Rigor and standardization of extracellular vesicle research: Paving the road towards robustness

Nieuwland, Rienk; Falcón‐Pérez, Juan Manuel; Théry, Clotilde; Witwer, Kenneth W.

doi:10.1002/jev2.12037

Cited by 55 publications

(45 citation statements)

References 30 publications

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“…Exacerbated by the current pandemic scenario, this topic will arguably gain broad relevance to the analytical community. Clinical sample preparation is indeed known to have a profound impact on the isolation of EVs and related contaminants [ 6 ]. A full awareness of the effect of any additional sample pre-analytical treatment should be consequently raised among EVs users, particularly due to the fact that an interlaboratory consensus on protocols for SARS-CoV2 serum inactivation protocols is far from being reached.…”

Section: Discussionmentioning

confidence: 99%

“…Human blood is an important source of accessible EVs originating from various body cell types with significant interest as potential diagnostic biomarkers, yet their isolation represents a considerable challenge. EVs from biological samples are indeed tremendously complex analytes, and the well-known influence of pre-analytical practices on downstream EVs use has been driving the need for standardization and rigor criteria largely before the COVID crisis, as highlighted by the International Society for Extracellular Vesicles (ISEV) community [ 6 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

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Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Frigerio

Musicò

Brucale

et al. 2021

Cells

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Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Frigerio

Musicò

Brucale

et al. 2021

Cells

View full text Add to dashboard Cite

show abstract

“…At the same time that major advances were achieved in the understanding of the role of EVs in cellular communication and homeostasis, as well as of their clinical use as biomarkers or for therapeutic purposes, a large variety of technologies and strategies for isolation and characterization of extracellular vesicles were designed, developed, and improved [ 8 , 91 , 92 ]. Because comprehensive reviews of the available methods for EV analysis describing their working principles, improvements, and limitations were recently published elsewhere [ 13 , 93 , 94 , 95 ], here we will highlight the techniques more frequently used for studying EVs secreted by human macrophages ( Table 2 ).…”

Section: Methods For Isolation and Characterization Of Evs From Human Macrophagesmentioning

confidence: 99%

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction

Arteaga-Blanco

Bou-Habib

2021

IJMS

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The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host–pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.

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“…Specifically, there remain several fundamental challenges the field must come to terms with; low yield of production, scalable and standardized EV generation, standardized dose and potency monitoring, determination and quantification of molecular bioactivity for regulatory purposes, and unsatisfactory targeting capacity. Fortunately, international efforts to address aspects of these are ongoing ( Lener et al, 2015 ; Reiner et al, 2017 ; Witwer et al, 2019 ; Gandham et al, 2020 ; Nguyen et al, 2020 ; Nieuwland et al, 2020 ; Rankin-Turner et al, 2021 ). In this section, we outline the challenges faced in EV therapeutic development, with an emphasis on the updated research and technologies offering avenues for preclinical and clinical advancement ( Figure 1 ).…”

Section: Challenges To Further Development Of Ev Therapiesmentioning

confidence: 99%

Development of Extracellular Vesicle Therapeutics: Challenges, Considerations, and Opportunities

Claridge

Lozano

Poh

et al. 2021

Front. Cell Dev. Biol.

113

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Extracellular vesicles (EVs) hold great promise as therapeutic modalities due to their endogenous characteristics, however, further bioengineering refinement is required to address clinical and commercial limitations. Clinical applications of EV-based therapeutics are being trialed in immunomodulation, tissue regeneration and recovery, and as delivery vectors for combination therapies. Native/biological EVs possess diverse endogenous properties that offer stability and facilitate crossing of biological barriers for delivery of molecular cargo to cells, acting as a form of intercellular communication to regulate function and phenotype. Moreover, EVs are important components of paracrine signaling in stem/progenitor cell-based therapies, are employed as standalone therapies, and can be used as a drug delivery system. Despite remarkable utility of native/biological EVs, they can be improved using bio/engineering approaches to further therapeutic potential. EVs can be engineered to harbor specific pharmaceutical content, enhance their stability, and modify surface epitopes for improved tropism and targeting to cells and tissues in vivo. Limitations currently challenging the full realization of their therapeutic utility include scalability and standardization of generation, molecular characterization for design and regulation, therapeutic potency assessment, and targeted delivery. The fields’ utilization of advanced technologies (imaging, quantitative analyses, multi-omics, labeling/live-cell reporters), and utility of biocompatible natural sources for producing EVs (plants, bacteria, milk) will play an important role in overcoming these limitations. Advancements in EV engineering methodologies and design will facilitate the development of EV-based therapeutics, revolutionizing the current pharmaceutical landscape.

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Rigor and standardization of extracellular vesicle research: Paving the road towards robustness

Cited by 55 publications

References 30 publications

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction

Development of Extracellular Vesicle Therapeutics: Challenges, Considerations, and Opportunities

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