Advances in extracellular vesicle isolation methods: a path towards cell-type specific EV isolation

Abstract

Extracellular vesicles are small, heterogenous, phospholipid-rich vesicles that are secreted by all cells into the extracellular space. They play a key role in intercellular communication because they can transport a variety of biomolecules such as proteins, lipids, and nucleic acids between cells. As categorized by the International Society of Extracellular Vesicles (ISEV), the term EV encompasses different sub-types, including exosomes, microvesicles, and apoptotic bodies, which differ in their size, origin, and cargo. EVs can be isolated from biological fluids such as blood, urine, and cerebrospinal fluid, and their biomolecular content can be analyzed to monitor the progression of certain diseases. Therefore, EVs can be used as a new source of liquid biomarkers for advancing novel diagnostic and therapeutic tools. Isolating and analyzing EVs can be challenging due to their nanoscopic size and low abundance. Several techniques have been developed for the isolation and characterization of EVs, including ultracentrifugation, density gradient separation, size-exclusion chromatography, microfluidics, and magnetic bead-based/affinity methods. This review highlights advances in EV isolation techniques in the last decade and provides a perspective on their advantages, limitations, and potential application to cell-type specific EV isolation in the future.

Keywords

Extracellular vesicles / biomarkers / diagnostics / isolation methods / cell-type specific

References

[1] Salomon C,Erdbrügger U.Extracellular vesicles and their emerging roles as cellular messengers in endocrinology: an endocrine society scientific statement.Endocr Rev2022;43:441-68

[2] Doyle L.Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis.Cells2019;8:727. PMCID:PMC6678302

[3] Shami-Shah A,Walt DR.Ultrasensitive protein detection technologies for extracellular vesicle measurements.Mol Cell Proteomics2023;22:100557. PMCID:PMC10326690

[4] Shao H,Castro CM,Weissleder R.New technologies for analysis of extracellular vesicles.Chem Rev2018;118:1917-50 PMCID:PMC6029891

[5] Wolf P.The nature and significance of platelet products in human plasma.Brit J Haemat1967;13:269-88

[6] Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles.Nat Rev Mol Cell Biol2018;19:213-28

[7] Zhang Y,Liu H.Exosomes: biogenesis, biologic function and clinical potential.Cell Biosci2019;9:19 PMCID:PMC6377728

[8] Abels ER.Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake.Cell Mol Neurobiol2016;36:301-12. PMCID:PMC5546313

[9] Gurung S,Touramanidou L.The exosome journey: from biogenesis to uptake and intracellular signalling.Cell Commun Signal2021;19:47. PMCID:PMC8063428

[10] Robbins PD.Regulation of immune responses by extracellular vesicles.Nat Rev Immunol2014;14:195-208 PMCID:PMC4350779

[11] Xu R,Chen M,Greening DW.Extracellular vesicles in cancer-implications for future improvements in cancer care.Nat Rev Clin Oncol2018;15:617-38

[12] Saeedi S,Nagy C.The emerging role of exosomes in mental disorders.Transl Psychiatry2019;9:122. PMCID:PMC6438960

[13] Thompson AG,Heman-Ackah SM.Extracellular vesicles in neurodegenerative disease-pathogenesis to biomarkers.Nat Rev Neurol2016;12:346-57.

[14] Chang W,Antonyak MA.Extracellular vesicles and their roles in cancer progression. In: Robles-flores M, editor. Cancer cell signaling. New York: Springer US; 2021. pp. 143-70. PMCID:PMC8008708

[15] Murphy DE,Brouwer M.Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking.Exp Mol Med2019;51:1-12. PMCID:PMC6418170

[16] Kodam SP.Diagnostic and therapeutic potential of extracellular vesicles.Technol Cancer Res Treat2021;20:15330338211041203 PMCID:PMC8504225

[17] Revenfeld AL,Nielsen MH,Varming K.Diagnostic and prognostic potential of extracellular vesicles in peripheral blood.Clin Ther2014;36:830-46

