{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T14:41:47Z","timestamp":1791988907486,"version":"4.4.0"},"reference-count":30,"publisher":"Springer Science and Business Media LLC","issue":"5","license":[{"start":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T00:00:00Z","timestamp":1745193600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T00:00:00Z","timestamp":1745193600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/100011941","name":"Gobierno del Principado de Asturias","doi-asserted-by":"publisher","award":["PA-21-PF-BP20-067"],"award-info":[{"award-number":["PA-21-PF-BP20-067"]}],"id":[{"id":"10.13039\/100011941","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100011941","name":"Gobierno del Principado de Asturias","doi-asserted-by":"publisher","award":["IDE\/2024\/000752"],"award-info":[{"award-number":["IDE\/2024\/000752"]}],"id":[{"id":"10.13039\/100011941","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["PI20\/01122"],"award-info":[{"award-number":["PI20\/01122"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["PI20\/01122"],"award-info":[{"award-number":["PI20\/01122"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["PI21\/01177"],"award-info":[{"award-number":["PI21\/01177"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006382","name":"Universidad de Oviedo","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100006382","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microchim Acta"],"published-print":{"date-parts":[[2025,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:p>Exosomes are a subpopulation of nanosized extracellular vesicles (EVs), formed by a lipid bilayer and naturally secreted by cells. They transport RNA, microRNAs, bioactive proteins, and lipids and play an important role in intercellular communication. Exosomes are a promising alternative cell-free therapy in regenerative medicine, immunotherapy, and drug delivery. The implementation of new exosomes treatments requires knowledge of its concentration, purity, and full characterization. However, comparing different studies is highly challenging due to the lack of validated methodologies for isolation and determination, as well as a lack of well-characterized exosomes reference standards. In this work, human uterine cervical mesenchymal stem cell (hUCESC) EVs have been isolated by ultracentrifugation and characterized, discussing the current limitations of characterization methods. First, total protein assays are heavily influenced by free-protein and lipid contaminations which confirm the need of employing several methods for vesicular protein determination. This purity variation seems heavily influenced by the vesicles origin as more complex mediums originate more matrix interferences. Size exclusion high performance liquid chromatography has been demonstrated as a new methodology for purity assessment of hUCESC-EVs and commercial EVs (adipose stem cells and human serum). The results found low purity in the commercial exosomes highlighting that protein and lipid purity must be included in the commercial EVs. Finally, the combination of this chromatography method\u00a0with total protein assays proved that particle concentration could be estimated using vesicular protein concentration.<\/jats:p>\n          <jats:p>\n            <jats:bold>Graphical abstract<\/jats:bold>\n          <\/jats:p>","DOI":"10.1007\/s00604-025-07147-4","type":"journal-article","created":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T16:17:44Z","timestamp":1745252264000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Limitations and challenges in the characterization of extracellular vesicles from stem cells and serum"],"prefix":"10.1007","volume":"192","author":[{"given":"Sara","family":"Escudero-Cernuda","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noemi","family":"Eiro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda","family":"Fraile","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco J.","family":"Vizoso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bel\u00e9n","family":"Fern\u00e1ndez-Colomer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda Luisa","family":"Fern\u00e1ndez-S\u00e1nchez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,21]]},"reference":[{"issue":"7","key":"7147_CR1","doi-asserted-by":"publisher","first-page":"855","DOI":"10.1002\/stem.3016","volume":"37","author":"A Andrzejewska","year":"2009","unstructured":"Andrzejewska A, Lukomska B, Janowski