Table 1 Methods for isolating and characterizing EVs

According to a survey conducted by the International Society for Extracellular Vesicles (ISEV), the current methods commonly employed for the isolation and purification of EVs include differential centrifugation, density gradient centrifugation, filtration, size-exclusion chromatography, and immunological approaches, with differential centrifugation comprising 81% of the total [40]. It is advisable to employ a combination of isolation methods to clearly segregate subpopulations of vesicles based on their size, density, or composition, thus enhancing credibility. Differential centrifugation initially employs low-speed centrifugation to separate cell debris and larger particles from the matrix, followed by high-speed centrifugation to precipitate EVs from cellular metabolites and other substances [41]. While this method is widely used, it comes with certain drawbacks, such as time-consuming procedures, the requirement of substantial initial sample volumes, the need for expensive equipment, and relatively low EVs extraction purity [42, 43]. Recently, microfluidics, as an emerging method for EVs isolation, has been gaining prominence. Microfluidic technology offers numerous advantages, including cost-effectiveness and high sensitivity. The objective of this technology is to capture, filter, separate, and purify exosomes based on their physical and chemical properties [44, 45]. This emerging approach provides researchers with additional choices

Methods commonly used to identify and assess EVs primarily involve the characterization of their physicochemical properties, specifically particle size, concentration, morphology, and cargo-carrying capabilities. Physical analyses encompass techniques such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), electron microscopy, and tunable resistive pulse sensing (tRPS). Both NTA and DLS operate on similar principles, relying on the detection of scattered light signals generated by particle Brownian motion within a specific range. Subsequently, these signals are captured using microscopy or other collection devices, and the Stokes–Einstein equation is then employed to calculate particle size and concentration [46, 47]. Biochemical analyses are typically conducted through methods such as flow cytometry, immunoblotting, or proteomic analysis, providing data regarding the components found within the isolated vesicles. Flow cytometry, for instance, is generally considered suitable for counting EVs ranging from 300 to 500 nm in size [48]. However, analysis of smaller-sized EVs can be accomplished using antibody-coated magnetic beads. Immunoblotting, on the other hand, identifies EVs by the specific binding of antigen and antibody. It is imperative to note that, as outlined by ISEV, in order to validate the EV properties and purity of an EV preparation, it is necessary to analyze transmembrane or GPI-anchored proteins located in the outer membrane of prokaryotic cells and cytoplasmic or periplasmic proteins to demonstrate the presence of EVs and evaluate their purity from common contaminants [49]