Comparing isolation methods of human saliva-derived exosomes

ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC).

exosome

Characterization of salivary exosomes isolated by two methods. Electron micrographs of exosomes isolated from saliva by ExoQuick-TCTM (EQ) (A) and the classical ultracentrifugation (UC) method (B) demonstrating small vesicles of different sizes (ranging between 30–120 nm) with lipid bilayer membranes. Electron micrographs stained with 10-nm gold-conjugated anti-CD63 antibody and uranyl acetate counterstaining in EQ (C–E) and UC (F–H) preparations.

Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC.

Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles.

ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04).

Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ.

In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.

Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, Vered M. (2015) Human saliva-derived exosomes: comparing methods of isolation. J Histochem Cytochem 63(3):181-9. [abstract]

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