Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called "liquid biopsies". Obtaining a high-quality EV sample with minimum contaminants is crucial for proteomic analyses using LC-MS/MS or other techniques. However, the EV content in various body fluids largely differs, which may hamper subsequent analyses. Here, we present a comparison of extracellular vesicle yields from blood plasma, cerebrospinal fluid, and seminal plasma using an experimental pig model. Pigs are widely used in biomedical research as large animal models with anatomy and physiology close to those of humans and enable studies (e.g., of the nervous system) that are unfeasible in humans. EVs were isolated from body fluids by differential centrifugation followed by ultracentrifugation. EVs were characterized according to protein yields and to the quality of the isolated vesicles (e.g., size distribution, morphology, positivity for exosome markers). In our experimental setting, substantial differences in EV amounts were identified among body fluids, with the seminal plasma being the richest EV source. The yields of pellet proteins from ultracentrifugation of 1 mL of porcine body fluids may help to estimate body fluid input volumes to obtain sufficient samples for subsequent proteomic analyses.

Department of Pediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Ke Karlovu 2 12109 Prague Czech Republic

Institute of Animal Physiology and Genetics of the Czech Academy of Sciences Rumburska 89 27721 Libechov Czech Republic

Institute of Experimental Medicine of the Czech Academy of Sciences Videnska 1083 14220 Prague Czech Republic

Zobrazit více v PubMed

Théry C., Witwer K.W., Aikawa E., Alcaraz M.J., Anderson J.D., Andriantsitohaina R., Antoniou A., Arab T., Archer F., Atkin-Smith G.K. 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. 2018;7:1535750. doi: 10.1080/20013078.2018.1535750. PubMed DOI PMC

Colombo M., Raposo G., Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 2014;30:255–289. doi: 10.1146/annurev-cellbio-101512-122326. PubMed DOI

Kalra H., Drummen G.P.C., Mathivanan S. Focus on Extracellular Vesicles: Introducing the Next Small Big Thing. Int. J. Mol. Sci. 2016;17:170. doi: 10.3390/ijms17020170. PubMed DOI PMC

Yáñez-Mó M., Siljander P.R.-M., Andreu Z., Zavec A.B., Borràs F.E., Buzas E.I., Buzas K., Casal E., Cappello F., Carvalho J., et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles. 2015;4:27066. doi: 10.3402/jev.v4.27066. PubMed DOI PMC

Mathieu M., Martin-Jaular L., Lavieu G., Théry C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat. Cell Biol. 2019;21:9–17. doi: 10.1038/s41556-018-0250-9. PubMed DOI

Li W., Li C., Zhou T., Liu X., Liu X., Li X., Chen D. Role of exosomal proteins in cancer diagnosis. Mol. Cancer. 2017;16:145. doi: 10.1186/s12943-017-0706-8. PubMed DOI PMC

Shi M., Sheng L., Stewart T., Zabetian C.P., Zhang J. New windows into the brain: Central nervous system-derived extracellular vesicles in blood. Prog. Neurobiol. 2019;175:96–106. doi: 10.1016/j.pneurobio.2019.01.005. PubMed DOI PMC

Caruso Bavisotto C., Scalia F., Marino Gammazza A., Carlisi D., Bucchieri F., Conway de Macario E., Macario A.J.L., Cappello F., Campanella C. Extracellular Vesicle-Mediated Cell−Cell Communication in the Nervous System: Focus on Neurological Diseases. Int. J. Mol. Sci. 2019;20:434. doi: 10.3390/ijms20020434. PubMed DOI PMC

Ghafarian F., Pashirzad M., Khazaei M., Rezayi M., Hassanian S.M., Ferns G.A., Avan A. The clinical impact of exosomes in cardiovascular disorders: From basic science to clinical application. J. Cell. Physiol. 2018 doi: 10.1002/jcp.27964. PubMed DOI

EL Andaloussi S., Mäger I., Breakefield X.O., Wood M.J.A. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 2013;12:347–357. doi: 10.1038/nrd3978. PubMed DOI

Campanella C., Caruso Bavisotto C., Logozzi M., Marino Gammazza A., Mizzoni D., Cappello F., Fais S. On the Choice of the Extracellular Vesicles for Therapeutic Purposes. Int. J. Mol. Sci. 2019;20:236. doi: 10.3390/ijms20020236. PubMed DOI PMC

