Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods
Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are s...
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| Format: | Article |
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MDPI AG
2022-03-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/10/4/785 |
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| author | Vedrana Krušić Alić Mladenka Malenica Maša Biberić Siniša Zrna Lara Valenčić Aleksandar Šuput Lada Kalagac Fabris Karmen Wechtersbach Nika Kojc Mario Kurtjak Natalia Kučić Kristina Grabušić |
| author_facet | Vedrana Krušić Alić Mladenka Malenica Maša Biberić Siniša Zrna Lara Valenčić Aleksandar Šuput Lada Kalagac Fabris Karmen Wechtersbach Nika Kojc Mario Kurtjak Natalia Kučić Kristina Grabušić |
| author_sort | Vedrana Krušić Alić |
| collection | DOAJ |
| description | Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins. |
| first_indexed | 2024-03-09T11:07:11Z |
| format | Article |
| id | doaj.art-7395201b79dd4cf0bdf657cf085529e0 |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-09T11:07:11Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-7395201b79dd4cf0bdf657cf085529e02023-12-01T00:53:18ZengMDPI AGBiomedicines2227-90592022-03-0110478510.3390/biomedicines10040785Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography MethodsVedrana Krušić Alić0Mladenka Malenica1Maša Biberić2Siniša Zrna3Lara Valenčić4Aleksandar Šuput5Lada Kalagac Fabris6Karmen Wechtersbach7Nika Kojc8Mario Kurtjak9Natalia Kučić10Kristina Grabušić11Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, CroatiaDepartment of Medical Chemistry, Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Rijeka, 51000 Rijeka, CroatiaGeneral Hospital Pula, 52100 Pula, CroatiaGeneral Hospital Pula, 52100 Pula, CroatiaDepartment of Anaesthesia and Intensive Care Medicine, Clinical Hospital Centre Rijeka, 51000 Rijeka, CroatiaGeneral Hospital Pula, 52100 Pula, CroatiaGeneral Hospital Pula, 52100 Pula, CroatiaInstitute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, SloveniaInstitute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, SloveniaAdvanced Materials Department, Jožef Stefan Institute, 1000 Ljubljana, SloveniaDepartment of Physiology and Immunology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, CroatiaDepartment of Physiology and Immunology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, CroatiaExtracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.https://www.mdpi.com/2227-9059/10/4/785extracellular vesiclescerebrospinal fluidtraumatic brain injurysize exclusion chromatographyCD9 proteinCD81 protein |
| spellingShingle | Vedrana Krušić Alić Mladenka Malenica Maša Biberić Siniša Zrna Lara Valenčić Aleksandar Šuput Lada Kalagac Fabris Karmen Wechtersbach Nika Kojc Mario Kurtjak Natalia Kučić Kristina Grabušić Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods Biomedicines extracellular vesicles cerebrospinal fluid traumatic brain injury size exclusion chromatography CD9 protein CD81 protein |
| title | Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods |
| title_full | Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods |
| title_fullStr | Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods |
| title_full_unstemmed | Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods |
| title_short | Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods |
| title_sort | extracellular vesicles from human cerebrospinal fluid are effectively separated by sepharose cl 6b comparison of four gravity flow size exclusion chromatography methods |
| topic | extracellular vesicles cerebrospinal fluid traumatic brain injury size exclusion chromatography CD9 protein CD81 protein |
| url | https://www.mdpi.com/2227-9059/10/4/785 |
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