The work is supported by the NHLBI/NIH grants HL103868 (to P.C.) and HL137076 (to P.C.), the American Heart Association Grant-in-Aid (to P.C.), and the Samuel Oschin Comprehensive Cancer Institute (SOCCI) Lung Cancer Research Award (to P.C.).&#160;We would like to express our great appreciation to the Smidt Heart Institute at Cedars-Sinai Medical Center that provides us a Nanosight machine for EV nanoparticle tracking analysis.

The utilization of animals and all animal procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at Cedars-Sinai Medical Center (CSMC).
1. Murine Bronchoalveolar Lavage Fluid (BALF) Collection and Preparation
<ol>
	<li>BALF collection
	<ol>
		<li>Euthanize mice with a cocktail of ketamine (300 mg/kg) and xylazine (30 mg/kg) via the intraperitoneal route followed by cervical dislocation.</li>
		<li>Insert a 22 G angiocatheter into the trachea. Attach an insulin syringe containing 1 mL (mL) of ice-cold sterile Dulbecco&#8217;s phosphate buffer saline (DPBS) and instill 1 mL of DBPS into both lungs through the angiocatheter.</li>
		<li>Slowly withdraw the syringe plunger to retrieve BALF and dispense the BALF into a 50-mL conical tube. Keep the BALF on ice.</li>
		<li>Repeat steps 1.1.2 and 1.1.3 3x (4x in total in each mouse). 
		Note: Approximately 0.8 mL is generally retrieved per milliliter of instillation. Also, the following steps can be performed for individual mice (i.e., 3 mL of BALF), but pooling multiple BALF samples will allow the isolation of a larger batch of EVs for consistency in downstream experiments.</li>
	</ol>
	</li>
	<li>BALF preparation
	<ol>
		<li>Pool BALF from 25 mice and divide it into two equal sets (~35 mL per aliquot).</li>
		<li>Centrifuge the BALF at 400 x g, at 4 &#176;C for 5 min, to remove cells and other cellular debris and collect the supernatant.</li>
		<li>Centrifuge the supernatant at 1,500 x g, at 4 &#176;C for 10 min, to remove cell debris. Collect the supernatant and proceed to the EV isolation steps.</li>
	</ol>
	</li>
</ol>
2. Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid
NOTE: In this study, two EV isolation techniques, namely UFC and ultracentrifugation with buoyant used to isolate EVs from BALF. The detailed protocol of each method is described below.
<ol>
	<li>Ultrafiltration centrifugation (UFC) enrichment method 
	NOTE: This method was modified from a previously described protocol10.
	<ol>
		<li>Filter the supernatant from step 1.2.3 through a 0.2 &#956;m sterile syringe filter and keep the filtered BALF on ice. 
		NOTE: This is a size exclusion step whereby only vesicles smaller than 200 nm are collected.</li>
		<li>Equilibrate the 100 kDa MWCO centrifugal filter unit with sterile DPBS for 10 min. Centrifuge the centrifugal unit at 1,500 x g for 10 min at 4 &#176;C to discard the DPBS. 
		CAUTION: Once the membrane in the filter device is equilibrated with DPBS, the membrane must be kept wet at all time until the device is used.</li>
		<li>Fill the filter unit with 15 mL of BALF sample from step 2.1.1 and centrifuge at 3,000 x g for 30 min at 4 &#176;C. The flow-through BALF can be discarded or collected into a separate canonical tube and stored at -80 &#176;C for future use.</li>
		<li>Repeat step 2.1.3 for the remaining 0.2 &#181;m-filtered BALF. 
		Note: It took three repetitions of centrifugations to sufficiently concentrate the BALF EVs from the original starting volume of 35 mL. This resulted in 1&#8211;1.5 mL of retentate.</li>
		<li>Wash the retentate with 14 mL of sterile DPBS by a gently pipetting repetitively. Centrifuge the filter unit at 3,000 x g, at 4 &#176;C for 30 min, to remove the DPBS and to concentrate the EV retentate.</li>
		<li>Collect the concentrated BALF-derived EVs from the filter device by inserting a pipettor into the bottom of the filter device and withdrawing the sample using a side-to-side sweeping motion to ensure total recovery.</li>
