Scheme 3.

Exosome processing and characterization flowchart. The exosome separation portion of the workflow includes common processing approaches with yields and purity levels. The characterization portion of the flowchart contains quantitative characterization, qualitative characterization, single vesicle characterization, and topology. The workflow starts with measuring and recording parameters of the starting materials such as cell count, fluid volume, and quantity of non‐EV molecules. Next, an appropriate processing method is selected based on the starting material and the target yield and purity. After exosome isolation, characterization begins with quantitation of the purity and yield of particles, proteins, lipids, and nucleic acids. If the sample does not meet the desired purity and yield, additional processing is required. Appropriate methods are then selected for bulk and single vesicle characterization to finish the characterization process.