Isolation and substrate dependence on extracellular vesicle characterisation using atomic force microscopy

Abstract Extracellular vesicles are nano- to micro-sized structures that carry biomolecules between cells to coordinate cellular activity and communication. Isolation and characterisation must be standardised to better understand the role of extracellular vesicles and how they can be used for disease diagnosis. Here we use atomic force microscopy to determine the physical differences between extracellular vesicles isolated using two different methods. Extracellular vesicles were isolated using two standardised methods, vacuum filtration and syringe filtration. In addition, extracellular vesicles were immobilised to plain mica and amino-functionalised mica to observe differences in adhesion onto substrates with different hydrophobicity. The application of atomic force microscopy enabled the study of vesicle adhesion, size distribution and morphology on the two different surfaces. It was found that both the isolation method and the substrate had a considerable effect on the physical properties of the extracellular vesicles, such as root mean square roughness values and size distribution. This demonstrates the ability to use atomic force microscopy to gain a more detailed understanding of the physical features of extracellular vesicles and the influence of different isolation methods on their morphology.