How to store EVs

By the time an EV sample reaches our service lab, it has usually had quite a journey. It was isolated, resuspended in something, put into a tube, held in a fridge or freezer for a while, then packed on ice packs or dry ice and sent across the country or overseas. Any of those steps can change the particle count and size distribution that eventually gets measured.

We are frequently asked about storage and shipping conditions for samples sent to our NTA service. Is PBS fine? Should it go on dry ice? Can it sit in the fridge over the weekend? Published work answers some of these reasonably well and leaves others open. The primary studies used different EV sources, isolation methods and readouts, and reviews by Jeyaram and Jay and, more recently, Ahmadian and colleagues give a good overview of how varied they are.^1,2

Here we cover what happens to EVs at different temperatures and in different buffers, how much that matters for each of the main downstream applications, and what we suggest for samples that have to travel.

What Happens to EVs in a Tube

EVs are small, lipid-bound particles carrying a surface coat of proteins and glycans. In a buffer they change in several distinct ways, and these are worth distinguishing because they affect the data differently.

• Adsorption to surfaces. Vesicles stick to tube walls, most noticeably when the sample is dilute and the buffer contains no protein. Evtushenko and colleagues found that purified EVs held in PBS at 4 °C in ordinary polypropylene tubes lost about half their particles within 48 hours, and traced roughly two thirds of that loss to the tube walls.³
• Aggregation and fusion. Particles clump or merge, which lowers the count and shifts the size distribution upwards. When two differently labelled EV populations were mixed before storage, double-positive events appeared after freeze-thaw, consistent with fusion, although aggregation could not be ruled out.⁴
• Leakage and degradation of cargo. Proteins leave the vesicles or are degraded, and in airway EVs the set lost at 4 °C differed from the set lost at -80 °C.⁵
• Fragmentation. Ice formation can damage membranes and generate smaller particles, which can raise the apparent particle count.⁶
• pH shifts. Sodium phosphate buffers can acidify sharply as they freeze, because one buffer salt crystallises out before the other,^7,8 and carbon dioxide from dry ice can enter tubes and acidify samples on thawing.⁹

Some of these lower particle counts and some raise them. A stored sample can therefore give a reasonable-looking NTA result that no longer reflects the original preparation.

Temperature

The studies below differ in EV source, isolation method and how function was measured, and they do not all agree. The table sets out the main findings for each storage condition.

Ahmadian and colleagues reviewed 50 storage studies in 2024 and concluded that rapid freezing followed by a constant -80 °C gave the best preservation of EV numbers and cargo.² The -20 °C finding from LÅ‘rincz and colleagues matters in practice, because the quickest check most laboratories run is a particle count, and at -20 °C the count stayed normal while function was lost.¹⁰

Repeated freeze-thaw in PBS increased particle counts in the study by Bosch and colleagues⁶ and reduced them in the study by Gelibter and colleagues.⁴ Fragmentation raises counts and aggregation or fusion lowers them, so both results are plausible in different preparations. In either case, a second NTA reading describes a different sample from the first.

Airway EVs from lavage fluid, neutrophil EVs, plasma EVs and cell culture EVs may not share one stability profile, and it is not yet clear how much of the variation between studies comes from EV source and how much from isolation method and buffer.

The Buffer Matters as Much as the Temperature

PBS is the default resuspension buffer in most EV laboratories, but the evidence suggests it is a poor storage buffer. Görgens and colleagues compared buffer formulations over periods of up to two years and found that EVs stored in PBS showed drastically reduced recovery at every temperature tested, starting within days, with purer preparations affected most. Using PBS as a diluent reduced recovery within minutes.¹³

Their lead formulation, PBS-HAT, is PBS supplemented with 25 mM HEPES, 0.2% human serum albumin and 25 mM trehalose. It preserved EVs at -80 °C for up to two years, protected them through several freeze-thaw cycles, and markedly improved recovery when used as a diluent.¹³ Kawai-Harada and colleagues tested a version with bovine serum albumin in place of human albumin and found less variation in size and particle number at 4 °C and -80 °C, with the targeting function of engineered EVs retained.¹⁵

The three additives address different problems. Albumin coats tube walls and vesicle surfaces and reduces adsorption losses. Trehalose protects membranes during freezing; Bosch and colleagues found that 25 mM trehalose narrowed the size distribution and prevented the freeze-thaw changes seen in PBS alone.⁶ HEPES provides buffering that does not depend on phosphate. Sodium phosphate buffers can drop by as much as three pH units during freezing as disodium phosphate crystallises out,^7,8 though the size of the effect in dilute PBS depends on concentration and cooling rate.

