Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/16/12704 |
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| author | Juan Alfonso Martínez-Greene Margarita Gómez-Chavarín María del Pilar Ramos-Godínez Eduardo Martínez-Martínez |
| author_facet | Juan Alfonso Martínez-Greene Margarita Gómez-Chavarín María del Pilar Ramos-Godínez Eduardo Martínez-Martínez |
| author_sort | Juan Alfonso Martínez-Greene |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T23:52:22Z |
| format | Article |
| id | doaj.art-6707464ec8d044dd8e97124b209dc024 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T23:52:22Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-6707464ec8d044dd8e97124b209dc0242023-11-19T01:27:53ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-08-0124161270410.3390/ijms241612704Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion ChromatographyJuan Alfonso Martínez-Greene0Margarita Gómez-Chavarín1María del Pilar Ramos-Godínez2Eduardo Martínez-Martínez3Laboratory of Cell Communication and Extracellular Vesicles, National Institute of Genomic Medicine (INMEGEN), Mexico City 14610, MexicoPhysiology Department, School of Medicine, National Autonomous University of Mexico, Mexico City 04510, MexicoElectron Microscopy Laboratory, National Institute of Cancer, Mexico City 14080, MexicoLaboratory of Cell Communication and Extracellular Vesicles, National Institute of Genomic Medicine (INMEGEN), Mexico City 14610, MexicoIn 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.https://www.mdpi.com/1422-0067/24/16/12704extracellular vesiclestissue-derived extracellular vesiclesliveradipose tissuedensity gradientsize exclusion chromatography |
| spellingShingle | Juan Alfonso Martínez-Greene Margarita Gómez-Chavarín María del Pilar Ramos-Godínez Eduardo Martínez-Martínez Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography International Journal of Molecular Sciences extracellular vesicles tissue-derived extracellular vesicles liver adipose tissue density gradient size exclusion chromatography |
| title | Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography |
| title_full | Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography |
| title_fullStr | Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography |
| title_full_unstemmed | Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography |
| title_short | Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography |
| title_sort | isolation of hepatic and adipose tissue derived extracellular vesicles using density gradient separation and size exclusion chromatography |
| topic | extracellular vesicles tissue-derived extracellular vesicles liver adipose tissue density gradient size exclusion chromatography |
| url | https://www.mdpi.com/1422-0067/24/16/12704 |
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