Comparison of Methods for Concentration Assessment of Extracellular Vesicles Isolated from Different Biological...

Area of Science:

• Biomedical Research
• Nanotechnology
• Analytical Chemistry

Background:

• Accurate quantification of extracellular vesicles (EVs) is crucial for biomedical research but remains challenging.
• Existing methods are often unreliable due to non-vesicular nanoparticles and biological contaminants in samples.
• Quantitative evaluation of EVs from sources like uterine aspirates and gastric juice is largely unexplored.

Purpose of the Study:

• To comparatively analyze three EV quantification methods: total protein content, nanoparticle tracking analysis (NTA), and esterase activity.
• To assess these methods in EVs isolated from diverse biological fluids including blood plasma, ascitic fluid, uterine aspirates, gastric juice, and cancer cell-conditioned media.
• To identify method-specific limitations and sample-dependent variations in EV quantification.

Main Methods:

• Isolation of EVs from blood plasma, ascitic fluid, uterine aspirates, gastric juice, and conditioned media (ovarian and non-small cell lung cancer cells).
• Quantification using total protein content measurement.
• Quantification using Nanoparticle Tracking Analysis (NTA).
• Quantification using esterase activity assay (commercial FluoroCet kit).

Main Results:

• All three methods showed strong correlation for EVs from conditioned media, validating their use for in vitro studies with purified samples.
• Discrepancies were observed between methods for blood plasma, ascitic fluid, and uterine aspirates, likely due to non-vesicular nanoparticles.
• EVs from gastric juice showed strong correlation between protein content and esterase activity, suggesting unique composition.

Conclusions:

• The choice of EV quantification method significantly impacts results and depends on the biological fluid source.
• A multifactorial approach considering sample origin and method limitations is necessary for reliable EV quantification.
• Findings provide a basis for developing standardized EV quantification protocols, especially for clinical samples.