		<li>Aliquot the BALF-derived EVs and store them at -80 &#176;C for further particle quantification and characterization (see step 3).</li>
	</ol>
	</li>
	<li>Ultracentrifugation (UC) with buoyant density gradient centrifugation (DGC) 
	NOTE: The following protocol was modified from the previously described protocol24.
	<ol>
		<li>Transfer the supernatant from step 2.1.1 into a 37-mL ultracentrifuge tube and centrifuge the sample at 10,000 x g for 30 min at 4 &#176;C using ultracentrifuge. Collect the supernatant and centrifuge at 100,000 x g, at 4 &#176;C for 60 min. While the EV pellets are centrifuged, prepare different concentrations of buoyant density gradient buffers (Table 1) for step 2.2.3.</li>
		<li>Discard the supernatant and resuspend the EV pellets in 200 &#956;L of DPBS.</li>
		<li>Mix the EV suspension with 300 &#956;L of 50% iodixanol working solution (Table 1) and transfer it to the 15 mL ultracentrifuge tube. On top of the 50% iodixanol-EV mixture suspension, sequentially layer the following buffer solution in the order from the bottom to the top: 30% iodixanol (4.5 mL), 25% of iodixanol (3 mL), 15% iodixanol (2.5 mL), and 5% iodixanol (6 mL). Centrifuge at 100,000 x g, at 4 &#176;C for 230 min. 
		NOTE: The buoyant density gradient is based on the percent of iodixanol scaling with the highest concentration (50%) at the bottom to the lowest concentration (5%) at the top. To generate different concentrations of iodixanol, various amounts of homogenization medium (Table 1) were mixed with working solution (50% iodixanol).</li>
		<li>Collect the 15% and 25% fraction and dilute them in sterile DPBS to bring up the volume to 15 mL. Transfer them to a new small ultracentrifuge tube and centrifuge at 100,000 x g, at 4 &#176;C for 60 min.</li>
		<li>Discard the supernatant and resuspend the EV pellets in 50 &#956;L of sterile DPBS for further characterization.</li>
	</ol>
	</li>
</ol>
3. Extracellular Vesicle Quantification
NOTE: The BALF-derived EVs recovery yield is quantitated with two metrics.
<ol>
	<li>Nanoparticle tracking analysis (NTA) measurement
	<ol>
		<li>Dilute the EV sample at 1:200&#8211;1:500 in 1 mL of DPBS and load the sample into an insulin syringe. Attach a sample syringe to a syringe pump and begin to measure the particle numbers and size (see Table of Materials).</li>
		<li>Set the camera level at 14 and the detection threshold at 1 for all sample measurements. Five repetitive measurements with 1,500 frames in 30 s were recorded for each sample, with a delay of 20 s between reads. Combine and average the data for final concentration and size reports. 
		NOTE: For accurate capturing of all particles, adjust the camera level as appropriate to visualize all particles and use similar settings for all sample measurements in each experiment.</li>
	</ol>
	</li>
	<li>Protein quantification 
	NOTE: The bicinchoninic acid (BCA) protein assay was used to measure the protein concentration of the BALF-derived EVs.
	<ol>
		<li>Solubilize the EV samples in 1x lysis buffer.</li>
		<li>Quantify the amount of protein in the BALF-derived EVs per BCA standard protocol using colorimetric detection by measuring the absorbance at 560 nm in a plate reader.</li>
	</ol>
	</li>
</ol>
4. Detection of BALF-derived Extracellular Vesicles
NOTE: Commonly known exosome surface marker proteins (TSG101, CD63, CD81, and CD9) were used to verify EV recoveries by SDS-PAGE and immunoblotting and flow cytometry analysis.
<ol>
	<li>SDS-PAGE and immunoblotting
	<ol>
		<li>Dissolve an equal amount of EV proteins of each sample with a blotting loading buffer (lithium dodecyl sulfate) and 50 mM dithiothreitol (DTT) in a 1.6 mL tube. Heat the samples at 70 &#176;C for 10 min.</li>
		<li>Load the samples from step 4.1.1 into a 4&#8211;12% Bis-Tris Plus acrylamide gel and run electrophoresis (150 volts, 35 mA) for 35 min.</li>
