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 adenocar...

<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 border="0" cellpadding="0" cellspacing="0" style="width:1017px;" width="1017">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</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 ...

This protocol provides a rigorous and reproducible technique for extraction and isolation of small extracellular vesicles from whole tissue specimens for downstream ex-vivo analyses. With amongst the highest levels of purity currently achievable, this method facilitates the direct morphologic, immunophenotypic, and deep characterization of interstitial vesicles secreted into various tissue specimens, including tumors. Here, we demonstrate the isolation of tissue EVs from brain and lung tumor specimens.However, this technique can be applied to studies using diverse, benign, or other tumor specimens as well. To begin, prepare 10 milliliters of dissociation buffer in Hibernate-E medium for every 0.4 to 1.0 gram of tissue. Add whole fresh or frozen tissue to the buffer in a 50 milliliter tube, and incubate in a warm water bath at 37 degrees celsius for 20 minutes.After this, add protease and phosphatase inhibitors for a final 1x concentration in the dissociation buffer. Pour the solution with the tissue into a loose fit downs homogenizer. Use approximately 30 slow strokes per sample to gently dissociate the tissue.Then, transfer the dissociated tissue and buffer solution to a 50 milliliter conical tube. Centrifuge at 500x g and at four degrees celsius for five minutes to pellet the cells and the remaining fibers or cohesive tissue fragments. Transfer the supernatant to a clean 50 milliliter conical tube, and centrifuge at 2, 000x g and at four degrees celsius for 10 minutes to pellet and discard the large cellular debris.Transfer this supernatant to a clean 50 milliliter conical tube, and centrifuge at 10, 000x g and at four degrees celsius for 40 minutes to pellet any undesired larger vesicles or small apoptotic bodies. Decant the supernatant through a 0.45 micrometer filter into a clean 12 milliliter ultracentrifugation tube. Next, ultracentrifuge the sample at 100, 000x g and at four degrees celsius for two hours, to pellet small EVs.Decant the supernatant and leave the ultracentrifugation tubes inverted for five to 10 minutes, tapping frequently to remove any residual liquid on the sides of the tubes. Then, resuspend the EV pellet in 1.5 milliliters of 0.25 molar sucrose buffer. Cover the tubes with Parafilm, and then vortex the EVs into solution.Rock the ultracentrifuge tubes for 10 to 15 minutes at room temperature. And then vortex once more. Briefly centrifuge the tubes at a speed less than 1, 000x g to recover the liquid suspension at the bottom of the tube.If needed, store the suspension at four degrees celsius overnight. First, add 1.5 milliliters of 60%iodixanol to the 1.5 milliliters of the sucrose tris buffer that contains the EVs to create a final solution containing 30%iodixanol. Pipette up and down several times to mix the solution thoroughly.Transfer this solution to the bottom of a 5.5 milliliter ultracentrifugation tube. Next, mix the 60%iodixanol stock with ultra pure water to prepare at least 1.5 milliliters of both a 20%and a 10%iodixanol solution. Using a syringe and an 18 gauge needle, measure 1.3 milliliters of the 20%iodixanol solution and carefully layer it on top of the bottom gradient.Keep the bevel of the needle in contact with the inside of the tube, just above the meniscus and add the solution drop wise to avoid mixing the layers at the density interface. Then, layer 1.2 milliliters of the 10%iodixanol solution on top of the 20%layer, using the same technique. Carefully balance and load the ultracentrifugation tubes into rotor buckets.Set the acceleration and deceleration speeds of a swing bucket rotor to the minimum rates, and centrifuge at 268, 000x g and at four degrees celsius for 50 minutes. While the sample is being centrifuged, label 10 1.5 milliliter microcentrifuge tubes for each sample that will correspond with fractions one through 10 of the density gradient. Once the centrifugation is complete, gently remove the tubes from the rotor buckets and place them into a stable holder.Pipette 10 serial fractions of 490 microliters from the top of the gradient into the corresponding tubes. Using a refractometer, measure the refractive indices of the fractions. Then, transfer each fraction to a clean 12 milliliter ultracentrifugation tube.Add five milliliters of 1x PBS to each tube and pipette up and down slowly to mix. Add an additional six milliliters of 1x PBS to the top of the tube, and carefully mix again. Ultracentrifuge the tubes at 100, 000x g and at four degrees celsius to re-pellet the small vesicles.Decant the supernatant and tap the tubes dry before lysing the vesicles for protein analysis or reresuspending the EVs for morphologic analysis. To lyse the EVs for protein analysis, add 40 microliters of strong lysis buffer containing protease inhibitor to the EV pellets. Place Parafilm over each tube, and vortex vigorously.Next, rock the tubes for 20 minutes at room temperature and vortex again. Briefly centrifuge the sample at 1, 000x g for 30 to 60 seconds to recover the entire sample volume. Transfer each sample to a new 1.5 milliliter microcentrifuge tube, and store it at a temperature between 20 and 80 degrees celsius until ready for further processing.To prepare the purified lysates for immunoblot analysis, add 5x Laemmli sample buffer to the samples for a final concentration of 1x. Boil the sample at 95 degrees celsius for five to 10 minutes. Then, load an equal volume of fractions one through 10 into a 10%SDS page gel.Load an equal mass of tissue homogenate. Perform electrophoresis and Western blot analysis to confirm EV proteins in the purified lysates and compare relative EV abundance in fractions. In this study, extracellular vesicles are extracted and purified from whole tissue.Following ultracentrifugation of the 10 to 30%iodixanol gradient, a population of light EVs can be seen migrating up to fraction two, while a population of dense EVs can be seen migrating up to fraction five, depending on tissue type. Representative immunoblots of the gradient fractions exhibit the efficient separation and purification of small tumor derived EVs in fraction five. Notably, lung tumor specimens appear enriched in dense EVs compared to light EVs that were previously harvested from whole brain tissue.Representative nano particle tracking analysis and electron microscopy of tissue-derived vesicles in the predominant vesicle containing fraction demonstrate the enrichment and preservation of whole vesicles. Consistent with the known size and structure of small EVs. Interstitial EVs represent promising targets for the development of novel diagnostic or prognostic biomarker assays.This technique provides researchers with tools for the extraction and purification of vesicles for downstream utility. Following extraction of whole or lysed tissue EVs, broad characterization of vesicles including proteomic, genomic, and lipidomic analyses may be performed. Additionally, samples may be used for more targeted approaches.Vesicles isolated directly from tissue specimens can provide further insight into disease mechanisms, including tumor genesis, and may provide important diagnostic or prognostic tools in the future.

extraction-of-extracellular-vesicles-from-whole-tissue

Research

JoVE Journal

Cancer Research

14.6K Views.  Florida State University College of Medicine. This protocol provides a rigorous and reproducible technique for extraction and isolation of small extracellular vesicles from whole tissue specimens for downstream ex-vivo analyses. With amongst the highest levels of purity currently achievable, this method facilitates the direct morphologic, immunophenotypic, and deep characterization of interstitial vesicles secreted into various tissue specimens, including tumors. Here, we demonstrate the isolation of tissue EVs from brain and lung tumor ...