Isolation of extracellular vesicles with combined enrichment methods

Sabine Bartel,Rainer Bischoff,Janine Stam,Justina C. Wolters

doi:10.1016/j.jchromb.2021.122604

Abstract

Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield.This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation.From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.

Highlights

In the last decade, extracellular vesicles (EVs) have been attracting a large scientific interest as indicated by the exponential growth in the number of publications per year
We performed a systematic literature analysis over the year 2019 to screen for the use and benefits of combined EV isolation methods in terms of EV purity and EV yield, subdivided per biological material
The first part of our analyses looked at the current use of EV isolation methods and showed that about 60% of all EV isolations were based on combined methods

Summary

Introduction

Extracellular vesicles (EVs) have been attracting a large scientific interest as indicated by the exponential growth in the number of publications per year (see Fig. 1). EVs are of interest because almost all cells from all organisms can secrete EVs into the extracellular space with a cargo that consists of cell-specific proteins, RNA’s and lipids, their composition is not necessarily entirely similar to the parental cell, since some components are enriched while others are absent [1]. The term ’EVs’ should be seen as an umbrella term for “all nano particles that are naturally secreted by cells, encapsulated by a lipid bilayer and unable to replicate by themselves” as defined in 2018 in the minimal information for studies of extracellular vesicles (MISEV). In 1981, the term “exosome” was coined and in 1987, this term was used for the first time to address “all cell-secreted vesicles that are derived from multivesicular bodies” [4,5]. EVs may be named after their specific cargo because not all EVs contain the same cargo [8]

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