Potential and Challenges of Specifically Isolating Extracellular Vesicles from Heterogeneous Populations

Overview

Journal Sci Rep

Specialty Science

Date 2021 Jun 3

PMID 34079007

Citations 36

Authors

Susann Allelein

Paula Medina-Perez

Ana Leonor Heitor Lopes

Sabrina Rau

Gerd Hause

Andreas Kolsch

Dirk Kuhlmeier

Affiliations

Soon will be listed here.

Abstract

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

Citing Articles

The emerging role of extracellular vesicle RNAs as mediators of cardiometabolic diseases: from pathophysiology to clinical applications.

Limpitikul W, Garcia-Contreras M, Das S Curr Opin Physiol. 2025; 40.

PMID: 40060218 PMC: 11887618. DOI: 10.1016/j.cophys.2024.100764.

Size distribution of extracellular vesicles in pretreatment ascites and plasma is correlated with primary treatment outcome in advanced high-grade serous carcinoma.

Herzog M, Verdenik I, Kobal B, cerne K Sci Rep. 2025; 15(1):4500.

PMID: 39915670 PMC: 11802836. DOI: 10.1038/s41598-025-88707-9.

The Role of Exosomes in Cancer Progression and Therapy.

Sergazy S, Seydahmetova R, Gulyayev A, Shulgau Z, Aljofan M Biology (Basel). 2025; 14(1).

PMID: 39857258 PMC: 11763171. DOI: 10.3390/biology14010027.

Filter-Aided Extracellular Vesicle Enrichment (FAEVEr) for Proteomics.

Pauwels J, Van de Steene T, Van de Velde J, De Muyer F, De Pauw D, Baeke F Mol Cell Proteomics. 2025; 24(2):100907.

PMID: 39842778 PMC: 11872570. DOI: 10.1016/j.mcpro.2025.100907.

A comparative analysis of small extracellular vesicle (sEV) micro-RNA (miRNA) isolation and sequencing procedures in blood plasma samples.

Abeysinghe P, Turner N, Mitchell M Extracell Vesicles Circ Nucl Acids. 2024; 5(1):119-137.

PMID: 39698410 PMC: 11648519. DOI: 10.20517/evcna.2023.55.

References

Skog J, Wurdinger T, van Rijn S, Meijer D, Gainche L, Sena-Esteves M . Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008; 10(12):1470-6. PMC: 3423894. DOI: 10.1038/ncb1800. View

Minciacchi V, Freeman M, Di Vizio D . Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. Semin Cell Dev Biol. 2015; 40:41-51. PMC: 4747631. DOI: 10.1016/j.semcdb.2015.02.010. View

Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D . Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med. 1998; 4(5):594-600. DOI: 10.1038/nm0598-594. View

Colombo M, Raposo G, Thery C . Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014; 30:255-89. DOI: 10.1146/annurev-cellbio-101512-122326. View

van Niel G, DAngelo G, Raposo G . Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018; 19(4):213-228. DOI: 10.1038/nrm.2017.125. View

Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee J, Lotvall J . Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007; 9(6):654-9. DOI: 10.1038/ncb1596. View

Yanez-Mo M, Siljander P, Andreu Z, Bedina Zavec A, Borras F, Buzas E . Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015; 4:27066. PMC: 4433489. DOI: 10.3402/jev.v4.27066. View

Heidrich I, Ackar L, Mohammadi P, Pantel K . Liquid biopsies: Potential and challenges. Int J Cancer. 2020; 148(3):528-545. DOI: 10.1002/ijc.33217. View

Minciacchi V, Zijlstra A, Rubin M, Di Vizio D . Extracellular vesicles for liquid biopsy in prostate cancer: where are we and where are we headed?. Prostate Cancer Prostatic Dis. 2017; 20(3):251-258. PMC: 5569339. DOI: 10.1038/pcan.2017.7. View

10.

Quandt D, Dieter Zucht H, Amann A, Wulf-Goldenberg A, Borrebaeck C, Cannarile M . Implementing liquid biopsies into clinical decision making for cancer immunotherapy. Oncotarget. 2017; 8(29):48507-48520. PMC: 5564665. DOI: 10.18632/oncotarget.17397. View

11.

Thery C, Witwer K, Aikawa E, Alcaraz M, Anderson J, Andriantsitohaina R . Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2019; 7(1):1535750. PMC: 6322352. DOI: 10.1080/20013078.2018.1535750. View

12.

Harding C, Heuser J, Stahl P . Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol. 1984; 35(2):256-63. View

13.

Pan B, Teng K, Wu C, Adam M, Johnstone R . Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985; 101(3):942-8. PMC: 2113705. DOI: 10.1083/jcb.101.3.942. View

14.

Zhang H, Freitas D, Kim H, Fabijanic K, Li Z, Chen H . Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018; 20(3):332-343. PMC: 5931706. DOI: 10.1038/s41556-018-0040-4. View

15.

Akers J, Gonda D, Kim R, Carter B, Chen C . Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol. 2013; 113(1):1-11. PMC: 5533094. DOI: 10.1007/s11060-013-1084-8. View

16.

Tricarico C, Clancy J, DSouza-Schorey C . Biology and biogenesis of shed microvesicles. Small GTPases. 2016; 8(4):220-232. PMC: 5680703. DOI: 10.1080/21541248.2016.1215283. View

17.

Willms E, Johansson H, Mager I, Lee Y, Blomberg K, Sadik M . Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep. 2016; 6:22519. PMC: 4773763. DOI: 10.1038/srep22519. View

18.

Zabeo D, Cvjetkovic A, Lasser C, Schorb M, Lotvall J, Hoog J . Exosomes purified from a single cell type have diverse morphology. J Extracell Vesicles. 2017; 6(1):1329476. PMC: 5505001. DOI: 10.1080/20013078.2017.1329476. View

19.

Konoshenko M, Lekchnov E, Vlassov A, Laktionov P . Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. Biomed Res Int. 2018; 2018:8545347. PMC: 5831698. DOI: 10.1155/2018/8545347. View

20.

Tauro B, Greening D, Mathias R, Ji H, Mathivanan S, Scott A . Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 2012; 56(2):293-304. DOI: 10.1016/j.ymeth.2012.01.002. View