The authors thank Dr. Richard Nowakowski and the Florida State University Laboratory Animal Resources for providing and caring for the animals used to develop this protocol, respectfully. We thank Xia Liu, Dr. Rakesh Singh, and the Florida State University Translational Science Laboratory for assistance with the mass spectrometry work used for downstream vesicle characterization, as well as the FSU Biological Science Imaging Resource facility for the usage of the transmission electron microscope in this study. Finally, we thank Dr. Mandip Sachdeva (Florida A&#38;M University) for donation of the tumor specimens used in the development of this method. This study was supported by grants from the Florida Department of Health Ed and Ethel Moore Alzheimer&#39;s Disease Research Program awarded to D.G.M. and J.M.O (6AZ11) and the National Cancer Institute of the National Institutes of Health under Award Number R01CA204621 awarded to D.G.M.

Whole brains were obtained with approval from the Institutional Animal Use and Care Committee (IACUC) of the Florida State University. A total of twelve mouse brains (3 brains from each age group: 2, 4, 6, and 8 months) from a C57BL/6&#8239;J background were used for EV extraction, as previously described16. Lung tumor specimens were generously donated by Dr. Mandip Sachdeva under approval of the Florida Agricultural and Mechanical University IACUC. Lung tumors were derived from the human adenocarcinoma cell line H1975 grown in immunodeficient Balb/c nu/nu nude mice. Data from two representative tumor replicates are highlighted in this study.
NOTE: A schematic overview of the vesicle isolation and purification method is provided in Figure 1.
1. Tissue Dissociation and Differential Centrifugation
<ol>
	<li>Prepare 10 mL of dissociation buffer (10 mg of papain; 5.5 mM L-cysteine; 67 &#181;M 2-mercaptoethanol; 1.1 mM EDTA) in Hibernate-E medium per approximately 0.4-1.0 g of tissue. Note that all solutions used for EV enrichment and purification should be diluted in ultrapure filtered water.</li>
	<li>Add whole fresh or frozen tissue to dissociation buffer in a 50 mL conical tube, and incubate in a warm water bath at 37 &#176;C for 20 min. Tissue may be cut into smaller fragments before incubation if needed.</li>
	<li>Following the incubation, add protease and phosphatase inhibitors for a final 1x concentration to the dissociation buffer containing the tissue.</li>
	<li>Pour the solution containing the tissue into a loose-fit Dounce homogenizer, and gently dissociate the tissue using approximately 30 slow strokes per sample. The number of strokes may be adjusted based on tissue consistency.</li>
	<li>Transfer dissociated tissue in buffer to a 50 mL conical tube and centrifuge at 500 x g for 5 min at 4 &#176;C to pellet the cells and remaining fibrous or cohesive tissue fragments.</li>
	<li>Transfer the supernatant to a clean 50 mL conical tube and centrifuge at 2,000 x g for 10 min at 4 &#176;C to pellet and discard large cellular debris.</li>
	<li>Transfer the supernatant again to a clean 50 mL conical tube and centrifuge at 10,000 x g for 40 min at 4 &#176;C to pellet undesired larger vesicles or small apoptotic bodies. 
	NOTE: This pellet can be saved for additional study of larger vesicles if desired.</li>
	<li>Decant the supernatant through a 0.45 &#181;m filter into a clean 12 mL ultracentrifugation tube. 
	NOTE: Densely fibrotic tissue samples may not be easily filtered. These samples can be filtered through a 40 &#181;m cell strainer, then passed through serially smaller needles (18 G, 20 G, 22 G) before filtering through the 0.45 &#181;m filter.</li>
	<li>Ultracentrifuge the sample at 100,000 x g for 2 h at 4 &#176;C to pellet small EVs.</li>
	<li>Decant the supernatant and leave the ultracentrifugation tubes inverted for 5-10 min, tapping frequently to remove residual liquid on the sides of tubes. Residual fluid can also be aspirated using an aspirating vacuum pipette.</li>
	<li>Re-suspend EV pellet in 1.5 mL of 0.25 M sucrose buffer (10 mM Tris, pH 7.4). It is important to ensure full re-suspension of the pellet in this step. To do so, cover the tubes with parafilm before vortexing EVs into solution. Rock the ultracentrifuge tubes for 15-20 min at room temperature before a final vortex mix.</li>
	<li>Briefly centrifuge the tubes at a speed no more than 1,000 x g to recover the liquid suspension at the bottom of the tube.</li>
