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

Oğuz Kaan Kırbaş,Ayla Burçin Asutay,Hülya Ölmez,Bihter Ozdemir,Pakize Neslihan Taşlı,Dilek Öztürkoğlu,Fikrettin Şahin,Beyza Mat,Batuhan Turhan Bozkurt,Zeynep İşlek

doi:10.1038/s41598-019-55477-0

Oğuz Kaan Kırbaş, Ayla Burçin Asutay + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-55477-0

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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.

Highlights

Due to their role as natural messengers, Extracellular Vesicles (EVs) have been the focus of many studies investigating their potential as disease biomarkers and carriers for drugs and nucleic acid complexes, such as CRISPR-Cas[93]
We present a modification of the previously described Aqueous two-phase systems (ATPS) EV isolation method that optimizes the workflow of EV production
Washing steps of the ATPS-EV isolation method is capable of removing phytochemicals and other contaminants from the final isolate of EVs which may interfere with certain assays and studies

Summary

Introduction

Due to their role as natural messengers, EVs have been the focus of many studies investigating their potential as disease biomarkers and carriers for drugs and nucleic acid complexes, such as CRISPR-Cas[93]. Cargo that EVs carry -proteins and nucleic acids, especially miRNAs- make them a valuable source of diagnostic markers[12,13,14,15] Many of these studies have focused on using circulating EVs to diagnose different types of cancer, especially for types of cancer that are harder to diagnose. While these applications of EVs are shown to have great potential www.nature.com/scientificreports for a new generation of diagnosis and treatment options, many of these currently struggle to enter practice. Polymeric precipitation of EVs, which allows their isolation at normal centrifugation speeds, results with contaminating proteins and other extracellular vesicles[16] These isolation methods are incapable of producing EVs at a larger scale, which would reduce the production costs of EV-based therapeutics. Washing steps of the ATPS-EV isolation method is capable of removing phytochemicals and other contaminants from the final isolate of EVs which may interfere with certain assays and studies

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