M (2009) Mesenchymal stem cells: from roots to boost. Stem Cells 37(7):855\u2013864. https:\/\/doi.org\/10.1002\/stem.3016","journal-title":"Stem Cells"},{"key":"7147_CR2","doi-asserted-by":"publisher","first-page":"34","DOI":"10.21037\/sci.2019.08.11","volume":"6","author":"P Saeedi","year":"2019","unstructured":"Saeedi P, Halabian R, Imani Fooladi AA (2019) A revealing review of mesenchymal stem cells therapy, clinical perspectives and modification strategies. Stem Cell Investig 6:34. https:\/\/doi.org\/10.21037\/sci.2019.08.11","journal-title":"Stem Cell Investig"},{"issue":"7","key":"7147_CR3","doi-asserted-by":"publisher","first-page":"3576","DOI":"10.3390\/ijms22073576","volume":"22","author":"S Fern\u00e1ndez-Francos","year":"2021","unstructured":"Fern\u00e1ndez-Francos S, Eiro N, Costa LA, Escudero-Cernuda S, Fern\u00e1ndez-S\u00e1nchez ML, Vizoso FJ (2021) Mesenchymal stem cells as a cornerstone in a galaxy of intercellular signals: basis for a new era of medicine. Int J Mol Sci 22(7):3576. https:\/\/doi.org\/10.3390\/ijms22073576","journal-title":"Int J Mol Sci"},{"key":"7147_CR4","doi-asserted-by":"publisher","first-page":"652970","DOI":"10.3389\/fbioe.2021.652970","volume":"9","author":"M Sandon\u00e0","year":"2021","unstructured":"Sandon\u00e0 M, Di Pietro L, Esposito F, Ventura A, Silini AR, Parolini O et al (2021) Mesenchymal stromal cells and their secretome: new therapeutic perspectives for skeletal muscle regeneration. Front Bioeng Biotechnol 9:652970. https:\/\/doi.org\/10.3389\/fbioe.2021.652970","journal-title":"Front Bioeng Biotechnol"},{"key":"7147_CR5","doi-asserted-by":"publisher","unstructured":"L. PK, Kandoi S, Misra R, S. V, K. R, Verma RS (2019) The mesenchymal stem cell secretome: a new paradigm towards cell-free therapeutic mode in regenerative medicine. Cytokine Growth Factor Rev 46:1\u20139. https:\/\/doi.org\/10.1016\/j.cytogfr.2019.04.002.","DOI":"10.1016\/j.cytogfr.2019.04.002"},{"issue":"19","key":"7147_CR6","doi-asserted-by":"publisher","first-page":"7038","DOI":"10.3390\/ijms21197038","volume":"21","author":"P Ahangar","year":"2020","unstructured":"Ahangar P, Mills SJ, Cowin AJ (2020) Mesenchymal stem cell secretome as an emerging cell-free alternative for improving wound repair. Int J Mol Sci 21(19):7038. https:\/\/doi.org\/10.3390\/ijms21197038","journal-title":"Int J Mol Sci"},{"issue":"7","key":"7147_CR7","doi-asserted-by":"publisher","first-page":"1656","DOI":"10.3390\/ijms20071656","volume":"20","author":"G Sagaradze","year":"2019","unstructured":"Sagaradze G, Grigorieva O, Nimiritsky P, Basalova N, Kalinina N, Akopyan Z et al (2019) Conditioned medium from human mesenchymal stromal cells: towards the clinical translation. Int J Mol Sci 20(7):1656. https:\/\/doi.org\/10.3390\/ijms20071656","journal-title":"Int J Mol Sci"},{"key":"7147_CR8","doi-asserted-by":"publisher","first-page":"608","DOI":"10.3389\/fimmu.2014.00608","volume":"5","author":"OG de Jong","year":"2014","unstructured":"de Jong OG, van Balkom BWM, Schiffelers RM, Bouten CVC, Verhaar MC (2014) Extracellular vesicles: potential roles in regenerative medicine. Front Immunol 5:608. https:\/\/doi.org\/10.3389\/fimmu.2014.00608","journal-title":"Front Immunol"},{"issue":"4","key":"7147_CR9","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1038\/nrm.2017.125","volume":"19","author":"G Van Niel","year":"2018","unstructured":"Van Niel G, D\u2019Angelo G, Raposo G (2018) Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19(4):213\u2013228. https:\/\/doi.org\/10.1038\/nrm.2017.125","journal-title":"Nat Rev Mol Cell Biol"},{"issue":"1","key":"7147_CR10","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1186\/s13287-018-0791-7","volume":"9","author":"S Keshtkar","year":"2018","unstructured":"Keshtkar S, Azarpira N, Ghahremani MH (2018) Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther 9(1):63. https:\/\/doi.org\/10.1186\/s13287-018-0791-7","journal-title":"Stem Cell Res Ther"},{"key":"7147_CR11","doi-asserted-by":"publisher","first-page":"101093","DOI":"10.1016\/j.bonr.2021.101093","volume":"14","author":"VP Murali","year":"2021","unstructured":"Murali VP, Holmes CA (2021) Mesenchymal stromal cell-derived extracellular vesicles for bone regeneration therapy. Bone Rep 14:101093. https:\/\/doi.org\/10.1016\/j.bonr.2021.101093","journal-title":"Bone Rep"},{"key":"7147_CR12","doi-asserted-by":"publisher","first-page":"625221","DOI":"10.3389\/fcell.2021.625221","volume":"9","author":"A