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Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy

M. Kurtjak, S. Kereïche, D. Klepac, H. Križan, M. Perčić, V. Krušić Alić, T. Lavrin, M. Lenassi, K. Wechtersbach, N. Kojc, M. Vukomanović, S. Zrna, M. Biberić, R. Domitrović, K. Grabušić, M. Malenica

. 2022 ; 10 (6) : . [pub] 20220527

Jazyk angličtina Země Švýcarsko

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/bmc22017072

Grantová podpora
IP-2019-04-1511 (grant to K.G.) Croatian Science Foundation
uniri-biomed-18-279 (grant to M.M.), uniri-biomed-18-5 (grant to K.G.), uniri-prirod-18-299 (grant to D.K.), uniri-biomed-18-30 (grant to R.D.) University of Rijeka
P1-170 (grant to M.L.) and P3-054 (grant to N.K.) Slovenian Research Agency
Research Infrastructure for Campus-based Laboratories at the University of Rijeka project, (RC.2.2.06-0001) co-funded by the European Fund for Regional Development (ERDF) and Ministry of Science, Education and Sports of the Republic of Croatia University of Rijeka

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Citace poskytuje Crossref.org

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$a Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.
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