Enrichment protocols for human conjunctival extracellular vesicles and their characterization
Abstract The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study t...
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Nature Portfolio
2024-11-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-79481-1 |
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| author | Ismael Romero-Castillo Antonio López-García Yolanda Diebold Laura García-Posadas |
| author_facet | Ismael Romero-Castillo Antonio López-García Yolanda Diebold Laura García-Posadas |
| author_sort | Ismael Romero-Castillo |
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| description | Abstract The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment. |
| format | Article |
| id | doaj-art-9aef1dd226d84d67a96be9db7034bc48 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-11-01 |
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| spelling | doaj-art-9aef1dd226d84d67a96be9db7034bc482024-11-17T12:21:17ZengNature PortfolioScientific Reports2045-23222024-11-0114111310.1038/s41598-024-79481-1Enrichment protocols for human conjunctival extracellular vesicles and their characterizationIsmael Romero-Castillo0Antonio López-García1Yolanda Diebold2Laura García-Posadas3Ocular Surface Group, Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de ValladolidOcular Surface Group, Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de ValladolidOcular Surface Group, Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de ValladolidOcular Surface Group, Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de ValladolidAbstract The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment.https://doi.org/10.1038/s41598-024-79481-1ConjunctivaExtracellular vesiclesEV isolationOcular surfaceEpithelial cellsMesenchymal stromal cell EVs |
| spellingShingle | Ismael Romero-Castillo Antonio López-García Yolanda Diebold Laura García-Posadas Enrichment protocols for human conjunctival extracellular vesicles and their characterization Scientific Reports Conjunctiva Extracellular vesicles EV isolation Ocular surface Epithelial cells Mesenchymal stromal cell EVs |
| title | Enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| title_full | Enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| title_fullStr | Enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| title_full_unstemmed | Enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| title_short | Enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| title_sort | enrichment protocols for human conjunctival extracellular vesicles and their characterization |
| topic | Conjunctiva Extracellular vesicles EV isolation Ocular surface Epithelial cells Mesenchymal stromal cell EVs |
| url | https://doi.org/10.1038/s41598-024-79481-1 |
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