Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

Abstract

Background Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging. Scope of Review This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method. Various characteristics of individual methods, including isolation efficiency, EV yield, properties of isolated EVs, and labor consumption are compared. Major Conclusions A mixed population of vesicles is obtained in most studies of EVs for all used isolation methods. The properties of an analyzed sample should be taken into account when planning an experiment aimed at studying and using these vesicles. The problem of adequate EVs isolation methods still remains; it might not be possible to develop a universal EV isolation method but the available protocols can be used towards solving particular types of problems. General Significance With the wide use of EVs for diagnosis and therapy of various diseases the evaluation of existing methods for EV isolation is one of the key problems in modern biology and medicine.

Extracellular vesicles from oviductal isthmus and ampulla stimulate the induced acrosome reaction and signaling events associated with capacitation in bovine spermatozoa

“…In addition, EV maintain cell survival, and they also stimulate protein tyrosine phosphorylation and intracellular calcium increase as signaling events associated to sperm capacitation, also maintain cell survival. EV isolated from oviductal fluid obtained from the isthmus and ampulla regions show diameters of 30 to 500 nm, expressing the specific EV markers CD81 and HSP70, as similarly reported by others . The co‐incubation of spermatozoa with membrane fluorescent EV revealed that their incorporation by the cells takes place in a few minutes, reaching a maximum after 2 hours, becoming 62% sperm stained with different patterns.…”

“…EV from the ampulla showed 3‐ to 4‐fold higher total protein concentration than that obtained for the isthmus (data not shown). Western blot analysis (Figure D) revealed that the isolated EV from oviductal fluid were positive for CD81 and heat shock protein 70 (HSP70), two well characterized EV protein markers …”

Chicken seminal fluid lacks CD9‐ and CD44‐bearing extracellular vesicles

“…Among the many methods applied in different fluids and species (reviewed by Konoshenko, Lekchnov, Vlassov, & Laktionov, ) to isolate EVs (microvesicles and/or exosomes), immunoaffinity precipitation and capture is the method most often commercially offered (Exo‐Quick‐TC; System Biosciences) as being easy to perform and quick (Bai et al, ). However, due to the low density and small size of the exosomes, purity is low‐to‐poor and demands further screening of other exosome surface markers, such as CD44 for example (Alvarez‐Rodriguez, Ljunggren, et al, ).…”

Exosomes and cancer: From oncogenic roles to therapeutic applications

“…Another important challenge to the future use of exosomes in therapy is about optimization isolation and purification methods to produce exosomes with high quality and quantity, as well as desirable physicochemical features. The isolation methods can influence significantly on exosome properties including their density, shape, size, surface proteins, biochemical composition, and so on …”