µL drops of anti KRS antibody24 (1:100 in PBS containing 0.1% BSA) for 1 h (If necessary, this step should be carried out at 4 °C overnight.)</li>
	<li>Wash grids with five separate drops (50 µL) of PBS containing 0.1% BSA for 10 min each.</li>
	<li>Transfer grids to drops of 2nd antibody for 1 h (anti-rabbit IgG conjugated to 10 nm gold particle (1:100) in PBS containing 0.1% BSA).</li>
	<li>Wash grids with five separate drops (50 µL) of PBS containing 0.1% BSA each 10 min.</li>
	<li>Double-stain with 2% uranyl acetate for 20 min under dark conditions and with Reynold's lead citrate for 10 min, respectively.</li>
	<li>Click "Acquire" and then click "File" and "Save as" in CCD camera system under a TEM at 80 kV. Exposure time followed automatic settings.</li>
</ol>
<img alt="Sequential protein purification process; chromatography system; sample preparation; microanalysis tools." class="xfigimg" src="/files/ftp_upload/56482/56482fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Process of sample preparation and immunostaining. (A) Double fixed exosome is dehydrated and infiltrated with low viscosity embedding mixture resin. (B) Resin embedding. (C) Ultra-thin section with diamond knife. (D) Setup for Immuno-gold labelling. The grids with the ultrathin sections are put on the liquid droplets on a parafilm, which is placed with the wet paper towel. Each section is incubated with droplets of 50 - 100 µL antibody, then washed with buffer. (E) Double staining with uranyl acetate and lead citrate. The lid is covered with aluminum foil for heavy metal staining. (F) Staining solution is removed by filter paper. <a href="//cloudfront.jove.com/files/ftp_upload/56482/56482fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Negative Staining
<ol>
	<li>Glow-discharge the thin formvar/carbon film coated 200 mesh copper EM grids for 1 min by glow discharger.</li>
	<li>Fix purified exosomes with 1 mL of 2% Paraformaldehyde (PFA) for 5 min. 
	Caution: Paraformaldehyde fumes are toxic. All work should be done in a ventilated fume hood.</li>
	<li>Load 5 - 7 µL exosome suspension solution on the grid and incubate for 1 min. If the concentration of exosome is too high, dilute the concentration to 1/2 - 1/5.</li>
	<li>Immediately stain with the ~20 drops of filtered 1% uranyl acetate (UA) solution on the surface of the EM grid by syringe.</li>
	<li>Remove the excess UA solution on the grid by contacting the grid edge with filter paper.</li>
	<li>Quickly rinse the grid with a drop of water. This step will remove the excess staining solution.</li>
	<li>Place the grid on the table by holding with tweezers, and cover the grid partially with a culture dish to dry for 10 min under room temperature.</li>
	<li>Store the grid in an EM grid box for future observation by a TEM at 80 kV.</li>
</ol>
4. Whole Mount for Immunostaining
<ol>
	<li>Glow-discharge the thin formvar/carbon film coated 200 mesh copper EM grids for 30 s.</li>
	<li>Fix purified exosomes with 1 mL of 2% Paraformaldehyde (PFA) for 5 min. 
	Caution: Paraformaldehyde fumes are toxic. All work should be done in a ventilated fume hood.</li>
	<li>Load 5 - 7 µL fixed exosome solution on the grid and incubate for 5 min. Rinse with 100 µL of PBS three times each for 10 min.</li>
	<li>Treat grids with 50 µL of 0.05 M glycine for 10 min to quench free aldehyde groups.</li>
	<li>Transfer grids to a drop of blocking buffer (PBS containing 1% BSA) for 30 min.</li>
	<li>Incubate grids with 50 - 100 µL anti PD-L1 antibody (1:100 in PBS containing 0.1% BSA) for 1 h (if necessary, this step should be carried out at 4 °C overnight).</li>
	<li>Wash grid with five separate drops (50 µL) of PBS containing 0.1% BSA for 10 min each.</li>
	<li>Transfer grid to a drop of 2nd antibody for 1 h. Anti-mouse IgG conjugated to 9 - 11 nm gold particle diluted at 1:100 in PBS containing 0.1% BSA.</li>
	<li>Wash grid with five separate drops (50 µL) of PBS containing 0.1% BSA for 10 min each. Wash grid with two separate drops (50 µL) of DW. Perform negative staining with 2% uranyl acetate as described from 3.4 to 3.8.</li>
	<li>Store the grid in an EM grid box for future observation by a TEM at 80 kV.</li>
</ol>

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The authors have nothing to disclose.