Piffoux M., Nicolás-Boluda A., Mulens-Arias V., Richard S., Rahmi G., Gazeau F., Wilhelm C., Silva A.K.A. Extracellular vesicles for personalized medicine: The input of physically triggered production, loading and theranostic properties. Adv. Drug Deliv. Rev. 2018 doi: 10.1016/j.addr.2018.12.009. PubMed DOI

Schomberg D.T., Tellez A., Meudt J.J., Brady D.A., Dillon K.N., Arowolo F.K., Wicks J., Rousselle S.D., Shanmuganayagam D. Miniature Swine for Preclinical Modeling of Complexities of Human Disease for Translational Scientific Discovery and Accelerated Development of Therapies and Medical Devices. Toxicol. Pathol. 2016;44:299–314. doi: 10.1177/0192623315618292. PubMed DOI

Perleberg C., Kind A., Schnieke A. Genetically engineered pigs as models for human disease. Dis. Model. Mech. 2018;11 doi: 10.1242/dmm.030783. PubMed DOI PMC

Baxa M., Hruska-Plochan M., Juhas S., Vodicka P., Pavlok A., Juhasova J., Miyanohara A., Nejime T., Klima J., Macakova M., et al. A Transgenic Minipig Model of Huntington’s Disease. J. Huntingt. Dis. 2013;2:47–68. doi: 10.3233/JHD-130001. PubMed DOI

Navarro R., Juhas S., Keshavarzi S., Juhasova J., Motlik J., Johe K., Marsala S., Scadeng M., Lazar P., Tomori Z., et al. Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study. J. Neurotrauma. 2012;29:499–513. doi: 10.1089/neu.2011.2076. PubMed DOI PMC

Borovanský J., Horák V., Elleder M., Fortýn K., Smit N.P., Kolb A.M. Biochemical characterization of a new melanoma model–the minipig MeLiM strain. Melanoma Res. 2003;13:543–548. doi: 10.1097/00008390-200312000-00001. PubMed DOI

Foster B.P., Balassa T., Benen T.D., Dominovic M., Elmadjian G.K., Florova V., Fransolet M.D., Kestlerova A., Kmiecik G., Kostadinova I.A., et al. Extracellular vesicles in blood, milk and body fluids of the female and male urogenital tract and with special regard to reproduction. Crit. Rev. Clin. Lab. Sci. 2016;53:379–395. doi: 10.1080/10408363.2016.1190682. PubMed DOI

Xu R., Greening D.W., Zhu H.-J., Takahashi N., Simpson R.J. Extracellular vesicle isolation and characterization: Toward clinical application. J. Clin. Invest. 2016;126:1152–1162. doi: 10.1172/JCI81129. PubMed DOI PMC

Coumans F.A.W., Brisson A.R., Buzas E.I., Dignat-George F., Drees E.E.E., El-Andaloussi S., Emanueli C., Gasecka A., Hendrix A., Hill A.F., et al. Methodological Guidelines to Study Extracellular Vesicles. Circ. Res. 2017;120:1632–1648. doi: 10.1161/CIRCRESAHA.117.309417. PubMed DOI

Goetzl E.J., Boxer A., Schwartz J.B., Abner E.L., Petersen R.C., Miller B.L., Kapogiannis D. Altered lysosomal proteins in neural-derived plasma exosomes in preclinical Alzheimer disease. Neurology. 2015;85:40–47. doi: 10.1212/WNL.0000000000001702. PubMed DOI PMC

Fiandaca M.S., Kapogiannis D., Mapstone M., Boxer A., Eitan E., Schwartz J.B., Abner E.L., Petersen R.C., Federoff H.J., Miller B.L., et al. Identification of pre-clinical Alzheimer’s disease by a profile of pathogenic proteins in neurally-derived blood exosomes: A case-control study. Alzheimers Dement. J. Alzheimers Assoc. 2015;11:600–607.e1. doi: 10.1016/j.jalz.2014.06.008. PubMed DOI PMC

Soung Y.H., Ford S., Zhang V., Chung J. Exosomes in Cancer Diagnostics. Cancers. 2017;9:8. doi: 10.3390/cancers9010008. PubMed DOI PMC