	<li>Proceed to the gradient purification steps below, or if needed, store EV suspension at 4 &#176;C overnight.</li>
</ol>
2. Density Gradient Purification
<ol>
	<li>Add 1.5 mL of 60% iodixanol (stock solution in water) to the 1.5 mL sucrose/Tris buffer containing EVs to create a final solution containing 30% iodixanol. Pipette up and down several times to mix solution thoroughly. Be cautious to avoid losing solution in the pipette tip, as the high iodixanol concentration is quite viscous.</li>
	<li>Transfer the 30% iodixanol buffer solution containing EVs to the bottom of a 5.5 mL ultracentrifugation tube.</li>
	<li>Prepare at least 1.5 mL of 20% and 10% iodixanol solutions per sample in ultrapure water from the 60% iodixanol stock solution.</li>
	<li>Measure 1.3 mL of 20% iodixanol and carefully layer on top of the bottom gradient layer using a syringe and an 18 G needle. To avoid mixing the layers at the density interface, keep the bevel of the needle in contact with the inside of the ultracentrifugation tube just above the meniscus and add the solution dropwise.</li>
	<li>Layer 1.2 mL of 10% iodixanol solution on top of the 20% iodixanol layer using the same technique as above.</li>
	<li>Carefully balance and load the ultracentrifugation tubes into rotor buckets and centrifuge in a swing-bucket rotor at 268,000 x g for 50 min at 4 &#176;C. Set the acceleration and deceleration speeds of the centrifuge to the minimum rates allowed to avoid disruption of the density layers.</li>
	<li>Label ten 1.5 mL microcentrifuge tubes for each sample to correspond to fractions 1 through 10 of the density gradient. Fraction 1 is designated as the topmost fraction that will be first removed, while fraction 10 is the bottom fraction last removed.</li>
	<li>Once the gradient ultracentrifugation has completed, gently remove the tubes from the rotor buckets and place in a stable holder. A visible band of vesicles will often be seen in the fraction of interest. Pipette ten serial fractions of 490 &#181;L from the top of the gradient into the corresponding tubes. There will be a small amount of fluid remaining at the bottom of the tube that likely contains contaminating proteins. This sample can be discarded, or retained as fraction 11 if desired.</li>
	<li>Measure the refractive indices of fractions using a refractometer. This can also be performed with a control gradient run in parallel if sample conservation is necessary. Pipette 20-30 &#181;L of each fraction containing iodixanol onto the refractometer surface, and estimate the density by comparing the refractive indices to known iodixanol densities. Note that fractions can be stored overnight in the iodixanol-containing solution if needed before proceeding to the next ultracentrifugation wash step.</li>
	<li>Transfer each fraction to a clean 12 mL ultracentrifugation tube. Add 5 mL of 1x phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to the tube and mix by pipetting slowly up and down. 
	NOTE: PBS added to the samples should be particle-free, ensured by filtering sterile PBS through a 0.22 &#181;m filter and storing at room temperature to avoid salt precipitation.</li>
	<li>Add an additional 6 mL of 1x PBS to the top and again mix carefully.</li>
	<li>Ultracentrifuge the tubes at 100,000 x g for 2 h at 4 &#176;C to re-pellet small vesicles. Decant the supernatant and tap the tubes dry before lysis of vesicles for protein analysis (Step 3) or re-suspension of EVs for morphologic analysis (Step 5).</li>
</ol>
3. Lysis and Immunoblot Confirmation of EV Proteins
NOTE: If preservation of whole vesicles for morphologic characterization or biological activity is desired, researchers can proceed directly to Step 5. In initial experiments, or before mass spectrometry analysis, all fractions recovered should be analyzed by immunoblot to confirm fractions with EV proteins.
<ol>
	<li>To lyse EVs for protein analysis, add 40 &#181;L of strong lysis buffer (5% SDS, 10 mM EDTA, 120 mM Tris-HCl pH 6.8, 2.5% 2-mercaptoethanol, 8 M urea) with the addition of protease inhibitor to EV pellet in ultracentrifuge tube. 
	NOTE: If non-reducing conditions are desired for antibody detection of proteins, substitute ultrapure water for 2-mercaptoethanol in the strong lysis buffer.</li>
