{"id":27904,"date":"2025-10-16T14:28:31","date_gmt":"2025-10-16T06:28:31","guid":{"rendered":"https:\/\/www.rocker.com.tw\/?post_type=application&p=27904"},"modified":"2026-04-21T15:12:12","modified_gmt":"2026-04-21T07:12:12","slug":"27904-2","status":"publish","type":"application","link":"https:\/\/www.rocker.com.tw\/en\/application\/27904-2\/","title":{"rendered":"EVs Research Workflow"},"content":{"rendered":"

EVs Research Beginner\u2019s Guide: 4 Steps from Sample to Results<\/span><\/strong><\/h1>\n

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Extracellular vesicles (EVs) have gained increasing attention in biology and medicine due to their great potential as biomarkers and therapeutic tools. However, the heterogeneity and complexity of EVs also bring many challenges to beginners.<\/p>\n

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Table of Contents<\/strong><\/span><\/h2>\n

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Why Read This First?<\/a>
\nWhat Are Extracellular Vesicles and Exosomes?<\/a>
\nEVs Research Workflow<\/a>
\nCommon Pitfalls for Beginners<\/a>
\nFAQ<\/a><\/p>\n<\/div>\n

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<\/a>Why Read This First?<\/strong><\/span><\/h2>\n

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  1. Based on MISEV2023 guidelines, this article provides clear and practical guidance for newcomers.<\/li>\n
  2. This serves as a \u201cworkflow navigation\u201d<\/strong> to help you understand the fundamental steps of EV research.<\/li>\n
  3. For in-depth technical details, refer to these articles:
    \n    \u2022 How to Purify Exosomes Using Tangential Flow Filtration (TFF)?<\/strong><\/a><\/p>\n

    \u2022 <\/a>From Discovery to Validation: Why Do We Need \u201cExosome Standards\u201d? (in Chinese)<\/a><\/strong><\/p>\n<\/li>\n<\/ol>\n

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    <\/a>What Are Extracellular Vesicles and Exosomes?<\/span><\/span><\/strong><\/h2>\n

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    Extracellular vesicles (EVs)<\/strong> are particles released from cells, delimited by a lipid bilayer, and incapable of self-replication. Early studies classified EVs into three main types based on their biogenesis and size:<\/p>\n

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    Exosomes<\/p>\n<\/th>\n

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    Microvesicles<\/p>\n<\/th>\n

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    Apoptotic Bodies<\/p>\n<\/th>\n<\/tr>\n

    \u00a0<\/td>\n\"\"<\/td>\n\"\"<\/td>\n\"\"<\/td>\n<\/tr>\n
    Size<\/th>\n40\u2013120 nm<\/td>\n100\u20131000 nm<\/td>\n1000\u20135000 nm<\/td>\n<\/tr>\n
    Biogenesis<\/th>\nFrom multivesicular bodies (MVB), via endosomal pathway<\/td>\nPlasma membrane, via outward budding<\/td>\nPlasma membrane, during apoptosis<\/td>\n<\/tr>\n
    Markers & Composition<\/th>\nTetraspanins (CD9, CD63, CD81), Alix, Tsg101, flotillin-1<\/td>\nSelectins, Integrins<\/td>\nDNA, histones, organelles, nuclear fractions, Annexin V<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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    However, according to the International Society for Extracellular Vesicles (ISEV) in MISEV2018 and MISEV2023, if the biogenesis is unknown, it is recommended to use operational terms based on physical characteristics, surface markers, cellular origin, or morphology<\/strong>, such as sEV (small EVs), lEV (large EVs), or CD63+ EVs.<\/p>\n

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    Exosomes<\/strong> refer specifically to EVs derived from the endosomal system and released via multivesicular bodies (MVBs). However, since it is often difficult to directly prove the biogenesis, the term should not be used without evidence. Their typical size ranges from 40\u2013120 nm, and they are characterized by tetraspanins (CD9, CD63, CD81). Exosomes carry molecular information such as DNA, RNA, lipids, and proteins from parent cells, influencing recipient cells\u2019 function and participating in many physiological and pathological processes.<\/p>\n