[18] Wiklander OPB,Lötvall J,El Andaloussi S.Advances in therapeutic applications of extracellular vesicles.Sci Transl Med2019;11:eaav8521. PMCID:PMC7104415

[19] Lötvall J,Hochberg F.Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.J Extracell Vesicles2014;3:26913. PMCID:PMC4275645

[20] Konoshenko MY,Vlassov A V., Laktionov PP.Biomed Res Int2018;2018:8545347 PMCID:PMC5831698

[21] Théry C,Aikawa E.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 Vesicles2018;7:1535750 PMCID:PMC6322352

[22] Rikkert LG,Caro J.Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.Front Oncol2020;10:608. PMCID:PMC7287034

[23] Norman M,Trieu W.L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.Nat Methods2021;18:631-4

[24] Ter-Ovanesyan D,Lazarovits R.Framework for rapid comparison of extracellular vesicle isolation methods.Elife2021;10:e70725 PMCID:PMC8651285

[25] Lee K.Loss of lipid to plastic tubing.J Lipid Res1971;12:635-6

[26] Fleury JB.Microplastics destabilize lipid membranes by mechanical stretching.Proc Natl Acad Sci U S A2021;118:e2104610118 PMCID:PMC8346836

[27] Evtushenko EG,Lazarev VN,Khomyakova E.Adsorption of extracellular vesicles onto the tube walls during storage in solution.PLoS One2020;15:e0243738 PMCID:PMC7769454

[28] Ramirez MI,Gadelha C.Technical challenges of working with extracellular vesicles.Nanoscale2018;10:881-906

[29] Auber M.An estimate of extracellular vesicle secretion rates of human blood cells.J of Extracellular Bio2022;1:e46

[30] Li Y,Li Q.EV-origin: enumerating the tissue-cellular origin of circulating extracellular vesicles using exLR profile.Comput Struct Biotec2020;18:2851-9 PMCID:PMC7588739

[31] Gudbergsson JM,Skov MN.Systematic review of factors influencing extracellular vesicle yield from cell cultures.Cytotechnology2016;68:579-92 PMCID:PMC4960200

[32] Kowal EJK,Regev A.Extracellular vesicle isolation and analysis by western blotting.Methods Mol Biol2017;1660:110-9.

[33] Momen-heravi F.Isolation of extracellular vesicles by ultracentrifugation. In: Kuo WP, Jia S, editors. Extracellular vesicles. New York: Springer; 2017. pp. 25-32.

[34] Ismail N,Dakhlallah D.Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.Blood2013;121:984-95. PMCID:PMC3567345

[35] Bryzgunova OE,Skvortsova TE.Comparative study of extracellular vesicles from the urine of healthy individuals and prostate cancer patients.PLoS One2016;11:e0157566 PMCID:PMC4909321

[36] Momen-Heravi F,Alian S.Current methods for the isolation of extracellular vesicles.Biol Chem2013;394:1253-62 PMCID:PMC7075462

[37] Taylor DD.Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.Methods2015;87:3-10

[38] Karimi N,Jang SC.Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.Cell Mol Life Sci2018;75:2873-86. PMCID:PMC6021463

[39] Onódi Z,Terézia Nagy C.Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma.Front Physiol2018;9:1479 PMCID:PMC6206048

[40] Rosado M,G .Bexiga M, G. Jones J, Manadas B, Anjo SI. Advances in biomarker detection: alternative approaches for blood-based biomarker detection. Elsevier; 2019. pp. 141-99.

[41] Araùjo M,Stasyk T.Isolation of endocitic organelles by density gradient centrifugation. in: posch a, editor. 2d page: sample preparation and fractionation. Totowa: Humana Press; 2008. pp. 317-31.