	<li>Ensure complete re-suspension of pellet and lysis of EVs in buffer by placing parafilm over the ultracentrifugation tube, vortexing vigorously, then rocking for 20 min at room temperature before a final vortex.</li>
	<li>Briefly centrifuge at 1,000 x g for 30-60 s to recover the entire sample volume. Transfer the sample to a new 1.5 mL microcentrifuge tube and store at -20 &#176;C to -80 &#176;C until further processing.</li>
	<li>To prepare purified lysates for immunoblot analysis, add 5x Laemmli sample buffer (10% SDS, 250 mM Tris pH 6.8, 1 mg/mL bromophenol blue, 0.5 M DTT, 50% glycerol, 5% 2-mercaptoethanol) to the samples for a final concentration of 1x. 
	NOTE: If non-reducing conditions are desired, replace the DTT and 2-mercaptoethanol with ultrapure water.</li>
	<li>If whole tissue homogenate controls are desired, lyse the tissue in the urea-containing strong lysis buffer detailed above with protease inhibitor, then centrifuge at 10,000 x g for 10 min to discard the remaining extracellular matrix, whole cells, or intact cellular debris. Add 5x Laemmli sample buffer to the tissue homogenate for a final concentration of 1x.</li>
	<li>Boil the sample buffer containing EV or tissue homogenate samples at 95 &#176;C for 5-10 min, then load equal volume (from the equivalent of 0.1-0.3 g of starting tissue material; 1-2 &#181;g of purified EV protein) of fractions 1-10 into a 10% SDS-PAGE gel. Load equal mass of tissue homogenate. Greater volume of samples may be needed for detection by less sensitive antibodies.</li>
	<li>Proceed with gel electrophoresis and Western blot analysis as previously detailed21 to confirm EV proteins in the purified lysates and compare relative EV abundance in fractions. 
	NOTE: Once EV protein has been reproducibly demonstrated in consistent fractions, the final ultracentrifugation wash described in Step 2.12 may be limited to gradient fractions of interest.</li>
</ol>
4. EV Protein Quantification
<ol>
	<li>Use a detergent- and urea- compatible fluorescence-based assay if sensitive quantification of EV proteins is desired for further analysis (see Table of Materials). Note that some fluorescence-based assays are compatible with the bromophenol blue present in Laemmli sample buffer, facilitating direct lysis of the EV pellet (in Step 2.12) in this buffer if preferred.</li>
	<li>If the aforementioned fluorescence-based protein quantification kit is used, spot 1 &#181;L of sample or standard in at least duplicate on the provided filter paper, and continue protein quantification according to manufacturer&#39;s instructions.</li>
</ol>
5. Characterization of EV Morphology
<ol>
	<li>To examine whole vesicles after the final centrifugation wash in Step 2.12, thoroughly re-suspend EV pellet in particle-free 1x PBS. Store EVs at 4 &#176;C for no longer than one week until processing to avoid freeze-thaw cycles. 
	NOTE: It is beneficial to have previously confirmed fractions consistently containing enriched EV protein by immunoblot analysis to limit morphological analyses to fractions of interest.</li>
	<li>Perform nanoparticle tracking analysis on whole EV samples to determine the concentration and size of vesicles in preparations, as previously detailed22.</li>
	<li>Characterize EV morphology and confirm size measurements by electron microscopy of vesicles, if desired. EM grids can be prepared from vesicle suspensions following Step 5.123, or alternatively can be processed as block preparations24 from pellets following Step 2.12.</li>
</ol>
6. In-gel Purification and Trypsin Digestion for Mass Spectrometry Analysis
<ol>
	<li>If characterization of EV proteomes by mass spectrometry is desired, load equal mass (at least 10 &#181;g of protein) of fractions containing EVs into a pre-cast polyacrylamide gel to separate and purify proteins. Manufacturer supplied pre-cast gels are preferred to reduce the levels of potential contaminating proteins (e.g., keratin) being introduced into the samples.</li>
	<li>Run the sample at least 0.5 inches into the polyacrylamide gel for protein purification, then fix and stain with Coomassie dye, as previously described in detail25.</li>
	<li>Cut the lanes into 1 mm3 cubes for trypsin digestion, as previously detailed19,26.</li>
	<li>Load 5 &#181;L of digested peptide sample into LC-MS/MS instrument for separation on the analytical column and subsequent analysis by mass spectrometry according to parameters specified in detail16.</li>
</ol>