This article presents a protocol for observing the detailed exosome&#39;s structure and labeling of its specific proteins. Negative staining has been considered as the best method for exosome imaging17. This conventional technique has shown exosomes&#39; cup-shaped structure. However, this cup shape is a form of artefact that can occur due to the drying process. Cryo-TEM results have shown that exosomes have a perfectly spherical structure in aqueous solution25,<...

Extracellular vesicles (EVs) are lipid bilayer vesicles secreted by cells, and their size ranges between 30 and 300 nm. EVs were first reported in 1978, with evidence from vesicles of patients with Hodgkin&#39;s disease1. These vesicles were reported to be 40 to 120 nanometers in size. In 1980, it was reported that EVs are involved in the coagulation system and thrombosis, formation of a blood clot inside a blood vessel in cancer patients2. After 30 years, EVs have been reported to be an important factor to promote tumor invasion, immune escape, and angiogenesis3. In addition, the functions of EVs have been studied as regulators in cellular interactions in the areas of inflammation, immune disorder, neurological disease, and cancer4. Since EVs contain specific biomolecules such as protein, mRNA, and microRNA5, their potential application in diagnostics and therapeutics has been analyzed6,7. EVs are a category composed of subgroups including exosomes, prostasomes, oncosomes, dexosomes, microparticles, promininosomes, argosomes, and exosome-like vesicles, depending on their cellular origin and biological function3. In addition, based on their biogenesis, these EVs can be divided into microvesicles (shed microvesicles, 100 - 1000 nm) and exosomes (30 - 300 nm)8,9. Among these, the exosome has been reported as a cell communicator for immune response10, cancer11,12, and infectious disease13.
Interest in the exosomes as a biomarker for early diagnosis is increasing rapidly, and the purification and characterization of exosomes must be accompanied with molecular imaging techniques. The varying sizes and morphologies of exosomes, depending on their origin and function14, can be distinguished by microscopy techniques with high resolution, such as electron microscopy. Most exosomes were visualized by negative stained Transmission Electron Microscopy (TEM)15,16,17, and these results were confirmed by immunolabeling of a specific protein in whole mount vesicle18. Several research groups have reported the structure through Scanning Electron Microscopy, Atomic Force Microscopy19,20, and Cryo-TEM21,22. However, while these techniques are useful for studying the exosome structure, they are insufficient for observing the position of specific proteins located inside the exosome. Therefore, we introduced a protocol for imaging exosomes with a specific protein&#39;s labelling. We applied block preparation, ultrathin sectioning, and immunostaining for ascertaining the protein&#39;s detailed location in the exosome. This was compared with negative staining and whole mount immunostaining, which is traditionally used for characterization of the exosome.

<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

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

Currently, exosomes are classified into size and shape categories by transmission electron microscopy. Figure 2 shows negative stained exosome and immune-labelled exosome in whole mount status. Figure 3 shows sectioned exosome and immuno-labelled exosomes after thin sectioning. Immuno-gold staining using antibodies of specific proteins is used to positively identify an exosome and classify the types of proteins in the exosome. Th...

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Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

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

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Research

JoVE Journal

Biology

38.9K Views.  Asan Medical Center. Dit protocol beschrijft de verschillende technieken die nodig zijn voor de transmissie-elektronenmicroscopie, met inbegrip van negatieve kleuring, uiterst dunne segmenteren voor gedetailleerde structuur en immuno-goud etikettering om te bepalen van de standpunten van bepaalde eiwitten in exosomes.

Video: 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;

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

Biochemistry

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).

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

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.

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

Bioengineering

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.

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

Medicine

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.

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.

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

Microneedle Patches for Sustained Delivery of Exosomes in Biomedical Applications

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.

Author Spotlight: Innovative Microneedle-Based Strategies for Enhanced Exosome Delivery and Stability

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.

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.

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

Refining Techniques for Murine Spleen-Derived Exosome Isolation

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Negative Staining of Purified Exosomes: A Procedure to Visualize Exosome Morphology Using Transmission Electron Microscopy

Source: Jung, M. K. et al. <a href="https://www.jove.com/video/56482" target="_blank">Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> J. Vis. Exp. (2018)
This video describes a procedure for negative staining of purified exosomes. The negatively stained exosomes are imaged using transmission electron microscopy. This technique helps visualize the morphology of exosomes.

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
This video describes a ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

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