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Background <p>Extracellular vesicles (EVs) are membrane-bound particles crucial for intercellular communication and serve as promising biomarkers for diseases, including cancer. Isolating and characterizing specific EV subpopulations, particularly those in plasma/serum, enhances biomarker precision and supports targeted therapies. Cancer-derived EVs often express unique surface markers, enabling distinction from other EVs. Accurate sorting of tumor-associated EVs provides insights into cancer progression, metastasis, and treatment response.</p> Results <p>This study presents a robust method for isolating and sorting CD9 + plasma EVs as a proof-of-concept for broader EV subpopulation analyses. Plasma EVs were isolated via sucrose cushion ultracentrifugation, optimizing purity and yield. Flow cytometry with fluorescence threshold triggering was fine-tuned to detect and sort CD9 + EVs, with instrument calibration and parameter adjustments mitigating swarming and improving sorting accuracy. Size exclusion chromatography further enhanced efficiency by reducing background noise. Sorted CD9 + EVs retained size and marker expression, including Syntenin, Alix, Flotillin-1, and CD9, which were enriched post-sorting.</p> Conclusions <p>These advancements enable high-purity EV subpopulation isolation, facilitating applications such as identifying cancer biomarkers and developing EV-based targeted therapies.</p>

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Refining Flow Cytometry-based Sorting of Plasma-derived Extracellular Vesicles

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月20日

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  • Daniele Reverberi,
  • Maria Chiara Ciferri,
  • Nicole Rosenwasser,
  • Alessandro Catino,
  • Giuseppina Poppa,
  • Ilaria Giusti,
  • Vincenza Dolo,
  • Rodolfo Quarto,
  • Sara Santamaria,
  • Monica Colombo,
  • Simona Coco,
  • Roberta Tasso

摘要

Background

Extracellular vesicles (EVs) are membrane-bound particles crucial for intercellular communication and serve as promising biomarkers for diseases, including cancer. Isolating and characterizing specific EV subpopulations, particularly those in plasma/serum, enhances biomarker precision and supports targeted therapies. Cancer-derived EVs often express unique surface markers, enabling distinction from other EVs. Accurate sorting of tumor-associated EVs provides insights into cancer progression, metastasis, and treatment response.

Results

This study presents a robust method for isolating and sorting CD9 + plasma EVs as a proof-of-concept for broader EV subpopulation analyses. Plasma EVs were isolated via sucrose cushion ultracentrifugation, optimizing purity and yield. Flow cytometry with fluorescence threshold triggering was fine-tuned to detect and sort CD9 + EVs, with instrument calibration and parameter adjustments mitigating swarming and improving sorting accuracy. Size exclusion chromatography further enhanced efficiency by reducing background noise. Sorted CD9 + EVs retained size and marker expression, including Syntenin, Alix, Flotillin-1, and CD9, which were enriched post-sorting.

Conclusions

These advancements enable high-purity EV subpopulation isolation, facilitating applications such as identifying cancer biomarkers and developing EV-based targeted therapies.