Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity and it might be suitable for larger-scale discovery studies.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Profiling plasma extracellular vesicle by pluronic block‐copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion

Zhong

Rosenow

Xiao

et al. 2018

“…This approach yields pure, biologically active, EVs at concentrations of up to 10 14 EV/mL. CFF/Capto Core outperformed UC- and PEG-based isolation on a bevy of EVs characterization assays as endorsed by the international society for extracellular vesicles [35]. This approach allows for buffer-exchange and nucleic acid digestion, ensuring that non-EVs encapsulated nucleic acids are eliminated.…”

Section: Introductionmentioning

confidence: 99%

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

McNamara

Caro-Vegas

Costantini

et al. 2018

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.

“…ISEV ( F ) developed a massive open online course on EVs that is freely available [Massive Online Open Course (MOOC) ( G ), http://coursera.org/learn/extracelllular-vesicles, 12], published minimal requirements for studies on EVs [MISEV, 13] ( H ). ISEV endorses a knowledge base that has been launched to monitor the quality of pre-analytical data reporting [http://evtrack.org, 14] ( H ).…”

Section: Basic and Clinical Aspects Of Extracellular Vesiclesmentioning

confidence: 99%

Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles

Nieuwland

Falcón‐Pérez

Soekmadji

et al. 2018

The past decade has witnessed an exponential development in the field of extracellular vesicles. Sporadic observations have reached a critical level and the scientific community increasingly recognizes the potential biomedical significance of these subcellular structures present in all body fluids as significant components of the cellular secretome. The Educational Committee of the International Society for Extracellular Vesicles prepared two posters (“Basic aspects of extracellular vesicles” and “Clinical aspects of extracellular vesicles”) to provide essential pieces of information on extracellular vesicles at glance for anyone not familiar with the field.