Current methods for the isolation of extracellular vesicles

Abstract

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…Unlike shed large-sized microvesicles and apoptotic bodies, exosomes and small-sized microvesicles can be purified from biological fluids and cell culture-conditioned media using various techniques, including ultracentrifugation (UC), density-based separation, size-dependent methods such as ultrafiltration and size-exclusion chromatography, precipitation using polymers like ExoQuick TM , and immune-affinity capture methods [167,168]. To enable the therapeutic use of EVs, however, we urgently need more efficient and reliable methods for isolating large amounts of highly pure EVs.…”

Adiponectin is partially associated with exosomes in mouse serum

“…Next, the sample solutions containing smaller vesicles are diluted with PBS to reduce viscosity and subjected to ultracentrifugation at 100,000-200,000×g with fixed angle or swinging bucket rotor for about 2 times. However, the limitation of this method includes contamination from extravescular protein complexes, lipoprotein particles, and other contaminants (Momen-Heravi et al, 2013). Although the pellets are washed with PBS in order to remove these particles, it is difficult to completely remove them.…”

“…Exosomes are separated in different concentrations of sucrose solution, sucrose cushion, or iodixanol (OptiPrep TM ) at the density range of 1.1-1.9 g/ml (Momen- Heravi et al, 2013;Tauro et al, 2012;Witwer et al, 2013).…”

“…Low protein binding-size exclusion filters such as hydrophilized polyvinylidene difluoride membrane are used to isolate and enrich exosomes. However, this method may damage large vesicles and platelet and possibly cause the skewing result (Momen-Heravi et al, 2013;Witwer et al, 2013).…”

“…However, this method is not recommended for functional studies since elution of exosomes from the antibody and media seems to be difficult. It is also not recommended for large amount of samples (Chaput & Théry, 2010;Momen-Heravi et al, 2013;Witwer et al, 2013).…”