Brett S.I., Lucien F., Guo C., Williams K.C., Kim Y., Durfee P.N., Brinker C.J., Chin J.I., Yang J., Leong H.S. Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples. The Prostate. 2017;77:1335–1343. doi: 10.1002/pros.23393. PubMed DOI

Park Y.H., Shin H.W., Jung A.R., Kwon O.S., Choi Y.-J., Park J., Lee J.Y. Prostate-specific extracellular vesicles as a novel biomarker in human prostate cancer. Sci. Rep. 2016;6:30386. doi: 10.1038/srep30386. PubMed DOI PMC

Johnsen K.B., Gudbergsson J.M., Andresen T.L., Simonsen J.B. What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer. Biochim. Biophys. Acta Rev. Cancer. 2019;1871:109–116. doi: 10.1016/j.bbcan.2018.11.006. PubMed DOI

Laterra J., Keep R., Betz L.A., Goldstein G.W. Blood—Cerebrospinal Fluid Barrier. . . [(accessed on 29 March 2019)];Basic Neurochemistry: Molecular, Cellular and Medical Aspects. (6th Ed.). 1999 Available online: https://www.ncbi.nlm.nih.gov/books/NBK27998/

Kroksveen A.C., Opsahl J.A., Aye T.T., Ulvik R.J., Berven F.S. Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics. J. Proteom. 2011;74:371–388. doi: 10.1016/j.jprot.2010.11.010. PubMed DOI

Gámez-Valero A., Beyer K., Borràs F.E. Extracellular vesicles, new actors in the search for biomarkers of dementias. Neurobiol. Aging. 2019;74:15–20. doi: 10.1016/j.neurobiolaging.2018.10.006. PubMed DOI

Wu X., Zheng T., Zhang B. Exosomes in Parkinson’s Disease. Neurosci. Bull. 2017;33:331–338. doi: 10.1007/s12264-016-0092-z. PubMed DOI PMC

Selmaj I., Mycko M.P., Raine C.S., Selmaj K.W. The role of exosomes in CNS inflammation and their involvement in multiple sclerosis. J. Neuroimmunol. 2017;306:1–10. doi: 10.1016/j.jneuroim.2017.02.002. PubMed DOI

Otero-Ortega L., Laso-García F., Gómez-de Frutos M., Fuentes B., Diekhorst L., Díez-Tejedor E., Gutiérrez-Fernández M. Role of Exosomes as a Treatment and Potential Biomarker for Stroke. Transl. Stroke Res. 2018 doi: 10.1007/s12975-018-0654-7. PubMed DOI

Zagrean A.-M., Hermann D.M., Opris I., Zagrean L., Popa-Wagner A. Multicellular Crosstalk Between Exosomes and the Neurovascular Unit After Cerebral Ischemia. Therapeutic Implications. Front. Neurosci. 2018;12:811. doi: 10.3389/fnins.2018.00811. PubMed DOI PMC

Shankar G.M., Balaj L., Stott S.L., Nahed B., Carter B.S. Liquid biopsy for brain tumors. Expert Rev. Mol. Diagn. 2017;17:943–947. doi: 10.1080/14737159.2017.1374854. PubMed DOI PMC

Verheul C., Kleijn A., Lamfers M.L.M. Cerebrospinal fluid biomarkers of malignancies located in the central nervous system. Handb. Clin. Neurol. 2017;146:139–169. doi: 10.1016/B978-0-12-804279-3.00010-1. PubMed DOI

Sarko D.K., McKinney C.E. Exosomes: Origins and Therapeutic Potential for Neurodegenerative Disease. Front. Neurosci. 2017;11 doi: 10.3389/fnins.2017.00082. PubMed DOI PMC

Coleman B.M., Hill A.F. Extracellular vesicles--Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases. Semin. Cell Dev. Biol. 2015;40:89–96. doi: 10.1016/j.semcdb.2015.02.007. PubMed DOI

Saez F., Sullivan R. Prostasomes, post-testicular sperm maturation and fertility. Front. Biosci. Landmark Ed. 2016;21:1464–1473. doi: 10.2741/4466. PubMed DOI

Machtinger R., Laurent L.C., Baccarelli A.A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum. Reprod. Update. 2016;22:182–193. doi: 10.1093/humupd/dmv055. PubMed DOI PMC