		<li>Transfer proteins to a nitrocellulose membrane using a dry transfer method.</li>
		<li>Block the membrane with 5% skimmed milk for 60 min, rocking at room temperature (RT).</li>
		<li>Incubate the membrane with an antibody to an EV surface protein marker, Tumor Susceptibility Gene 101 (TSG101), at 1:500 dilution in 5% BSA in Tris-buffered saline Tween-20 (TBST) at 4 &#176;C, rocking overnight.</li>
		<li>The next day, wash the membrane 3x, 10 min each wash, in TBST buffer, and incubate it with anti-rabbit IgG, an HRP-linked antibody, at 1:5,000 dilution for 60 min at RT.</li>
		<li>Wash the membrane 3x, 10 min each wash, in TBST buffer. Develop the membrane with chemiluminescent HRP antibody detection reagent and image (see Table of Materials for imaging system).</li>
	</ol>
	</li>
	<li>Flow cytometry
	<ol>
		<li>Dilute BALF-derived EVs in 49 &#956;L of PEB staining buffer (PBS + 5 mM EDTA + 0.5% BSA, filtered through a 0.1 &#956;m syringe filter membrane).</li>
		<li>Add each of the following antibodies into each individual sample: 1) rat anti-mouse PE CD63 antibody (100 ng per reaction); 2) rat anti-mouse PE CD81 (500 ng); 3) rat anti-mouse FITC CD9 (200 ng). Incubate at 4 &#176;C, rocking for 60 min in the dark.</li>
		<li>Dilute the samples with 450 &#956;L of membrane-filtered PEB staining buffer and subject the samples to flow cytometry analysis (see Table of Materials)25.</li>
		<li>Adjust the flow cytometer settings as follows: 1) set all channels on hyper log (hlog); 2) set the trigger on SSC at 4; 3) turn off the secondary trigger. Run the samples in a low-speed setting and acquire at least 10,000 events in each sample.</li>
		<li>Perform low cytometry data analysis in each sample using analysis software (see Table of Materials).</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein,+microRNA,+and+mRNA+yields+using+different+methods+of+urinary+exosome+isolation+for+the+discovery+of+kidney+disease+biomarkers.">Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.</a> Kidney International. 82 (9), 1024-1032 (2012).</li><li>Bosch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+prevents+aggregation+of+exosomes+and+cryodamage.">Trehalose prevents aggregation of exosomes and cryodamage.</a> Scientific Reports. 6 (1), 329 (2016).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+progenitor+cell-derived+exosomes+prevent+cardiomyocytes+apoptosis+through+exosomal+miR-21+by+targeting+PDCD4.">Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4.</a> Cell Death &amp; Disease. 7 (6), e2277 (2016).</li><li>Agarwal, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental,+Systems+and+Computational+Approaches+to+Understanding+the+MicroRNA-Mediated+Reparative+Potential+of+Cardiac+Progenitor+Cell-Derived+Exosomes+From+Pediatric+Patients.">Experimental, Systems and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.</a> Circulation Research. 120 (4), 701-712 (2017).</li><li>Merchant, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfiltration+isolation+of+human+urinary+exosomes+for+characterization+by+MS.">Microfiltration isolation of human urinary exosomes for characterization by MS.</a> PROTEOMICS - Clinical Applications. 4 (1), 84-96 (2010).</li><li>Gouin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+method+for+identification+of+suitable+reference+genes+in+extracellular+vesicles.">A comprehensive method for identification of suitable reference genes in extracellular vesicles.</a> Journal of Extracellular Vesicles. 6 (1), 1347019 (2017).</li><li>Betsuyaku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Granule+Proteins+in+Bronchoalveolar+Lavage+Fluid+from+Subjects+with+Subclinical+Emphysema.">Neutrophil Granule Proteins in Bronchoalveolar Lavage Fluid from Subjects with Subclinical Emphysema.</a> American Journal of Respiratory and Critical Care Medicine. 159 (6), 1985-1991 (1999).</li></ol>

