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.

Advanced Materials Department Jožef Stefan Institute SI 1000 Ljubljana Slovenia

Centre for Micro and Nanosciences and Technologies University of Rijeka HR 51000 Rijeka Croatia

Department of Medical Chemistry Biochemistry and Clinical Chemistry Faculty of Medicine University of Rijeka HR 51000 Rijeka Croatia

Department of Physiology Immunology and Pathophysiology Faculty of Medicine University of Rijeka HR 51000 Rijeka Croatia

Faculty of Engineering University of Rijeka HR 51000 Rijeka Croatia

Institute of Biochemistry and Molecular Genetics Faculty of Medicine University of Ljubljana SI 1000 Ljubljana Slovenia

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University CZ 12800 Prague Czech Republic

Institute of Pathology Faculty of Medicine University of Ljubljana SI 1000 Ljubljana Slovenia

Pula General Hospital HR 52100 Pula Croatia

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