Exosomes require characterization and validation prior to therapeutic applications.
Current methods for exosome characterization primarily focus on the size, morphology, and cargo profile of exosomes. Size-oriented validation includes nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and tunable resistive pulse sensing (TRPS), while morphology-oriented analysis involves scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
In addition, cargo classification is further divided into proteomic, lipidomic, and genomic analyses, including Western blotting, ELISA, flow cytometry, mass spectrometry, and PCR. 36 Since each of the above characterization methods has its advantages and disadvantages, it is a global practice to combine analyses from three different aspects, for example, a combination of TEM, NTA, and Western blotting, to identify isolated exosomes.
For example, microscopy-based methods, such as SEM and TEM, can directly visualize surface topography and internal structure, respectively. However, TEM is not suitable for rapid measurement of large numbers of samples due to complex operations and tedious sample preparation.
NTA facilitates rapid detection and real-time monitoring of exosomes with higher resolution than flow cytometry. The main drawback of NTA is the difficulty in distinguishing exosomes from contaminating proteins. As a mature technique, Western blotting can qualitatively and quantitatively detect the expression of exosomal protein biomarkers, particularly in exosomes from cell culture media.
However, it is time-consuming and unsuitable for detecting exosomes from biological fluids.