The authors have nothing to disclose.

In the past few decades, scientists have unraveled the significances of EVs in cellular homeostasis. More importantly, EVs play major roles in many disease processes by modulating neighboring and distant cells through their bioactive cargo molecules1,21,22,26,27,28,29,<sup class="...

Exosomes are the smallest subset of EVs, 50&#8211;200 nm in diameter, and have multiple biological functions across a diverse array of signaling processes1,2,3,4,5. They govern cellular and tissue homeostasis primarily by facilitating intercellular communication through cargo molecules such as lipids, proteins, and nucleic acids6,7,8,9. One critical step in EV research is the isolation process. Differential ultracentrifugation (UC), with or without density gradient centrifugation (DGC), is considered the gold standard approach, but this method carries major limitations, including inefficient EV recovery rates and low scalability10,11,12, that restrict its best utilization to larger volume samples, such as cell culture supernatant or high exosome production specimens. The advantages and disadvantages of other methods, such as size exclusion by ultrafiltration or chromatography, immunoaffinity isolation by beads or columns, and microfluidics, are well described, and modern supplemental procedures have been developed to overcome and minimize technical limitations in each approach11,12,13,14,15. Others have shown that an ultrafiltration centrifugation (UFC) with a nanoporous membrane in the filter unit is an alternative technique that provides comparable purity to a UC method16,17,18. This technique could be considered as one of the alternative isolation methods.
Bronchoalveolar lavage fluid (BALF) contains EVs that possess numerous biological functions in various respiratory conditions19,20,21,22. Studying BALF-derived EVs entails some challenges due to the invasiveness of the bronchoscopy procedure in humans, as well as a limited amount of lavage fluid recovery. In small laboratory animals such as mice, only a few milliliters can be recovered in normal lung conditions, even less in inflamed or fibrotic lungs23. Consequently, collecting a sufficient amount of BALF for EV isolation by a differential ultracentrifugation for downstream applications may not be feasible. However, isolating correct EV populations is a crucial factor for studying EV biological functions. The delicate balance between efficiency and efficacy continues to be a challenge in well-established EV isolation methods.
In this current study, we demonstrate that a centrifugal ultrafiltration approach, utilizing a 100 kDa molecular weight cut-off (MWCO) nanomembrane filter unit, is suitable for small-volume biological specimen such as BALF. This technique is simple, efficient, and provides high purity and scalability to support the study of BALF-derived EVs.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra-15 centrifugal filters Ultracel-100K</td><td>Sigma-Millipore, St. Louis, MO</td><td>UFC910024</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Corning Cellgro, Manassas, VA</td><td>21-031-CV</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Millipore, St. Louis, MO</td><td>EMD8550</td><td></td></tr><tr><td>HEPES</td><td>Research Products International, Prospect, IL</td><td>75277-39-3</td><td></td></tr><tr><td>EDTA</td><td>Corning Cellgro, Manassas, VA</td><td>46-034-CI</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Millipore, St. Louis, MO</td><td>S3014-1KG</td><td></td></tr><tr><td>OptiPrep</td><td>Sigma-Millipore, St. Louis, MO</td><td>MKCD9753</td><td>Density Gradient Medium</td></tr><tr><td>Ketamine</td><td>VetOne, Boise, ID</td><td>13985-702-10</td><td></td></tr><tr><td>Xylazine</td><td>Akorn Animal Health, Lake Forest, IL</td><td>59399-110-20</td><td></td></tr><tr><td>Syringe 1 mL</td><td>BD Syringe, Franklin Lakes, NJ</td><td>309656</td><td></td></tr><tr><td>Angiocatheter 20 G</td><td>BD Syringe, Franklin Lakes, NJ</td><td>381703</td><td></td></tr><tr><td>Centrifuge tubes 15 mL</td><td>VWR, Radnor, PA</td><td>89039-666</td><td></td></tr><tr><td>Centrifuge tubes 50 mL</td><td>Corning Cellgro, Manassas, VA</td><td>430828</td><td></td></tr><tr><td>Bicinchonic acid (BCA) protein assay</td><td>Pierce, Thermo Fischer Scientific, Rockford, IL</td><td>23235</td><td></td></tr><tr><td>Rabbit anti-mouse TSG101 Antibody</td><td>AbCam, Cambridge, MA</td><td>AB125011</td><td></td></tr><tr><td>Rat anti-mouse PE-CD63 Antibody</td><td>Biolegend, San Diego, CA</td><td>143904</td><td></td></tr><tr><td>CD81</td><td></td><td></td><td></td></tr><tr><td>CD9</td><td></td><td></td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked antibody</td><td>Cell Signaling Technology, Danvers, MA</td><td>7074S</td><td></td></tr><tr><td>4x LDS</td><td></td><td></td><td></td></tr><tr><td>10x Reducing agent (Bolt)</td><td></td><td></td><td></td></tr><tr><td>10x Lysis buffer (Bolt)</td><td>Cell Signaling Technology, Danvers, MA</td><td></td><td></td></tr><tr><td>Bolt 4-12% Bis-Tris Plus acrylamide gel</td><td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td><td>NW04120</td><td></td></tr><tr><td>iBlot 2 Nitrocellulose mini stacks</td><td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td><td>IB23002</td><td></td></tr><tr><td>Chemiluminescent HRP antibody detection reagent HyGLO</td><td>Denville Scientific, Holliston, MA</td><td>E2400</td><td></td></tr><tr><td>Ultracentrifuge tubes 17 mL</td><td>Beckman Coulter, Pasadena, CA</td><td>337986</td><td></td></tr><tr><td>Ultracentrifuge tubes 38.5 mL</td><td>Beckman Coulter, Pasadena, CA</td><td>326823</td><td></td></tr><tr><td>Corning SFCA Syringe Filters 0.2 &amp;micro;m pore</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>09-754-13</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hamburg, Germany</td><td>-</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter, Pasadena, CA</td><td>-</td><td></td></tr><tr><td>Nanosight (NS300)</td><td>Malvern, Worcestershire, UK</td><td>-</td><td>To measure particle size distribution and particle concentration</td></tr><tr><td>MACSQuant Analyzer 10 flow cytometer</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>-</td><td></td></tr><tr><td>iBlot Transfer Apparatus</td><td>Thermo Fischer Scientific, Waltham, MA</td><td>-</td><td></td></tr><tr><td>Bio-Rad ChemiDoc MP Imaging System</td><td>Bio-Rad, Hercules, CA</td><td></td><td></td></tr><tr><td>FlowJo v. 10</td><td></td><td></td><td>Analysis software</td></tr></tbody></table>

