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.
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.
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.
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.
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