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.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.45 &#181;m filter</td>
			<td style="width:183px;">VWR</td>
			<td style="width:176px;">28145-505</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">12 mL ultracentrifuge tubes</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">5.5 mL ultracentrifuge tubes</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">344057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-Alix antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-7129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-Calnexin antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-11397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-CD63 antibody</td>
			<td style="width:183px;">Abcam</td>
			<td style="width:176px;">ab59479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-CD81 antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-Flotillin 2 antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-25507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-HSC70 antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-7298</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-Syntenin-1 antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-100336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">anti-TSG101 antibody</td>
			<td style="width:183px;">Santa Cruz</td>
			<td style="width:176px;">sc-7964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Dounce homogenizer</td>
			<td style="width:183px;">DWK Life Sciences</td>
			<td style="width:176px;">885300-0015</td>
			<td>Loose-fit pestle (clearance of 0.889&#8211;0.165&#8239;mm) used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">EZQ protein quantification kit</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:176px;">R33200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">FA-45-6-30 rotor&#160;</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:176px;">5820715006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">FEI CM120 Electron Microscope</td>
			<td style="width:183px;">TSS Microscopy</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">goat anti-rabbit IgG (Fab fragment)</td>
			<td style="width:183px;">Genetex</td>
			<td style="width:176px;">27171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">HALT phosphatase inhibitor (100x solution)</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:176px;">78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">HALT protease inhibitor (100x solution)</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:176px;">78438</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Hibernate E medium</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:176px;">A1247601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MLS-50 swinging-bucket rotor</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">367280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NanoSight LM10</td>
			<td style="width:183px;">Malvern</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima MAX-XP Benchtop Ultracentrifuge&#160;</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">393315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Optima XE-100 ultracentrifuge</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">A94516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Optiprep</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:176px;">D1556</td>
			<td>60% iodixanol in sterile water solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Q Exactive HF Mass Spectrometer</td>
			<td style="width:183px;">ThermoFisher Scientific</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit anti-goat IgG</td>
			<td style="width:183px;">Genetex</td>
			<td style="width:176px;">26741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit anti-mouse IgG</td>
			<td style="width:183px;">Genetex</td>
			<td style="width:176px;">26728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Refracto 30PX (refractometer)</td>
			<td style="width:183px;">Mettler Toledo</td>
			<td style="width:176px;">51324650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">S-4-104 rotor</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:176px;">5820759003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SW 41 Ti swinging-bucket Rotor</td>
			<td style="width:183px;">Beckman Coulter</td>
			<td style="width:176px;">333790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Tabletop 5804R centrifuge</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:176px;">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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14.7K 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

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.

This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

We present a protocol for the isolation and freeze-fracturing of extracellular vesicles (EVs) originating from cancerous urothelial cells. The freeze-fracture technique revealed the EVs' diameter and shape and-as a unique feature-the internal organization of the EV membranes. These are of immense importance in understanding how EVs interact with the recipient membranes.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular ...

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 ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...