Hern\u00e1ndez-Barranco","year":"2021","unstructured":"Hern\u00e1ndez-Barranco A, Nogu\u00e9s L, Peinado H (2021) Could extracellular vesicles contribute to generation or awakening of \u201csleepy\u201d metastatic niches? Front Cell Dev Biol 9:625221. https:\/\/doi.org\/10.3389\/fcell.2021.625221","journal-title":"Front Cell Dev Biol"},{"key":"7147_CR13","doi-asserted-by":"publisher","unstructured":"Kalluri R, LeBleu VS (2020) The biology, function, and biomedical applications of exosomes. Science (1979) 367(6478):eaau6977. https:\/\/doi.org\/10.1126\/science.aau6977.","DOI":"10.1126\/science.aau6977"},{"issue":"1","key":"7147_CR14","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1186\/s12951-018-0403-9","volume":"16","author":"EJ Bunggulawa","year":"2018","unstructured":"Bunggulawa EJ, Wang W, Yin T, Wang N, Durkan C, Wang Y et al (2018) Recent advancements in the use of exosomes as drug delivery. J Nanobiotechnology 16(1):81. https:\/\/doi.org\/10.1186\/s12951-018-0403-9","journal-title":"J Nanobiotechnology"},{"issue":"3","key":"7147_CR15","doi-asserted-by":"publisher","first-page":"228","DOI":"10.1038\/nmeth.4185","volume":"14","author":"J Van Deun","year":"2017","unstructured":"Van Deun J, Mestdagh P, Agostinis P, Akay \u00d6, Anand S, Anckaert J et al (2017) EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods 14(3):228\u2013232. https:\/\/doi.org\/10.1038\/nmeth.4185","journal-title":"Nat Methods"},{"issue":"10","key":"7147_CR16","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1016\/j.tibtech.2020.05.012","volume":"38","author":"S Gandham","year":"2020","unstructured":"Gandham S, Su X, Wood J, Nocera AL, Alli SC, Milane L et al (2020) Technologies and standardization in research on extracellular vesicles. Trends Biotechnol 38(10):1066\u20131098. https:\/\/doi.org\/10.1016\/j.tibtech.2020.05.012","journal-title":"Trends Biotechnol"},{"issue":"6","key":"7147_CR17","doi-asserted-by":"publisher","first-page":"824","DOI":"10.3390\/biom10060824","volume":"10","author":"E Serrano-Pertierra","year":"2020","unstructured":"Serrano-Pertierra E, Oliveira-Rodr\u00edguez M, Matos M, Guti\u00e9rrez G, Moyano A, Salvador M et al (2020) Extracellular vesicles: current analytical techniques for detection and quantification. Biomolecules 10(6):824. https:\/\/doi.org\/10.3390\/biom10060824","journal-title":"Biomolecules"},{"issue":"1","key":"7147_CR18","doi-asserted-by":"publisher","first-page":"1596016","DOI":"10.1080\/20013078.2019.1596016","volume":"8","author":"D Bachurski","year":"2019","unstructured":"Bachurski D, Schuldner M, Nguyen PH, Malz A, Reiners KS, Grenzi PC et al (2019) Extracellular vesicle measurements with nanoparticle tracking analysis\u2013an accuracy and repeatability comparison between NanoSight NS300 and ZetaView. J Extracell Vesicles 8(1):1596016. https:\/\/doi.org\/10.1080\/20013078.2019.1596016","journal-title":"J Extracell Vesicles"},{"issue":"4","key":"7147_CR19","doi-asserted-by":"publisher","first-page":"800","DOI":"10.1007\/s11095-017-2109-3","volume":"34","author":"K Bai","year":"2017","unstructured":"Bai K, Barnett GV, Kar SR, Das TK (2017) Interference from proteins and surfactants on particle size distributions measured by nanoparticle tracking analysis (NTA). Pharm Res 34(4):800\u2013808. https:\/\/doi.org\/10.1007\/s11095-017-2109-3","journal-title":"Pharm Res"},{"key":"7147_CR20","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1016\/j.exer.2016.06.022","volume":"149","author":"MA Bermudez","year":"2016","unstructured":"Bermudez MA, Sendon-Lago J, Seoane S, Eiro N, Gonzalez F, Saa J et al (2016) Anti-inflammatory effect of conditioned medium from human uterine cervical stem cells in uveitis. Exp Eye Res 149:84\u201392. https:\/\/doi.org\/10.1016\/j.exer.2016.06.022","journal-title":"Exp Eye Res"},{"key":"7147_CR21","unstructured":"HansaBioMed Life Sciences. Exosomes and EVs. https:\/\/hansabiomed.eu\/shop\/index.php?route=product\/category&path=18. Accessed 31 Aug 2024."