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Related Experiment Video

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Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants
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Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy.

Sagar Rayamajhi1, Benjamin K Gibbs1, Jared Sipes1

  • 1Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160, United States.

ACS Applied Bio Materials
|November 1, 2024
PubMed
Summary

Researchers developed a minimally invasive method to track extracellular vesicles (EVs) using PicoGreen (PG). This new technique allows for efficient EV tracking in various models without altering their natural function, aiding cell communication and biomarker discovery.

Keywords:
EV-trackingPicoGreen-based EV-labelingdsDNA intercalatordual-labeled EVsmCherry EVsminimally invasive EV-labeling

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Area of Science:

  • Cell Biology
  • Biotechnology
  • Nanotechnology

Background:

  • Extracellular vesicles (EVs) are crucial for intercellular communication, influencing normal and pathological processes.
  • Tracking EVs is vital for understanding cell communication, early cancer detection, and biomarker discovery.
  • Current EV tracking methods are often invasive, potentially altering EV function and limiting their use in functional studies.

Purpose of the Study:

  • To develop and validate a minimally invasive extracellular vesicle (EV) labeling and tracking strategy.
  • To assess the efficacy of PicoGreen (PG) for labeling EVs based on their associated dsDNA.
  • To evaluate the utility of PG-labeled EVs (PG-EVs) in various biological models.

Main Methods:

  • Utilized PicoGreen (PG), a dsDNA-binding fluorescent molecule, for minimally invasive EV labeling.
  • Characterized PG-EVs for stability and fluorescence properties.
  • Validated PG-EV tracking in 2D cell cultures and 3D organoid models, employing dual-labeling strategies including surface amine bioconjugation and donor cell engineering with fluorescent reporters (mCherry-tetraspanin).

Main Results:

  • PicoGreen (PG) effectively binds to dsDNA within small extracellular vesicles (50-200 nm), forming stable, fluorescent PG-EVs.
  • PG-EVs demonstrated efficient tracking with high signal-to-noise ratios, time- and concentration-dependent uptake, and successful navigation within 3D environments.
  • Orthogonal validation confirmed the reliability of PG-EV tracking compared to established labeling methods.

Conclusions:

  • PicoGreen (PG) offers a minimally invasive and effective strategy for labeling and tracking extracellular vesicles (EVs).
  • PG-EVs maintain their natural characteristics, enabling robust functional studies across diverse model systems.
  • This approach holds significant potential for advancing research in cell communication, disease mechanisms, and biomarker development.