[42] Fernandez-Martinez J,Rout MP.Density gradient ultracentrifugation to isolate endogenous protein complexes after affinity capture.Cold Spring Harb Protoc2016;2016:pdb.prot087957

[43] Palma J,Pigati L.MicroRNAs are exported from malignant cells in customized particles.Nucleic Acids Res2012;40:9125-38 PMCID:PMC3467054

[44] Aalberts M,van Adrichem NP.Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans.Biol Reprod2012;86:82.

[45] Yuana Y,Grootemaat A,Nieuwland R.Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.J Extracell Vesicles2014;3:23262 PMCID:PMC4090368

[46] Hogan MC,Woollard JR.Characterization of PKD protein-positive exosome-like vesicles.J Am Soc Nephrol2009;20:278-88 PMCID:PMC2637052

[47] Sidhom K,Saleem A.A review of exosomal isolation methods: is size exclusion chromatography the best option?.Int J Mol Sci2020;21:6466. PMCID:PMC7556044

[48] Kaddour H,Okeoma CM.The past, the present, and the future of the size exclusion chromatography in extracellular vesicles separation.Viruses2021;13:2272 PMCID:PMC8618570

[49] Mol EA,Doevendans PA,Vader P.Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.Nanomedicine2017;13:2061-5

[50] Konoshenko MY,Vlassov AV.Isolation of extracellular vesicles: general methodologies and latest trends.Biomed Res Int2018;2018:8545347 PMCID:PMC5831698

[51] Van Deun J,Li H.Integrated dual-mode chromatography to enrich extracellular vesicles from plasma.Adv Biosyst2020;4:e1900310 PMCID:PMC7606548

[52] Jung J,Yoon J.Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.Sci Rep2021;11:217 PMCID:PMC7804108

[53] Greening DW,Ji H,Simpson RJ.A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. In: Posch A, editor. Proteomic profiling. New York: Springer; 2015. pp. 179-209.

[54] Shih CL,Hsu SC.Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.N Biotechnol2016;33:116-22.

[55] Miyanishi M,Koike M,Kitamura T.Identification of Tim4 as a phosphatidylserine receptor.Nature2007;450:435-9

[56] Contreras-Naranjo JC,Ugaz VM.Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine.Lab Chip2017;17:3558-77. PMCID:PMC5656537

[57] Raju D,Badilescu S,Packirisamy M.Microfluidic platforms for the isolation and detection of exosomes: a brief review.Micromachines2022;13:730. PMCID:PMC9147043

[58] Ayala-mar S,Mata-gómez MA,González-valdez J.Electrokinetically driven exosome separation and concentration using dielectrophoretic-enhanced PDMS-Based microfluidics.Anal Chem2019;91:14975-82.

[59] Lewis J,Akers J,Heller M.A pilot proof-of-principle analysis Demonstrating Dielectrophoresis (DEP) as a glioblastoma biomarker platform.Sci Rep2019;9:10279 PMCID:PMC6635369

[60] Kanwar SS,Simeone DM.Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes.Lab Chip2014;14:1891-900

[61] Weng Y,Shan Y.Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling.Analyst2016;141:4640-6.

[62] García-Romero N,Rackov G.Polyethylene glycol improves current methods for circulating extracellular vesicle-derived DNA isolation.J Transl Med2019;17:75 PMCID:PMC6419425

[63] Gámez-Valero A,Carreras-Planella L,Beyer K.Size-Exclusion chromatography-based isolation minimally alters extracellular vesicles' characteristics compared to precipitating agents.Sci Rep2016;6:33641. PMCID:PMC5027519

[64] Konoshenko MY,Bryzgunova OE,Pyshnaya IA.Isolation of extracellular vesicles from biological fluids via the aggregation-precipitation approach for downstream miRNAs detection.Diagnostics2021;11:384 PMCID:PMC7996260

[65] Allelein S,Lopes ALH.Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations.Sci Rep2021;11:11585. PMCID:PMC8172572

[66] Couch Y,Di Vizio D.A brief history of nearly EV-erything-The rise and rise of extracellular vesicles.J of Extracellular Vesicle2021;10:e12144. PMCID:PMC8681215