Name: extraction of extracellular vesicles from whole tissue
Uploaded: 2019-02-07T14:30:00
Description: Watch this Scientific Journal Video about Extraction of Extracellular Vesicles from Whole Tissue at JoVE.com

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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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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			<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>
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			<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 ...

Research

JoVE Journal

Cancer Research

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 ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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 ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

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 ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

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

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue (Video) | JoVE 

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