Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications

Abstract

Extracellular vesicles, especially small extracellular vesicles (sEVs) are now accepted as important messengers in cell-to-cell communication and as a promising drug delivery platform. They are involved in nearly all physiological and pathological processes and are involved in disease diagnosis and therapy. However, their heterogeneity of physicochemical properties and functions is not fully understood, which hinders further clinical applications. To obtain highly bioactive sEVs with both high yield and purity, will certainly facilitate their future study and application. This review informs up-to-date research on frequently-used and cutting-edge technologies of sEVs isolation and makes a deep comparison and analysis of different methods, including their advantages, limitations and applications. Pending questions about the inherent property of these small vesicles as well as isolation strategies are discussed. Additionally, an overview of their applications in disease diagnosis and treatment, including some of the on-going clinical trials, are also reviewed.

Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study

“…Another factor which influences the experimental results of EV studies is the applied isolation protocol. Currently, without a consensus method that allows the recovery of a pure EV subpopulation, there are a series of isolation techniques based on different physical or biochemical properties of the target EV subpopulation [ 19 , 20 ]. One of the most frequently used sEV isolation methods is differential ultracentrifugation.…”

“…Today, there are a series of isolation methods based on EV size, density, marker expressions or their combinations. However, they result in quite different EV purities and yields [ 19 , 20 ]. Additionally, the storage conditions of EVs can also affect downstream analyses and functional studies [ 21 ].…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Potential Therapy for Diabetes Mellitus and Diabetic Complications

“…MSC-EVs can be isolated from the cell supernatants of MSC. According to the physical and chemical features of MSC-EVs, several isolation methods have been established such as ultracentrifugation [ 47 ], density gradient centrifugation [ 48 ], size exclusion chromatography [ 49 ], immunoaffinity capture [ 50 ], ultrafiltration [ 51 ], and polymer precipitation [ 52 ]. Each method has its own advantages and limitations ( Table 1 ).…”

Dapagliflozin‐Loaded Exosome Mimetics Facilitate Diabetic Wound Healing by HIF‐1α‐Mediated Enhancement of Angiogenesis

“…[ 23 ] The difficult purification, low yield, and high cost have blocked the biomedical applications of exosomes or extracellular vesicles. [ 24–26 ] Owing to the current limitations of exosome production, some studies have introduced an extrusion approach to preparing nanovesicles (NVs) as mimetics for exosomes. [ 23,27 ] For example, Cui et al.…”

“…[23] The difficult purification, low yield, and high cost have blocked the biomedical applications of exosomes or extracellular vesicles. [24][25][26] Owing to the current limitations of exosome production, some studies have introduced an extrusion approach to preparing nanovesicles (NVs) as mimetics for exosomes. [23,27] For example, Cui et al utilized a classical serial extrusion approach to generate NVs from human induced pluripotent stem cells (iPSCs)-derived ECs (iPS-ECs) to treat osteoporosis.…”