Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry

Area of Science:

• Biomedical engineering
• Nanotechnology
• Cell biology

Background:

• Extracellular vesicles (EVs) hold significant promise for biomedical applications.
• Clinical translation of EVs is hindered by the absence of standardized methods for isolation, characterization, and storage.
• High-sensitivity flow cytometry (HS-FCM) offers single-EV resolution for assessing EV isolation and storage.

Purpose of the Study:

• To compare the yield and purity of EV fractions isolated using two distinct methods.
• To evaluate the impact of storage conditions on EV concentration and fluorescent label stability.
• To assess the utility of HS-FCM in characterizing EV isolation and storage.

Main Methods:

• EVs were isolated from lipopolysaccharide-stimulated THP-1 cells using differential centrifugation/ultracentrifugation and exoEasy membrane affinity spin columns.
• EV characterization involved HS-FCM and nanoparticle tracking analysis (NTA).
• EV fractions were stored at 4°C and -80°C for up to one month to assess stability.

Main Results:

• Differences in EV yield and purity were observed between the two isolation methods.
• EV concentration remained stable under tested storage conditions (4°C and -80°C).
• Fluorescence intensity of labeled EVs stored at 4°C showed a decline within one day.

Conclusions:

• The choice of EV isolation method impacts yield and purity.
• While EV concentration is stable, fluorescent label integrity is compromised by short-term 4°C storage.
• HS-FCM is a valuable tool for evaluating EV isolation and storage protocols, guiding future method development for reproducible EV analysis.