Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography
Por:
Martinez-Greene, Juan Alfonso, Gomez-Chavarin, Margarita, Ramos-Godínez M.D.P., Martinez-Martinez, Eduardo
Publicada:
1 ago 2023
Resumen:
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.
Filiaciones:
Martinez-Greene, Juan Alfonso:
Laboratory of Cell Communication and Extracellular Vesicles, National Institute of Genomic Medicine (INMEGEN), Mexico City, 14610, Mexico
Gomez-Chavarin, Margarita:
Physiology Department, School of Medicine, National Autonomous University of Mexico, Mexico City, 04510, Mexico
Ramos-Godínez M.D.P.:
Electron Microscopy Laboratory, National Institute of Cancer, Mexico City, 14080, Mexico
Martinez-Martinez, Eduardo:
Laboratory of Cell Communication and Extracellular Vesicles, National Institute of Genomic Medicine (INMEGEN), Mexico City, 14610, Mexico
gold, All Open Access; Gold
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