How can exosomes be isolated and characterized?

Exosome isolation

Exosomes may be isolated using several different techniques such as ultracentrifugation, ultrafiltration, precipitation, immunity capture, and microfluidics. 

• Ultracentrifugation is an exosome isolation technique that involves increasing speeds of centrifugation to pellet out contaminants of different sizes from a biological fluid. In this process, exosomes are separated from other particles in the sample based on their size and density. 
• Ultrafiltration uses a membrane with very small pores to allow small-sized exosomes to pass through while holding larger particles back. This exosome isolation technique works on the size exclusion principle. 
• Precipitation techniques isolate exosomes using a poly-ethylene glycol (PEG) solution to enable exosome aggregation. This is followed by low-speed centrifugation resulting in precipitation and isolation of the exosomes. 
• In immunoaffinity-based isolation techniques, antibodies are used to bind to the exosomes, which are then isolated using a magnetic field or other separation methods. 
• Microfluidics isolate exosomes based on size, density and immunoaffinity using specially designed microfluidic devices. 

Exosome Characterization

After isolation, exosome characterization can be accomplished using a few common strategies: 

• Dynamic light scattering (DLS) analysis works by the selective enrichment of exosomes within a specific size range, based on their unique light scattering patterns. 
• Electron microscopy serves as a valuable tool for visualizing the morphology and size of vesicles. When used in combination with immune-labeling, electron microscopy enables the observation of specific exosome features such as surface proteins. 
• Mass spectrometry can be used for identifying protein cargo in exosomes, and to determine if any contaminants such as protein aggregates are present in the sample. 
• While conventional western blotting can also be used for exosome characterization, it has one major drawback. It can only detect if certain proteins are present in a sample but is unable to quantify the exosomes in the sample.  

Other exosome characterization techniques that are less frequently used include flow cytometry, atomic force microscopy, resistive pulse sensing, and optical single particle tracking.