A microfluidic platform for multi-marker profiling of extracellular vesicles from single-cell-derived clones

Area of Science:

• Biotechnology
• Cell Biology
• Nanotechnology

Background:

• Extracellular vesicles (EVs) are crucial intercellular communicators, but bulk analysis methods obscure their inherent heterogeneity.
• Understanding single-cell-derived EV profiles is vital for accurate biological interpretation and therapeutic applications.

Purpose of the Study:

• To develop a microfluidic platform for high-throughput, multi-marker profiling of EVs from single-cell-derived clones.
• To investigate the heterogeneity of EV marker expression and its correlation with cellular characteristics.

Main Methods:

• A semi-open microfluidic platform with 17,305 wells was designed to capture EVs from single-cell clones.
• EVs were immunolabeled for tetraspanin markers (CD9, CD63, CD81) and EpCAM, then analyzed via fluorescence microscopy and automated image analysis.
• Single-cell-derived PC3 clones were utilized to assess EV heterogeneity and marker co-expression.

Main Results:

• The platform enabled multi-marker profiling of EVs at a near single-EV level, linked to specific clonal lineages.
• Substantial heterogeneity in EV tetraspanin marker co-expression was observed, with four distinct profiles identified.
• EpCAM-positive EV fraction correlated with PC3 cell proliferation, unlike free EpCAM.

Conclusions:

• The developed platform effectively dissects cellular and EV heterogeneity at the single-cell lineage level.
• This approach provides a practical method for linking EV signatures to their single-cell origins.
• Findings highlight the importance of single-cell-based analysis for understanding EV biology.