Journal
BIOMEDICINES
Volume 10, Issue 6, Pages -Publisher
MDPI
DOI: 10.3390/biomedicines10061251
Keywords
extracellular vesicles; cerebrospinal fluid; size-exclusion chromatography; atomic force microscopy; cryogenic transmission electron microscopy; morphology
Categories
Funding
- Croatian Science Foundation [IP-2019-04-1511]
- University of Rijeka [uniri-biomed-18-279, uniri-biomed-18-5, uniri-prirod-18-299, uniri-biomed-18-30]
- Slovenian Research Agency (ARRS) [P1-170, P3-054]
- Research Infrastructure for Campus-based Laboratories at the University of Rijeka [RC.2.2.06-0001]
- European Fund for Regional Development (ERDF)
- Ministry of Science, Education and Sports of the Republic of Croatia
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Extracellular vesicles in biofluids have great diagnostic and prognostic potential. This study observed the morphology of EVs in human cerebrospinal fluid and identified different subpopulations with potential different biological functions.
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 10(10) particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 10(9) 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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