Freeze-fracture electron microscopy for extracellular vesicle analysis

Resnik, Nataša (author), Romih, Rok (author), Erdani-Kreft, Mateja (author), Hudoklin, Samo (author)

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

• Language: English
• Keywords: extracellular vesicles, electron microscopy, urothelial cells, cancer
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 13 str.
• Numbering: Vol. 187,
• PID: 20.500.12556/RUL-183558
• UDC: 616.6
• ISSN on article: 1940-087X
• DOI: 10.3791/63550
• COBISS.SI-ID: 125862659
• Publication date in RUL: 15.06.2026

Record is a part of a journal

• Title: Journal of visualized experiments
• Shortened title: J. vis. exp.
• Publisher: MYJoVE Corporation
• ISSN: 1940-087X
• COBISS.SI-ID: 6867284

Licences

• License: CC BY-NC-ND 3.0, Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
• Link: http://creativecommons.org/licenses/by-nc-nd/3.0/
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Secondary language

• Language: Slovenian
• Keywords: zunajcelični vezikli, elektronska mikroskopija, urotelijske celice