isolation of extracellular vesicles from murine bronchoalveolar lavage fluid using an ultrafiltration centrifugation technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

We performed EV isolation from mouse BALF using UFC and UC-DGC isolation methods on the same day. The UFC method required approximately 2.5&#8211;3 h, whereas the UC-DGC technique required 8 h of processing time. This did not include buffers and reagent preparation time. It should be noted that some other tasks could be performed during the long centrifugation periods. Nevertheless, the entire procedure lasted nearly an entire day for the UC-DGC isolation technique.
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Watch this Scientific Journal Video about Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique at JoVE.com

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

isolation-extracellular-vesicles-from-murine-bronchoalveolar-lavage

Research

JoVE Journal

Immunology and Infection

9.4K Views.  Cedars-Sinai Medical Center. Deze methode kan helpen bij het aanpakken van vragen met betrekking tot de efficiëntie, herstelopbrengst en zuiverheid van bronchoalveolaire lavage vloeistof afgeleid extracellulaire vesicles met behulp van een ultrafiltratie centrifugatie benadering. We hebben deze techniek direct vergeleken met een ultracentrifugatie- en dichtheidgradiënt zuiveringstechniek. Het belangrijkste voordeel van deze techniek is dat het eenvoudig, efficiënt en geschikt is voor kleine volumemonsters zoals murine...

Video: Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

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

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 ...

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

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

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

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

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

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

Bioengineering

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

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

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

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

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

Biology

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Cardiovascular Biomarker Studies Using Plasma Extracellular Vesicle Proteome

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

Medicine

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

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.

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

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Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Source: Urwitz, 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 demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

Source: Urwitz, S. N. et al. Extraction of Extracellular Vesicles from Whole Tissue. J. Vis. Exp. (2019)
This video demonstrates the density gradient ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

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