Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each.
University of Manitoba researchers provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. The researchers evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. They argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.
Extracellular vesicle (EV) biogenesis, subpopulations,
and conventional and novel methods of exosome isolation
EVs are categorized into three main types depending on their site of origin, density, expression of markers, and/or size. Apoptotic bodies (AB) are released by the blebbing of an apoptotic cell membrane (500–5000 nm); microvesicles (MV) are shed from the outward budding of the plasma membrane (100–1000 nm); and exosomes (EXO) are formed when multivesicular bodies fuse to the plasma membrane and release intraluminal vesicles (30–150 nm). EVs have variable: (1) protein expression profiles—EXOs are enriched with Fliotillin-1, ALIX, TSG101, CD81, CD63 and CD9 proteins, whereas MVs preferentially express MMP2 and ARF6; (2) lipidomic profiles—MVs are enriched with phosphatidylserine and cholesterol, EXOs with sphingomyelin and ceramide, and ABs by phosphatidylserine; and (3) distinct genomic and transcriptomic luminal cargo. Conventional methods of EV isolation include size-exclusion chromatography (SEC) and differential ultracentrifugation (dUC). SEC uses biofluids as a mobile phase against a porous stationary phase to differentially elute molecules with an inverse speed relation to their size—in other words, larger particles will elute first, followed by smaller vesicles that will enter and flow through the pores resulting in a longer path and thus increased elution time. dUC relies on the separation of EV subpopulations via gradually higher acceleration rates. More novel exosomal techniques also exist. Poly-ethylene glycol (PEG)-based precipitation uses a solution to facilitate a polymer-entrapped vesicle aggregate in large numbers. Immunoaffinity (IA) capture uses antibodies targeted against exosomal surface proteins to isolate specific vesicle population. Microfluidics (MF) technology uses chips with specific antibody-mediated binding to capture exosomes efficiently. Ultrafiltration (UF) is dependent on a filter of specific pore size that creates a vesicle-rich filtrate specific to the desired size.