Optimized Isolation of Extracellular Vesicles From Various Organic Sources Using Aqueous Two-Phase System

Abstract

From biomarkers to drug carriers, Extracellular Vesicles (EVs) are being used successfully in numerous applications. However, while the subject has been steadily rising in popularity, current methods of isolating EVs are lagging behind, incapable of isolating EVs at a high enough quantity or quality while also requiring expensive, specialized equipment. The “isolation problem” is one of the major obstacles in the field of EV research - and even more so for their potential, widespread use for clinical diagnosis and therapeutic applications. Aqueous Two-Phase Systems (ATPS) has been reported previously as a promising method for isolating EVs quickly and efficiently, and with little contaminants - however, this method has not seen widespread use. In this study, an ATPS-based isolation protocol is used to isolate small EVs from plant, cell culture, and parasite culture sources. Isolated EVs were characterized in surface markers, size, and morphological manner. Additionally, the capacity of ATPS-based EV isolation in removing different contaminants was shown by measuring protein, fatty acid, acid, and phenol red levels of the final isolate. In conclusion, we have shown that EVs originating from different biological sources can be isolated successfully in a cost-effective and user-friendly manner with the use of aqueous two-phase systems.

Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis

“…Garlic was minced and grounded in a laboratory blender to obtain the juice, which was then centrifuged at 300 g for 10 min. Following transfer of the supernatant into new tubes and SEVs isolation by using aqueous two-phase systems is performed as described 19 . In brief, 20 ml of garlic juice is mixed with ATPS-EV isolation solution in 1:1 ratio and centrifuged at 1000× g for 10 min.…”

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment

“…As a result, isolation approaches can vary between studies, depending on the downstream application and scientific question. Though some effort has been made to standardize the EV isolation methodologies and create best-practice guidelines (e.g., EV-TRACK) (34), the lack of community standards and the inability of current methods to isolate EVs with high enough quality and quantity have created a major "isolation problem" in the field of EV research (35,36). Highly purified EVs are needed to attribute a function or a biomarker to vesicles as compared with other excretory-secretory bioparticles, especially when evaluating the relative importance of EVs for a detected function.…”

Extracellular Vesicles-Based Drug Delivery Systems: A New Challenge and the Exemplum of Malignant Pleural Mesothelioma

“…Other approaches are precipitation methods, which exploit the variation of EVs solubility and/or their aggregation when placed in polymeric solutions, and lastly, methods utilizing affinity interactions, such as immunoaffinity techniques. Further information on technical issues on the isolation methods of EVs can be found in recent papers [89][90][91][92].…”