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The authors have nothing to disclose.

Much scientific interest has been generated with regards to the roles small EVs play in the tumor microenvironment, as well as in organ development, maturation, and function. Overall, this study provides an optimized workflow for the extraction of intact EVs from whole brain or tumor specimens. While here we simply demonstrate the applicability of this technique to lung tumor-derived EVs, this method could easily be adapted for work on other solid tissues, providing opportunities for further ex vivo characterization of s...

Small extracellular vesicles (EVs) include endosomal-derived exosomes and small membrane-shed microvesicles that are of broad biomedical interest. Small EVs are composed of a heterogeneous population of 50-250 nm membrane-bound vesicles containing biologically active proteins, lipids, and nucleic acids that are collectively believed to play important roles in a multitude of disease processes. Advancing research has particularly implicated these vesicles in neurodegenerative and prion disorders, infectious processes, autoimmune or inflammatory conditions, and tumor growth and metastasis1,2,3,4,5,6,7,8,9,10,11,12,13. Rapidly growing biomedical research into the significance of EVs in disease pathogenesis has generated parallel interest in developing reproducible and rigorous methods for the isolation and purification of these vesicles.
A historical and current challenge in EV characterization has been the inability to fully separate small EV subpopulations. This challenge is largely due to our limited understanding of the differing molecular mechanisms governing the biogenesis of distinct vesicles. Overlapping size, density, and biologic cargo between subpopulations further convolutes these distinctions. Part of this challenge has also been the use of widely differing enrichment techniques, providing inconsistency in downstream analysis of isolated vesicles across laboratories, and undermining the global effort to illuminate categorical EV populations.
It is worth noting that the majority of EV characterization has been performed from in vitro collection of cell culture supernatant, with more recent in vivo studies describing techniques to isolate vesicles from animal or human body fluids, including plasma, urine, and saliva. While EVs are present in large quantities in circulation, it also recognized that these vesicles play important roles in cell-to-cell communication events and are present in the interstitium of cellular tissues. In the context of cancer, interstitial EVs may be particularly important in modulating the tumor microenvironment for cancer cell seeding and metastatic growth14,15. Consequently, there is value in the development and optimization of techniques to extract vesicles from solid tissue specimens. These methods will provide a means to directly study organ- or tumor- derived EVs harvested from clinical specimens, including small biopsies and partial or full organ resections.
In this study, and in a previous report published by our laboratory16, we aim to address several major current concerns in EV enrichment methodology: 1) to describe a reproducible technique to isolate and purify EVs to the highest standards currently accepted in the field; 2) to attempt to isolate small EV subpopulations highly enriched in endosomal-derived exosomes; and 3) to provide a protocol for the extraction of these vesicles from solid tissue specimens for the purpose of further characterization.
Recently, Kowal and colleagues described a relatively small-scale iodixanol density gradient to separate and purify EV subpopulations with greater efficacy than comparable sucrose density gradients17. In the cited study, dendritic cell-derived EVs captured in a relatively light density fraction, consistent with a density of 1.1 g/mL, were highly enriched in endosomal proteins believed to be most consistent with a high proportion of exosomes present in this fraction. According to the authors, these &#34;bona fide&#34; exosomal proteins included tumor susceptibility gene 101 (TSG101), syntenin-1, CD81, ADAM10, EHD4, and several annexin proteins17. We later adapted this technique to succeed a method of tissue dissociation described by Perez-Gonzalez et al18 and a subsequent differential centrifugation protocol to isolate whole brain-derived EVs16. We also demonstrated the utility of this method in characterizing EV proteomes by combining a sequential protocol for downstream quantitative and comparative mass spectrometry of vesicular protein, previously described by our laboratory19. This work paralleled that from the Hill laboratory, in which EVs were enriched from the frontal cortex of brains20.
In this study, we elaborate on this technique and extend the application of the protocol recently published from our laboratory to the isolation of EVs from solid lung tumors. To our knowledge, this is the first study to describe a protocol to enrich EVs from ex vivo tumor specimens. Given the widespread interest in EVs as novel diagnostic biomarkers and their role in tumorigenesis, this method will likely prove valuable to a growing number of scientific researchers. From a clinical perspective, interstitial EVs could harbor great diagnostic value, particularly in specimens where histologic evaluation is limited. Our hope is that the method outlined here will provide a foundation for a reproducible technique to harvest EVs from fresh or frozen animal or human surgical specimens, paving the way for future work to uncover the significant roles in disease pathogenesis these small vesicles may play.