},{"key":"7147_CR22","doi-asserted-by":"publisher","first-page":"1199","DOI":"10.3389\/fphar.2018.01199","volume":"9","author":"GD Kusuma","year":"2018","unstructured":"Kusuma GD, Barabadi M, Tan JL, Morton DAV, Frith JE, Lim R (2018) To protect and to preserve: novel preservation strategies for extracellular vesicles. Front Pharmacol 9:1199. https:\/\/doi.org\/10.3389\/fphar.2018.01199","journal-title":"Front Pharmacol"},{"issue":"15","key":"7147_CR23","doi-asserted-by":"publisher","first-page":"e2103222","DOI":"10.1002\/advs.202103222","volume":"9","author":"JJ Lai","year":"2022","unstructured":"Lai JJ, Chau ZL, Chen SY, Hill JJ, Korpany KV, Liang NW et al (2022) Exosome processing and characterization approaches for research and technology development. Advanced Science 9(15):e2103222. https:\/\/doi.org\/10.1002\/advs.202103222","journal-title":"Advanced Science"},{"key":"7147_CR24","doi-asserted-by":"publisher","unstructured":"Webber J, Clayton A (2013) How pure are your vesicles? J Extracell Vesicles 2(1). https:\/\/doi.org\/10.3402\/jev.v2i0.19861.","DOI":"10.3402\/jev.v2i0.19861"},{"key":"7147_CR25","doi-asserted-by":"publisher","first-page":"114672","DOI":"10.1016\/j.ab.2022.114672","volume":"647","author":"T Liangsupree","year":"2022","unstructured":"Liangsupree T, Multia E, Saarinen J, Ruiz-Jimenez J, Kemell M, Riekkola ML (2022) Raman spectroscopy combined with comprehensive gas chromatography for label-free characterization of plasma-derived extracellular vesicle subpopulations. Anal Biochem 647:114672. https:\/\/doi.org\/10.1016\/j.ab.2022.114672","journal-title":"Anal Biochem"},{"issue":"17","key":"7147_CR26","doi-asserted-by":"publisher","first-page":"6257","DOI":"10.3390\/ijms21176257","volume":"21","author":"D Lucchetti","year":"2020","unstructured":"Lucchetti D, Battaglia A, Ricciardi-Tenore C, Colella F, Perelli L, De MR et al (2020) Measuring extracellular vesicles by conventional flow cytometry: dream or reality? Int J Mol Sci 21(17):6257. https:\/\/doi.org\/10.3390\/ijms21176257","journal-title":"Int J Mol Sci"},{"issue":"1","key":"7147_CR27","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1186\/s13287-024-03717-0","volume":"15","author":"N Eiro","year":"2024","unstructured":"Eiro N, Fraile M, Escudero-Cernuda S, Sendon-Lago J, Gonzalez LO, Fernandez-S\u00e1nchez ML et al (2024) Synergistic effect of human uterine cervical mesenchymal stem cell secretome and paclitaxel on triple negative breast cancer. Stem Cell Res Ther 15(1):121. https:\/\/doi.org\/10.1186\/s13287-024-03717-0","journal-title":"Stem Cell Res Ther"},{"issue":"3","key":"7147_CR28","doi-asserted-by":"publisher","first-page":"940","DOI":"10.1016\/j.celrep.2019.01.009","volume":"27","author":"Q Zhang","year":"2019","unstructured":"Zhang Q, Higginbotham JN, Jeppesen DK, Yang YP, Li W, McKinley ET et al (2019) Transfer of functional cargo in exomeres. Cell Rep 27(3):940-954.e6. https:\/\/doi.org\/10.1016\/j.celrep.2019.01.009","journal-title":"Cell Rep"},{"issue":"5","key":"7147_CR29","doi-asserted-by":"publisher","first-page":"1462","DOI":"10.1038\/s41596-023-00811-0","volume":"18","author":"Q Zhang","year":"2023","unstructured":"Zhang Q, Jeppesen DK, Higginbotham JN, Franklin JL, Coffey RJ (2023) Comprehensive isolation of extracellular vesicles and nanoparticles. Nat Protoc 18(5):1462\u20131487. https:\/\/doi.org\/10.1038\/s41596-023-00811-0","journal-title":"Nat Protoc"},{"key":"7147_CR30","doi-asserted-by":"publisher","first-page":"68","DOI":"10.3389\/fcvm.2017.00068","volume":"4","author":"MEM Parsons","year":"2017","unstructured":"Parsons MEM, McParland D, Szklanna PB, Guang MHZ, O\u2019Connell K, O\u2019Connor HD et al (2017) A protocol for improved precision and increased confidence in nanoparticle tracking analysis concentration measurements between 50 and 120 nm in biological fluids. Front Cardiovasc Med 4:68. https:\/\/doi.org\/10.3389\/fcvm.2017.00068","journal-title":"Front Cardiovasc Med"}],"container-title":["Microchimica Acta"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00604-025-07147-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00604-025-07147-4\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00604-025-07147-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,10]],"date-time":"2025-05-10T12:38:43Z","timestamp":1746880723000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00604-025-07147-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,21]]},"references-count":30,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2025,5]]}},"alternative-id":["7147"],"URL":"https:\/\/doi.org\/10.1007\/s00604-025-07147-4","relation":{},"ISSN":["0026-3672","1436-5073"],"issn-type":[{"value":"0026-3672","type":"print"},{"value":"1436-5073","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,21]]},"assertion":[{"value":"6 February 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"1 April 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"21 April 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}}],"article-number":"311"}}