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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.

SUBMITTER: Nakai W

PROVIDER: S-EPMC5034288 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Nakai Wataru W Yoshida Takeshi T Diez Diego D Miyatake Yuji Y Nishibu Takahiro T Imawaka Naoko N Naruse Ken K Sadamura Yoshifusa Y Hanayama Rikinari R

Scientific reports 20160923

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 develop ...[more]

PMID: 27659060

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Project description:BACKGROUND:Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC. METHODS:By using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science). RESULTS:Our data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit. CONCLUSIONS:We demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

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

Publications

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

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