<table><tbody><tr><td>0.45 &amp;micro;m filter</td><td>VWR</td><td>28145-505</td><td></td></tr><tr><td>12 mL ultracentrifuge tubes</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>5.5 mL ultracentrifuge tubes</td><td>Beckman Coulter</td><td>344057</td><td></td></tr><tr><td>anti-Alix antibody</td><td>Santa Cruz</td><td>sc-7129</td><td></td></tr><tr><td>anti-Calnexin antibody</td><td>Santa Cruz</td><td>sc-11397</td><td></td></tr><tr><td>anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>anti-CD81 antibody</td><td>Santa Cruz</td><td>sc-9158</td><td></td></tr><tr><td>anti-Flotillin 2 antibody</td><td>Santa Cruz</td><td>sc-25507</td><td></td></tr><tr><td>anti-HSC70 antibody</td><td>Santa Cruz</td><td>sc-7298</td><td></td></tr><tr><td>anti-Syntenin-1 antibody</td><td>Santa Cruz</td><td>sc-100336</td><td></td></tr><tr><td>anti-TSG101 antibody</td><td>Santa Cruz</td><td>sc-7964</td><td></td></tr><tr><td>EZQ protein quantification kit</td><td>ThermoFisher Scientific</td><td>R33200</td><td></td></tr><tr><td>FA-45-6-30 rotor&amp;nbsp;</td><td>Eppendorf</td><td>5820715006</td><td></td></tr><tr><td>FEI CM120 Electron Microscope</td><td>TSS Microscopy</td><td></td><td></td></tr><tr><td>goat anti-rabbit IgG (Fab fragment)</td><td>Genetex</td><td>27171</td><td></td></tr><tr><td>HALT phosphatase inhibitor (100x solution)</td><td>ThermoFisher Scientific</td><td>78420</td><td></td></tr><tr><td>HALT protease inhibitor (100x solution)</td><td>ThermoFisher Scientific</td><td>78438</td><td></td></tr><tr><td>Hibernate E medium</td><td>ThermoFisher Scientific</td><td>A1247601</td><td></td></tr><tr><td>MLS-50 swinging-bucket rotor</td><td>Beckman Coulter</td><td>367280</td><td></td></tr><tr><td>NanoSight LM10</td><td>Malvern</td><td></td><td></td></tr><tr><td>Optima MAX-XP Benchtop Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>393315</td><td></td></tr><tr><td>Optima XE-100 ultracentrifuge</td><td>Beckman Coulter</td><td>A94516</td><td></td></tr><tr><td>Optiprep</td><td>Sigma</td><td>D1556</td><td>60% iodixanol in sterile water solution</td></tr><tr><td>Q Exactive HF Mass Spectrometer</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>rabbit anti-goat IgG</td><td>Genetex</td><td>26741</td><td></td></tr><tr><td>rabbit anti-mouse IgG</td><td>Genetex</td><td>26728</td><td></td></tr><tr><td>Refracto 30PX (refractometer)</td><td>Mettler Toledo</td><td>51324650</td><td></td></tr><tr><td>S-4-104 rotor</td><td>Eppendorf</td><td>5820759003</td><td></td></tr><tr><td>SW 41 Ti swinging-bucket Rotor</td><td>Beckman Coulter</td><td>333790</td><td></td></tr><tr><td>Tabletop 5804R centrifuge</td><td>Eppendorf</td><td>22623508</td><td></td></tr></tbody></table>

