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Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

Guerreiro, Eduarda M ; Vestad, Beate ; Steffensen, Lilly Alice ; Aass, Hans Christian D ; Saeed, Muhammad ; Øvstebø, Reidun ; Costea, Daniela Elena ; Galtung, Hilde Kanli ; Søland, Tine M Ahmad, Aamir

PloS one, 2018-09, Vol.13 (9), p.e0204276-e0204276 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography
Autor: Guerreiro, Eduarda M ; Vestad, Beate ; Steffensen, Lilly Alice ; Aass, Hans Christian D ; Saeed, Muhammad ; Øvstebø, Reidun ; Costea, Daniela Elena ; Galtung, Hilde Kanli ; Søland, Tine M
Ahmad, Aamir
Assuntos: Adenocarcinoma ; Biochemistry ; Biology ; Biology and Life Sciences ; Biomarkers ; Biotechnology ; Blood ; Brain ; Cancer ; CD9 antigen ; Cell culture ; Cell growth ; Cell proliferation ; Cellular stress response ; Centrifuges ; Chromatography ; Clinical medicine ; Contamination ; Culture media ; Dentistry ; Diagnosis ; Electron microscopy ; Engineering and Technology ; Exosomes ; Extracellular vesicles ; Flow cytometry ; Hospitals ; Laboratories ; Melanoma ; Metastases ; Metastasis ; Methods ; Molecular weight ; Nanoparticles ; Oral squamous cell carcinoma ; Pancreas ; Pancreatic cancer ; Pancreatic carcinoma ; Pathology ; Physiology ; Production methods ; Proteins ; Proteomics ; Reagents ; Reproducibility ; Research and Analysis Methods ; Size exclusion chromatography ; Squamous cell carcinoma ; Transmission electron microscopes ; Transmission electron microscopy ; Ultrafiltration ; Vesicles
É parte de: PloS one, 2018-09, Vol.13 (9), p.e0204276-e0204276
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Current address: Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway
Current address: Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Competing Interests: The authors have declared that no competing interests exist.
Descrição: Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60-140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.
Editor: United States: Public Library of Science
Idioma: Inglês;Norueguês

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Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

Guerreiro, Eduarda M ; Vestad, Beate ; Steffensen, Lilly Alice ; Aass, Hans Christian D ; Saeed, Muhammad ; Øvstebø, Reidun ; Costea, Daniela Elena ; Galtung, Hilde Kanli ; Søland, Tine M Ahmad, Aamir

United States: Public Library of Science

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