Du J., Shen J., Wang Y., Pan C., Pang W., Diao H., Dong W. Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane. Oncotarget. 2016;7:58832–58847. doi: 10.18632/oncotarget.11315. PubMed DOI PMC

Macakova M., Bohuslavova B., Vochozkova P., Pavlok A., Sedlackova M., Vidinska D., Vochyanova K., Liskova I., Valekova I., Baxa M., et al. Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars. Neurodegener. Dis. 2016;16:245–259. doi: 10.1159/000443665. PubMed DOI

Théry C., Amigorena S., Raposo G., Clayton A. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Curr. Protoc. Cell Biol. 2006;30:3–22. doi: 10.1002/0471143030.cb0322s30. PubMed DOI

Chandler W.L. Microparticle counts in platelet-rich and platelet-free plasma, effect of centrifugation and sample-processing protocols. Blood Coagul. Fibrinolysis Int. J. Haemost. Thromb. 2013;24:125–132. doi: 10.1097/MBC.0b013e32835a0824. PubMed DOI

Baranyai T., Herczeg K., Onódi Z., Voszka I., Módos K., Marton N., Nagy G., Mäger I., Wood M.J., El Andaloussi S., et al. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. PloS ONE. 2015;10:e0145686. doi: 10.1371/journal.pone.0145686. PubMed DOI PMC

Bæk R., Søndergaard E.K.L., Varming K., Jørgensen M.M. The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray. J. Immunol. Methods. 2016;438:11–20. doi: 10.1016/j.jim.2016.08.007. PubMed DOI

Sódar B.W., Kovács Á., Visnovitz T., Pállinger É., Vékey K., Pocsfalvi G., Turiák L., Buzás E.I. Best practice of identification and proteomic analysis of extracellular vesicles in human health and disease. Expert Rev. Proteomics. 2017;14:1073–1090. doi: 10.1080/14789450.2017.1392244. PubMed DOI

Street J.M., Barran P.E., Mackay C.L., Weidt S., Balmforth C., Walsh T.S., Chalmers R.T.A., Webb D.J., Dear J.W. Identification and proteomic profiling of exosomes in human cerebrospinal fluid. J. Transl. Med. 2012;10:5. doi: 10.1186/1479-5876-10-5. PubMed DOI PMC

Manek R., Moghieb A., Yang Z., Kumar D., Kobessiy F., Sarkis G.A., Raghavan V., Wang K.K.W. Protein Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal Fluid Following Traumatic Brain Injury. Mol. Neurobiol. 2018;55:6112–6128. doi: 10.1007/s12035-017-0821-y. PubMed DOI PMC

Chiasserini D., van Weering J.R.T., Piersma S.R., Pham T.V., Malekzadeh A., Teunissen C.E., de Wit H., Jiménez C.R. Proteomic analysis of cerebrospinal fluid extracellular vesicles: A comprehensive dataset. J. Proteom. 2014;106:191–204. doi: 10.1016/j.jprot.2014.04.028. PubMed DOI

Piehl L.L., Fischman M.L., Hellman U., Cisale H., Miranda P.V. Boar seminal plasma exosomes: Effect on sperm function and protein identification by sequencing. Theriogenology. 2013;79:1071–1082. doi: 10.1016/j.theriogenology.2013.01.028. PubMed DOI

Perez-Patiño C., Barranco I., Parrilla I., Valero M.L., Martinez E.A., Rodriguez-Martinez H., Roca J. Characterization of the porcine seminal plasma proteome comparing ejaculate portions. J. Proteom. 2016;142:15–23. doi: 10.1016/j.jprot.2016.04.026. PubMed DOI

Pérez-Patiño C., Parrilla I., Barranco I., Vergara-Barberán M., Simó-Alfonso E.F., Herrero-Martínez J.M., Rodriguez-Martínez H., Martínez E.A., Roca J. New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers. J. Proteome Res. 2018;17:1065–1076. doi: 10.1021/acs.jproteome.7b00728. PubMed DOI

Koh Y.Q., Peiris H.N., Vaswani K., Meier S., Burke C.R., Macdonald K.A., Roche J.R., Almughlliq F., Arachchige B.J., Reed S., et al. Characterization of exosomes from body fluids of dairy cows1. J. Anim. Sci. 2017;95:3893–3904. doi: 10.2527/jas.2017.1727. PubMed DOI

Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington's Disease and Human Blood Plasma