extraction of extracellular vesicles from whole tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

A schematic overview of the tissue dissociation, differential centrifugation, and gradient purification of vesicles is displayed in Figure 1. The morphologic and immunophenotypic confirmation of gradient-purified EVs is highlighted in Figure 2. A diagram of reproducible densities following ultracentrifugation of the 10-30% iodixanol gradient is shown, with two distinct vesicle populations migrating upward to fraction 2 (light EVs...

Watch this Scientific Journal Video about Extraction of Extracellular Vesicles from Whole Tissue at JoVE.com

Extraction of Extracellular Vesicles from Whole Tissue

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

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Research

JoVE Journal

Cancer Research

14.9K Views.  Florida State University College of Medicine. Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Video: Extraction of Extracellular Vesicles from Whole Tissue

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Biochemistry

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Immunology and Infection

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.

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

Neuroscience

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 ...

Medicine

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Biology

Optimized Isolation of Skeletal Muscle-Derived EVs for Research and Therapeutics

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles (SkM-EVs) from rodent muscle tissues through mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It demonstrates consistency and reliability. The isolated SkM-EVs provide insights into muscle homeostasis and diseases, with potential applications as diagnostic biomarkers or therapeutic vehicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, ...

Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from one cell to another. EVs have been shown to travel short and far distances in the body and are tissue-specific. EVs are not only found in tissues, but they can also be found in practically all bodily fluids, such as tears, saliva, cerebral spinal fluid, blood, etc. Even though EVs can be collected non-invasively from tears and saliva, only small volumes can be collected at a time, which can cause issues in obtaining enough EVs to analyze proteins. The scanner discussed in this paper is a nanoparticle analyzer that provides a solution to&#160;this problem, allowing us to characterize and study the phenotype, size, and total particle count of EVs from as little as 1 &#181;L of biological fluid. This protocol will expand the knowledge of EVs from small volumes of samples that are difficult to extract from patients. This could enhance patient comfort and potentially identify new therapeutic targets for a range of diseases and disorders.

Here, we demonstrate the characterization method for extracellular vesicles (EVs) collected from biological fluids, such as tears and saliva, of human subjects. The scanner used in this method is capable of detecting the phenotype, size, and total particle count of EVs from 1 &#181;L of the sample.

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from ...

SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion

Source: Lemaire, Q. et al. <a href="https://www.jove.com/v/60118/characterization-immune-cell-derived-extracellular-vesicles-studying">Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the SDS-PAGE in-gel digestion method for protein extraction from extracellular vesicles or EVs. Once isolated, the obtained protein fragments can be used for proteomic analysis.

Ekstrakcja białek związanych z pęcherzykami zewnątrzkomórkowymi oparta na SDS-PAGE: procedura ekstrakcji białek z EV i przygotowania ich do trawienia w żelu

Source: Lemaire, Q. et al. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

Source: Hurwitz, S. N. et al. <a href="https://www.jove.com/v/59143/extraction-of-extracellular-vesicles-from-whole-tissue">Extraction of Extracellular Vesicles from Whole Tissue.</a> J. Vis. Exp. (2019)
This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

Ekstrakcja pęcherzyków zewnątrzkomórkowych: technika izolowania pęcherzyków zewnątrzkomórkowych z próbek całych tkanek za pomocą wirowania różnicowego

Source: Hurwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video describes the technique of extracting ...