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.
Based on MISEV2023 guidelines, this article provides clear and practical guidance for newcomers.
This serves as a “workflow navigation” to help you understand the fundamental steps of EV research.
For in-depth technical details, refer to these articles:
• How to Purify Exosomes Using Tangential Flow Filtration (TFF)?
Extracellular vesicles (EVs) 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:
Exosomes | Microvesicles | Apoptotic Bodies | |
|---|---|---|---|
 |  |  | |
| Size | 40–120 nm | 100–1000 nm | 1000–5000 nm |
| Biogenesis | From multivesicular bodies (MVB), via endosomal pathway | Plasma membrane, via outward budding | Plasma membrane, during apoptosis |
| Markers & Composition | Tetraspanins (CD9, CD63, CD81), Alix, Tsg101, flotillin-1 | Selectins, Integrins | DNA, histones, organelles, nuclear fractions, Annexin V |
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, such as sEV (small EVs), lEV (large EVs), or CD63+ EVs.
Exosomes 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 40–120 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’ function and participating in many physiological and pathological processes.
EVs research involves several key steps—from sample collection to analysis and storage—all of which affect EV quality.
Purpose: Obtain biological materials containing EVs and remove cells or contaminants as early as possible.
Materials: EVs can be collected from cell-conditioned medium, bacteria, blood, urine, milk, and more.
General Recommendations:
• Report sample source, volume/weight, collection, and storage details.
• Remove cells early to avoid artifacts or compositional changes caused by damaged cells.
• Pre-process samples before storage to eliminate interfering substances.
• Avoid repeated freeze–thaw cycles.
• Document all storage conditions, preservatives, cryoprotectants, temperatures, durations, and thawing procedures.
* For sample-specific recommendations, refer to MISEV2023.
Purpose: Isolate and concentrate EVs from biological materials while minimizing non-EV contaminants such as free proteins or debris.
Methods: Common techniques include differential ultracentrifugation (dUC), density gradient (DG), ultrafiltration (UF), size exclusion chromatography (SEC), and precipitation methods.
General Recommendations:
• Choose methods according to sample type, research goal, and required purity/yield.
• Combine complementary techniques for higher purity.
• Pre-concentrate large volumes (e.g., urine, milk) before isolation.
• Prefer kits with transparent protocols.
• Report purity and recovery data.
• Provide full methodological details for reproducibility.
• Evaluate enrichment fold and yield after each step.
Purpose: Confirm that isolated particles are indeed EVs by analyzing size, morphology, and biomarkers.
Methods: Quantify particle concentration, protein/lipid/RNA content, and morphological features.
General Recommendations:
• Characterize EVs on three levels: (1) physical properties, (2) EV markers, and (3) non-EV contaminants.
• Use orthogonal methods with different detection limits.
• Report instruments, parameters, and data analysis methods.
• Define yield relative to biological source (cell count, volume, or tissue weight).
• Quantify particle count, protein/lipid content.
• Verify EV subtype-specific components when needed.
• Report LOD (limit of detection).
• Use standards, negative controls, and cross-platform validation.
Purpose: Maintain EV integrity and stability for downstream use. Improper storage can alter EV properties, stability, and functionality.
General Recommendations:
• Report all storage details (preservatives, temperature, duration, thaw cycles).
• Avoid repeated freeze–thaw cycles; aliquot when possible.
• Rapid freezing and thawing are recommended to preserve EV morphology and function.
Due to their complexity, EVs research often traps beginners in common mistakes:
A1: ISEV recommends using the general term “EVs” when subcellular origin is not confirmed, and naming based on physical characteristics, biochemical composition, or cellular origin to avoid confusion.
A2: No single method yields completely pure EVs. Choose based on sample type and research purpose. Combining complementary purification techniques can improve both yield and purity.
A3: Validate through multiple approaches: (1) physical properties (size/concentration), (2) EV-specific markers (transmembrane or GPI-anchored proteins), and (3) negative markers (non-EV co-isolated proteins) to assess purity.
This article was originally published by our partners Rocker Scientific. View the original article here
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