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    <\/a>EVs Research Workflow<\/strong><\/span><\/h2>\n

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    EVs research involves several key steps\u2014from sample collection to analysis and storage\u2014all of which affect EV quality.<\/p>\n

    \"EVs<\/p>\n

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    1. Collection and Pre-Processing<\/strong><\/span><\/h3>\n

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    Purpose:<\/strong> Obtain biological materials containing EVs and remove cells or contaminants as early as possible.<\/p>\n

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    Materials:<\/strong> EVs can be collected from cell-conditioned medium, bacteria, blood, urine, milk, and more.<\/p>\n

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    General Recommendations:<\/strong>
    \n\u2022 Report sample source, volume\/weight, collection, and storage details.
    \n\u2022 Remove cells early to avoid artifacts or compositional changes caused by damaged cells.
    \n\u2022 Pre-process samples before storage to eliminate interfering substances.
    \n\u2022 Avoid repeated freeze\u2013thaw cycles.
    \n\u2022 Document all storage conditions, preservatives, cryoprotectants, temperatures, durations, and thawing procedures.
    \n* For sample-specific recommendations, refer to MISEV2023.<\/span><\/p>\n

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    2. EV Separation and Concentration<\/strong><\/span><\/h3>\n

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    Purpose:<\/strong> Isolate and concentrate EVs from biological materials while minimizing non-EV contaminants such as free proteins or debris.<\/p>\n

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    Methods:<\/strong> Common techniques include differential ultracentrifugation (dUC), density gradient (DG), ultrafiltration (UF), size exclusion chromatography (SEC), and precipitation methods.<\/p>\n

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    General Recommendations:<\/strong>
    \n\u2022 Choose methods according to sample type, research goal, and required purity\/yield.
    \n\u2022 Combine complementary techniques for higher purity.
    \n\u2022 Pre-concentrate large volumes (e.g., urine, milk) before isolation.
    \n\u2022 Prefer kits with transparent protocols.
    \n\u2022 Report purity and recovery data.
    \n\u2022 Provide full methodological details for reproducibility.
    \n\u2022 Evaluate enrichment fold and yield after each step.<\/p>\n

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    Further Reading:<\/strong> How to Purify Exosomes Using TFF?<\/a><\/p>\n

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    3. EV Characterization<\/strong><\/span><\/h3>\n

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    Purpose:<\/strong> Confirm that isolated particles are indeed EVs by analyzing size, morphology, and biomarkers.<\/p>\n

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    Methods:<\/strong> Quantify particle concentration, protein\/lipid\/RNA content, and morphological features.<\/p>\n

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    General Recommendations:<\/strong>
    \n\u2022 Characterize EVs on three levels: (1) physical properties, (2) EV markers, and (3) non-EV contaminants.
    \n\u2022 Use orthogonal methods with different detection limits.
    \n\u2022 Report instruments, parameters, and data analysis methods.
    \n\u2022 Define yield relative to biological source (cell count, volume, or tissue weight).
    \n\u2022 Quantify particle count, protein\/lipid content.
    \n\u2022 Verify EV subtype-specific components when needed.
    \n\u2022 Report LOD (limit of detection).
    \n\u2022 Use standards, negative controls, and cross-platform validation.<\/p>\n

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    Further Reading:<\/strong> Why Do We Need Exosome Standards? <\/a>(in Chinese)<\/a><\/p>\n

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    4. Storage<\/strong><\/span><\/h3>\n

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    Purpose:<\/strong> Maintain EV integrity and stability for downstream use. Improper storage can alter EV properties, stability, and functionality.<\/p>\n

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    General Recommendations:<\/strong>
    \n\u2022 Report all storage details (preservatives, temperature, duration, thaw cycles).
    \n\u2022 Avoid repeated freeze\u2013thaw cycles; aliquot when possible.
    \n\u2022 Rapid freezing and thawing are recommended to preserve EV morphology and function.<\/p>\n

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    <\/a>Common Pitfalls for Beginners<\/span><\/strong><\/h2>\n

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    Due to their complexity, EVs research often traps beginners in common mistakes:<\/p>\n

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