A comprehensive technical system covering sample preparation, EV isolation, multi-dimensional characterization, cargo analysis, functional validation and mechanistic dissection has been established around the biogenesis, isolation, characterization, molecular composition and biological functions of extracellular vesicles (EVs). It provides systematic research support for basic exosome studies, disease mechanism investigation and translational applications.

1.1 Cell Source & Sample Preparation

Select cells or biological samples of different origins according to research objectives, and establish standardized workflows for EV collection and sample processing.

Cell sources: mesenchymal stem cells (MSCs), immune cells, tumor cells, epithelial cells, stem cells, primary cells

Sample types: cell culture supernatant, conditioned medium, blood, urine, saliva, cerebrospinal fluid, tissue-derived samples

1.2 EV Isolation & Enrichment

Select appropriate isolation and enrichment strategies based on sample origin, EV properties and downstream research requirements to achieve efficient recovery and further purification of EVs.

Major techniques: ultracentrifugation, size-exclusion chromatography (SEC), precipitation, ultrafiltration, immunoaffinity capture, density gradient centrifugation

1.3 Multi-Dimensional EV Characterization

Comprehensively characterize EV samples from multiple dimensions including particle size & concentration, morphological structure and protein markers to evaluate basic physicochemical properties and EV-associated features.

Including: particle size and concentration analysis, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), morphological analysis (transmission electron microscopy (TEM), used to observe typical vesicular morphology and ultrastructure of EVs), protein marker analysis (Western Blot, ELISA / immunoassays).

Common EV-associated markers: CD9, CD63, CD81, TSG101, ALIX. Non-EV proteins are used as controls to assess EV sample profiles and potential co-isolated contaminants from multiple perspectives.

1.4 Analysis of EV Cargo and Molecular Composition

EVs carry proteins, lipids and diverse nucleic acid molecules. Analysis of EV cargo via multi-omics and molecular biology techniques helps decipher biological information carried by EVs and their potential functions.

Ø Proteins (protein expression analysis, ELISA, proteomics)

Ø RNA (miRNA, mRNA, lncRNA, circRNA, RNA sequencing)

Ø DNA (EV-associated DNA, DNA sequencing)

Ø Lipids (lipid composition analysis, lipidomics)

By comparing variations in EV cargo across different sources, treatment conditions or disease states, potential functional molecules and disease-related biomarkers can be further screened.

1.5 Biological Functional Validation

Evaluate the biological activity and functional effects of EVs at the cellular level through experiments including recipient cell uptake, cellular behaviors and immune-inflammatory responses.

Ø Cellular uptake (fluorescent labeling, confocal imaging, flow cytometry): assess EV-recipient cell interactions and intracellular delivery processes.

Ø Cellular functions (cell proliferation, cell migration, cell viability, cell differentiation, cell apoptosis)

Ø Immunomodulation (cytokine detection, macrophage polarization, T cell function, inflammatory signaling pathways)

Ø Tissue repair-related functions (scratch wound assay, Transwell migration assay, angiogenesis assay, extracellular matrix analysis): evaluate the regulatory effects of EVs on recipient cell behaviors and tissue microenvironment using diverse functional models.

1.6 Mechanistic Dissection and Multi-Omics Research

On the basis of EV characterization and functional validation, combined with transcriptomics, proteomics and signaling pathway analysis, the molecular links between EV cargo and functional alterations in recipient cells can be further dissected.

Overview of the EV Research Workflow. A streamlined workflow covering sample preparation, EV isolation and enrichment, multidimensional characterization, cargo analysis, biological function validation, and mechanism and multi-omics analysis.

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