Title:Comparison of the Characteristics of Circulating Small
Extracellular Vesicles Isolated by Ultracentrifugation and a
Commercial Kit
Volume: 19
Issue: 4
Author(s): Reza Afrisham, Vida Farrokhi, Roya Moradi and Shaban Alizadeh*
Affiliation:
- Department of Hematology and Transfusion Sciences, School of Allied Medical
Sciences, Tehran University of Medical Sciences, Tehran, Iran
Keywords:
Exosome, extracellular vesicles, ultracentrifugation, extracellular vesicles extraction kit, extracellular vesicles isolation, commercial kit, plasma.
Abstract:
Introduction: The market offers a wide range of extracellular vesicles (EVs)
isolation products, but their lack of standardization is a concern. Therefore, it is important
to carefully assess the quality of the EVs obtained using these products to patent
the ideal method. In this study, we compared the EXOCIB kit with the ultracentrifuge
method, which is considered the gold standard for small EV isolation.
Methods: After overnight fasting, small plasma EVs were extracted from four individuals
using both the ultracentrifuge and the EXOCIB kit methods. The pooled EVs were
then compared for the presence of the cluster of differentiation 63 (CD63) protein using
the western blot analysis, and their size and zeta potential were performed by Dynamic
Light Scattering (DLS). In addition, the size and morphology of small EVs were determined
by using the Transmission Electron Microscopy (TEM) technique.
Results: An average hydrodynamic size of 135.7 nm and a zeta potential of -6.33 Mv at
25°C was found for small EVs isolated by the ultracentrifuge, whereas the kit method resulted
in small EVs with a hydrodynamic size of 102.8 nm and a zeta potential of -0.907.
Notably, the size of the particles in the kit samples was smaller compared to those obtained
through the ultracentrifuge (P < 0.001). The western blot method confirmed the
expression of CD63 in both methods, so the ultracentrifuge yielded small EVs with a
higher level of purity compared to the kit-based approach (P = 0.036).
Conclusion: The DLS findings revealed the existence of vesicles within the appropriate
size range for small EVs like exosomes in both isolation techniques. The results of the
western blot analysis, in conjunction with DLS, displayed that the ultracentrifuge method
extracted small EVs with a greater degree of purity than the kit-based approach.
