A novel affinity-based method for the isolation of highly purified extracellular vesicles

Abstract

Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.

Ceramide and Exosomes: A Novel Target in Cancer Biology and Therapy

“…Immunoprecipitation using antibodies against exosome surface proteins can alleviate this problem and even separate exosomes derived from different cell types. However, the yield is usually lower than with centrifugation techniques and often compromises a quantitative analysis of exosome function (Brett et al, 2017; Momen-Heravi et al, 2013; Nakai et al, 2016; Szatanek, Baran, Siedlar, & Baj-Krzyworzeka, 2015). Recently, great strides were made in the field of exosome isolation introducing new techniques such as ultrafiltration and microfluidics-based separation.…”

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes

“…Using antibodies raised against the mammalian proteins, neither CD81 nor CD63 displayed any immunoreactive bands in A1 cells or EVs. Our Western blot results were further verified using FACS analysis with EV capture beads in which Tim4 was used to bind all vesicles containing exposed phosphatidyl serine [46] and then probed for surface protein phenotypes with labelled antibodies. High levels of TSP1 were quantitated in A1-EVs (~99%) with a lower abundance observed in NHDF-EVs at ~47% (Figure 5(i)).…”

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

“…A few studies have also reported that PS expression on the membrane leaflet is more abundant in cancer cells and cancer cell derived exosomes compared to those from healthy controls. In order to detect and quantify the PS expression on cells or vesicle surfaces, several proteins have been tested and specific binding affinities of Tim4 and annexin V to PS have been proved. One of the most widely studied PS‐binding molecules, annexin V, is a 35.8 kDa protein which binds PS in a calcium‐dependent manner .…”