4 Reasons Why Storage Conditions Matter for Extracellular Vesicle Research

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Four reasons why storage conditions matter to the world of extracellular vesicle research.

The optimisation of sample storage conditions for extracellular vesicle (EV)-based research and applications is aimed at one common goal: obtaining results that reflect the original physiological state of EVs. As biofluid storage conditions can impact EV concentration and composition, the isolation and analysis of fresh samples is often preferable – but not always practical.<super-script>1,2<super-script> Consequently, collection and storage protocols are needed: both for the preservation of samples from the time of collection until they are processed for EV separation, and for the time between EV isolation and analysis.  

The issue of EV storage is complex, with many different factors involved; there are more than 30 types of mammalian biofluids, each containing multiple EV subtypes – and, EV components will be differentially affected by storage conditions.<super-script>3<super-script> Given the scope of these complexities, MISEV2018 does not provide recommendations for all possible scenarios.<super-script>3<super-script> Instead, ISEV encourages the development of such guidelines under the MISEV umbrella. Furthermore – as very few large-scale studies are available to guide recommendations for storage conditions – researchers should evaluate the effects of their processing and storage methods on their own EV populations of interest.  

To help researchers consider possible sources of variation during storage, we released an app note titled: How to Store Extracellular Vesicles: A Comprehensive Guide on EV Storage Across a Variety of Samples. The app note contains a wealth of published, up-to-date resources, and discusses considerations for storing a range of sample types. Considerations for EV storage are covered, including tips for storing EVs prior to EV isolation, as well as pre-analytical considerations post-EV isolation. Be sure to download the full app note and keep an eye out for future blog posts which highlight some of these considerations. For now, we share four reasons why storage conditions are important for EV research.

  1. For consistency across EV literature  

Appropriate and standardised pre-analytical treatment of samples helps other researchers interpret their results. Already, there are many variables to consider, including differences in methods for EV isolation and characterisation. If there are major inconsistencies in how samples are stored, it becomes even more difficult to compare findings with those of others.  

  1. Leveraging valuable biobank samples  

Understanding the influence of storage conditions on EVs is important when interpreting studies involving samples from biobanks. Already, biobanks adhere to strict regulations and have systems in place for tracking many storage variables, including temperature, storage period, freeze-thaw cycles, and thawing conditions.<super-script>4<super-script> Optimising these variables will allow biobanks and research groups to work together to find ways to store and select samples with EVs in mind. EV protein composition has been shown to differ between the choice of anticoagulant used in blood collection tubes, as well as between plasma and serum.<super-script>1<super-script> While these differences may appear to fall under the umbrella of sample collection, the EV subtypes present in each sample may be affected differently with storage – highlighting the need to control or report as many pre-analytical variables as possible when working with EVs.  


  1. To enable the clinical translation of EV-based therapeutics  

The efficacy and safety of a biotherapeutic product depends on the product’s ability to maintain its stability between the time of manufacture, through to drug administration. Therefore, if an EV product is to be a

relevant EV stability characteristics must be demonstrated using an appropriate storage framework, if a product is to be approved for human use. With research groups setting their sights on EV-based therapeutics, standardised storage methods are needed to support reproducible therapeutic outcomes and enable clinical translation.

  1. Standardising protocols for diagnostic performance

Ensuring optimal sample storage conditions is an important part of any diagnostic procedure, and critical to avoiding loss of diagnostic performance. For example, storing bronchoalveolar lavage fluid (BALF) samples at -80°C, rather than the recommended 4°C for up to 24 hours, impacts the culture sensitivity of etiologic bacteria and can therefore lead to an underestimation of diagnosis of pneumonia, a serious clinical condition.<super-script>5<super-script>  

Similarly, optimising the storage conditions of samples will be critical for settings of EV-based diagnostics, e.g., where diagnostic potential lies in measuring the number of specific circulating EVs and/or detecting a signature EV molecular cargo. The loss or ex vivo generation of certain populations of plasma EVs can be significantly impacted by minor changes in protocol, such as a single freeze-thaw cycle of plasma samples, long-term storage at -80°C, or through delays in the time between venipuncture and centrifugation.<super-script>1<super-script> Sample storage is also particularly important to consider when studying EV miRNA as biomarkers, as platelet-derived EVs can easily contaminate plasma samples under suboptimal storage, and subsequently confound measurements of circulating EV-miRNA.<super-script>6<super-script>

Download now: How to Store Extracellular Vesicles: A Comprehensive Guide on EV Storage Across a Variety of Samples


  1. Ayers L, Kohler M, Harrison P, et al. Measurement of circulating cell-derived microparticles by flow cytometry: Sources of variability within the assay. Thrombosis Research. 2011;127(4):370-377. doi:10.1016/j.thromres.2010.12.014
  2. Witwer KW, Buzás EI, Bemis LT, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Journal of Extracellular Vesicles. 2013;2(1):20360. doi:10.3402/jev.v2i0.20360
  3. Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 2018;7(1):1535750. doi:10.1080/20013078.2018.1535750
  4. Mora EM, Álvarez-Cubela S, Oltra E. Biobanking of exosomes in the era of precision medicine: are we there yet? International Journal of Molecular Sciences. 2015;17(1). doi:10.3390/ijms17010013
  5. Kneidinger N, Warszawska J, Schenk P, et al. Storage of bronchoalveolar lavage fluid and accuracy of microbiologic diagnostics in the ICU: a prospective observational study. Critical Care. 2013;17(4):R135. doi:10.1186/cc12814
  6. Mitchell AJ, Gray WD, Hayek SS, et al. Platelets confound the measurement of extracellular miRNA in archived plasma. Scientific Reports. 2016;6(1):32651. doi:10.1038/srep32651

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