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.
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Method Article
Extraction of Extracellular Vesicles from Whole Tissue
In This Article
Summary
Abstract
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.
Introduction
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,
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Protocol
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 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.
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Results
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.......
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Discussion
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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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&M University) for donation of the tumor specimens used in the development of this method. This study was su....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.45 µm filter | VWR | 28145-505 | |
| 12 mL ultracentrifuge tubes | Beckman Coulter | 331372 | |
| 5.5 mL ultracentrifuge tubes | Beckman Coulter | 344057 | |
| anti-Alix antibody | Santa Cruz | sc-7129 | |
| anti-Calnexin antibody | Santa Cruz | sc-11397 | |
| anti-CD63 antibody | Abcam | ab59479 | |
| anti-CD81 antibody | Santa Cruz | sc-9158 | |
| anti-Flotillin 2 antibody | Santa Cruz | sc-25507 | |
| anti-HSC70 antibody | Santa Cruz | sc-7298 | |
| anti-Syntenin-1 antibody | Santa Cruz | sc-100336 | |
| anti-TSG101 antibody | Santa Cruz | sc-7964 | |
| EZQ protein quantification kit | ThermoFisher Scientific | R33200 | |
| FA-45-6-30 rotor | Eppendorf | 5820715006 | |
| FEI CM120 Electron Microscope | TSS Microscopy | ||
| goat anti-rabbit IgG (Fab fragment) | Genetex | 27171 | |
| HALT phosphatase inhibitor (100x solution) | ThermoFisher Scientific | 78420 | |
| HALT protease inhibitor (100x solution) | ThermoFisher Scientific | 78438 | |
| Hibernate E medium | ThermoFisher Scientific | A1247601 | |
| MLS-50 swinging-bucket rotor | Beckman Coulter | 367280 | |
| NanoSight LM10 | Malvern | ||
| Optima MAX-XP Benchtop Ultracentrifuge | Beckman Coulter | 393315 | |
| Optima XE-100 ultracentrifuge | Beckman Coulter | A94516 | |
| Optiprep | Sigma | D1556 | 60% iodixanol in sterile water solution |
| Q Exactive HF Mass Spectrometer | ThermoFisher Scientific | ||
| rabbit anti-goat IgG | Genetex | 26741 | |
| rabbit anti-mouse IgG | Genetex | 26728 | |
| Refracto 30PX (refractometer) | Mettler Toledo | 51324650 | |
| S-4-104 rotor | Eppendorf | 5820759003 | |
| SW 41 Ti swinging-bucket Rotor | Beckman Coulter | 333790 | |
| Tabletop 5804R centrifuge | Eppendorf | 22623508 |
References
- Bobrie, A., Colombo, M., Raposo, G., Théry, C. Exosome secretion: molecular mechanisms and roles in immune responses. Traffic. 12 (12), 1659-1668 (2011).
- Théry, C., Zitvogel, L., Amigorena, S. Exosomes: composition, biogenesis and function. Nature Reviews Immunology.
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