Comprehensive isolation of extracellular vesicles and nanoparticles.

Area of Science:

• Extracellular vesicle (EV) and nanoparticle research
• Biomolecular isolation and characterization

Background:

• Growing recognition of the heterogeneity within extracellular vesicles (EVs).
• Discovery of nonvesicular extracellular nanoparticles (NVEPs) like exomeres and supermeres, which share cargo with EVs.
• Limited methods for distinguishing between EVs and NVEPs, hindering cargo attribution.

Purpose of the Study:

• To present a comprehensive protocol for the sequential isolation of distinct EV and NVEP populations.
• To enable accurate assignment of biomolecular cargo to its specific extracellular carrier.
• To facilitate research into EV and NVEP biogenesis, function, and biomarker discovery.

Main Methods:

• Sequential isolation using differential ultracentrifugation, filtration, concentration, and high-resolution density-gradient fractionation.
• Application to both cell culture medium and human plasma.
• Protocol completion time of approximately 72 hours.

Main Results:

• Purified fractions of large EVs, small EVs, nonvesicular fractions, exomeres, and supermeres obtained from a single starting material.
• Demonstrated high purity and reproducibility of the isolation method.
• Minimal expertise required for protocol execution.

Conclusions:

• The protocol provides a robust method for separating and purifying diverse extracellular nanoparticle populations.
• Enables precise assignment of biomolecules to specific extracellular compartments (EVs vs. NVEPs).
• Supports translational research for biomarker identification and basic science understanding of extracellular particle biology.