Analysis of individual extracellular vesicles by imaging flow cytometry

Area of Science:

• Cell Biology
• Biomarker Discovery
• Nanotechnology

Background:

• Extracellular vesicles (EVs), including exosomes and microvesicles, mediate intercellular communication and carry cell-specific signatures, making them potential biomarkers and drug delivery vehicles.
• Current methods like nanoparticle tracking analysis and Western Blot lack the resolution to analyze EV heterogeneity.
• Electron microscopy visualizes EV heterogeneity but is not suitable for complex, quantitative analysis.

Purpose of the Study:

• To present a protocol for analyzing single small EVs (sEVs) using imaging flow cytometry.
• To demonstrate the potential of imaging flow cytometry for dissecting EV heterogeneity at the single vesicle level.
• To study the expression of tetraspanins CD9, CD63, and CD81 on single sEVs from human mesenchymal stromal cells (MSCs).

Main Methods:

• Utilized imaging flow cytometry (FCM) for high-resolution analysis of single EVs.
• Applied FCM to sEVs derived from human mesenchymal stromal cell (MSC) supernatants.
• Quantified the expression of specific tetraspanin markers (CD9, CD63, CD81) on individual sEVs.

Main Results:

• Demonstrated that imaging FCM can detect and analyze sEVs in the 70-150nm range, overcoming limitations of classical FCM.
• Successfully characterized the expression profiles of CD9, CD63, and CD81 on single sEVs.
• Provided a validated protocol for single EV analysis using imaging FCM.

Conclusions:

• Imaging FCM is a powerful tool for analyzing the heterogeneity of small EVs at the single vesicle level.
• This method enables quantitative assessment of EV surface markers, crucial for biomarker and therapeutic applications.
• The developed protocol facilitates deeper understanding of EV biology and potential clinical utility.