Dataset Information

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

ABSTRACT: 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.

SUBMITTER: Krusic Alic V

PROVIDER: S-EPMC9032713 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Publications

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

Krušić Alić Vedrana V Malenica Mladenka M Biberić Maša M Zrna Siniša S Valenčić Lara L Šuput Aleksandar A Kalagac Fabris Lada L Wechtersbach Karmen K Kojc Nika N Kurtjak Mario M Kučić Natalia N Grabušić Kristina K

Biomedicines 20220327 4

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 inj ...[more]

PMID: 35453535

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Dataset Information

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

Publications

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

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