Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography

Author:

Martínez-Greene Juan Alfonso1ORCID,Gómez-Chavarín Margarita2,Ramos-Godínez María del Pilar3,Martínez-Martínez Eduardo1ORCID

Affiliation:

1. Laboratory of Cell Communication and Extracellular Vesicles, National Institute of Genomic Medicine (INMEGEN), Mexico City 14610, Mexico

2. Physiology Department, School of Medicine, National Autonomous University of Mexico, Mexico City 04510, Mexico

3. Electron Microscopy Laboratory, National Institute of Cancer, Mexico City 14080, Mexico

Abstract

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

Funder

Consejo Nacional de Ciencia y Tecnología

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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