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Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

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Human saliva-derived exosomes: comparing methods of isolation.

Ayelet Zlotogorski-Hurvitz¹, Dan Dayan¹, Gavriel Chaushu^2,3

¹Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel (AZH, DD, MV)

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society

|December 5, 2014

Summary

ExoQuick-TC (EQ) efficiently precipitates salivary exosomes from small volumes. However, this chemical method yields more biological impurities compared to ultracentrifugation (UC).

Keywords:

ExoQuick exosomes extracellular vesicles isolation saliva ultracentrifugation

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Area of Science:

Biochemistry
Nanotechnology
Biomarker Discovery

Background:

Exosomes in saliva are valuable biomarkers.
Current isolation methods like ultracentrifugation (UC) can be time-consuming and require large sample volumes.
Novel precipitation agents offer potential for faster, more accessible exosome isolation.

Purpose of the Study:

To evaluate ExoQuick-TC (EQ), a chemical precipitation agent, for isolating exosomes from human saliva.
To compare the morphological and molecular characteristics of exosomes isolated by EQ with those isolated by ultracentrifugation (UC).

Main Methods:

Saliva samples from healthy individuals were used.
Exosomes were precipitated using ExoQuick-TC (EQ) and ultracentrifugation (UC).
Morphological analysis was performed using electron microscopy and atomic force microscopy.
Molecular characterization involved ELISA and Western blotting for exosomal markers (CD63, CD9, CD81) and a non-exosomal marker (EGFR).

Main Results:

Electron microscopy confirmed vesicular nanoparticles with bilayer membranes in both EQ and UC preparations.
Atomic force microscopy revealed larger, aggregated nanoparticles with EQ, contrasting with the round UC nanoparticles.
ELISA showed a trend towards higher expression of exosomal markers with EQ, but significantly higher concentration of a non-exosomal marker (EGFR).
Western blotting indicated less pronounced exosomal marker bands in EQ preparations, suggesting higher protein impurities.

Conclusions:

ExoQuick-TC (EQ) is effective for precipitating salivary exosomes from small saliva volumes.
EQ isolation is associated with a higher degree of non-exosomal protein and microvesicle contamination compared to UC.
Further purification steps may be necessary when using EQ for sensitive downstream applications.