Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis

Abstract

Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…The polymeric precipitation technique is based on the use of polymers such as polyethylene glycol (PEG) to entrap EVs, providing a rapid isolation of EVs from biological samples or culture media [ 114 , 115 ]. Several commercial isolation kits have been produced for the application of PEG solutions to isolate exosomes, such as the most commonly used Exo-spin exosome purification kit, ExoQuick exosome precipitation solution and Invitrogen total exosome isolation kit [ 116 ]. In order to remove the contaminating proteins and separate EVs [ 117 ], polymer-induced precipitation is often supported by pre- and post-isolation steps, such as SEC or the use of Sephadex G-25 spin columns which retains the polymer from precipitated EVs [ 118 , 119 ].…”

Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

“…Although phlebotomy is considered a minimally invasive procedure, blood samples from carefully phenotyped research participants remain a limited resource. Therefore, the type of method used to isolate exosomes should be considered carefully (Macias et al, 2019). Ensuring that the preparation provides a high yield of exosomes from the smallest volume of plasma sample possible is one of the most important criteria, although the ease and speed of preparation are also paramount when considering the use of exosomes as biomarkers or diagnostics in clinical settings.…”

“…Our decision to use the ExoQuick ® ULTRA kit resulted from our testing many kits for EV preparation and comparing their relative enrichment of exosomes based on NTA assessment of size and concentration (Basic Protocol 3). This, along with a review of the literature for studies performing similar comparisons [for example, the work of Macias and colleagues, who performed an analysis of six different commercial kits (Macias et al, 2019)], formed the basis of our decision to use the ExoQuick ® UL-TRA precipitation approach. This choice was also based on the ExoQuick ® ULTRA kit providing investigators the ability to examine an exosome preparation from which albumin and IgG have been depleted, allowing researchers to answer questions regarding the physiological and pathological roles of exosomes in a biochemically cleaner manner.…”

“…Whereas supernatants from cells grown in culture are readily available for exosome isolation, plasma samples from phenotypically characterized human research participants and from animal-model experiments are often precious and scarce. Thus, when de-termining the optimal exosome isolation method, investigators must consider the sample source (Macias et al, 2019). Here, we compared a precipitation-based exosome isolation method using the Systems Biosciences ExoQuick ® ULTRA EV Isolation Kit for Serum and Plasma (Alternate Protocol 1) to ultracentrifugation (Basic Protocol 2), which is considered the gold-standard method for exosome isolation.…”

“…For example, we include a second centrifugation step during the preparation of our plasma samples in order to ensure that no white blood cells or platelets are present as contaminants in the preparations. The speed, ease of isolation, and yield of exosomes obtained when using precipitation-based isolation methods have been the main reasons why this field has expanded so rapidly, with many companies developing kits that promise all of these facets for the isolation of exosomes (Macias et al, 2019). We chose to compare a precipitation method using the ExoQuick ® ULTRA EV Isolation Kit for Serum and Plasma (Alternate Protocol 1) to the ultracentrifugation method (Basic Protocol 2) because ultracentrifugation is the historical gold standard in the field for preparing exosomes.…”