Van de Wakker and colleagues reached similar conclusions from a different angle. Recovery was higher with HEPES-buffered saline than with PBS during isolation, polypropylene tubes outperformed glass, and adding BSA or Tween 20 improved preservation without affecting EV function.¹²

Each additive also affects some downstream methods, as summarised below.

NTA calculates size from how fast particles diffuse, and the software assumes the viscosity of water unless told otherwise. A 10% glycerol solution is around 30% more viscous than water at room temperature, so vesicles suspended in it diffuse more slowly and are reported as roughly 30% larger than they are. The 25 mM trehalose in PBS-HAT makes a negligible difference. If a sample contains anything beyond simple saline, we need to know about it.

Albumin causes a different problem. Particle-to-protein ratios are widely used as a purity indicator, and a buffer containing 0.2% albumin makes the ratio meaningless. For samples going on to protein quantification or proteomics, trehalose and HEPES without albumin is a reasonable compromise. Some extra adsorption loss is likely, and low-binding tubes and a higher EV concentration will partly offset it.³

Storing the Sample Instead of the Isolate

Gelibter and colleagues compared eight storage strategies and concluded that, where storage cannot be avoided, it is better to store the biological matrix than the isolated EVs. When plasma EVs were stored after isolation, single-particle measurements showed a shift towards more positive surface charge, and that shift did not appear when the EVs were recovered from frozen plasma.⁴

Ge and colleagues reported a similar pattern for RNA. EV RNA recovered from plasma stored at 4 °C, -20 °C or -80 °C for periods of up to five years did not differ significantly from fresh samples, whereas the non-vesicular plasma RNA degraded at 4 °C.¹⁶ The study measured a small number of miRNAs, so it says more about the protective effect of the vesicle membrane than about every RNA species.

This only works if cells and platelets are removed before freezing. MISEV2023 notes that freezing cells generates EV-like fragments, so frozen whole blood or uncleared conditioned medium will contain particles that were never secreted EVs.¹⁷ Storing the matrix is also impractical for many samples. A litre of conditioned medium is awkward to freeze and ship, and many samples sent for NTA are already concentrated isolates.

How Storage Shows Up in Downstream Data

Each downstream method is sensitive to different storage artefacts, so the best storage condition depends on what the sample will be used for.

Functional readouts are the most vulnerable. In the neutrophil study, EVs stored at -20 °C kept their numbers and lost almost all of their antibacterial activity,¹⁰ and in airway EVs, storage at both 4 °C and -80 °C changed the protein content.⁵ Neither change would show up in a particle count, so for therapeutic or functional work, activity needs to be measured after storage.

Freeze-Drying for Transport

Dry ice is expensive, regulated for air transport, and awkward to source in some destinations. Freeze-drying removes that dependence. Charoenviriyakul and colleagues found that lyophilising B16BL6 melanoma EVs without a cryoprotectant caused aggregation. With trehalose present, protein and RNA were protected, pharmacokinetics in mice were largely unchanged, and loaded cargo kept its activity for about four weeks at 25 °C.¹⁴

Formulations do not necessarily transfer between EV types. Lyoprotectant concentration, the freezing step and the reconstitution buffer all affect the result, so a freeze-dried product should be characterised before drying and after reconstitution, by particle count, size distribution and whatever functional readout matters for the work.

Shipping Samples

For shipped samples, most of the risk sits in the few days in transit. Partial thawing is the commonest problem: a sample that softens in a box and refreezes on arrival has been through a freeze-thaw cycle, even though nobody opened it. Allow enough dry ice for a delay of at least a day, and send early in the week so that samples do not sit in a depot over a weekend.

Dry ice also brings a less obvious problem. Many screw-cap tubes let CO₂ through their seals at dry ice temperatures. The gas stays in the headspace while the sample is frozen and dissolves as it thaws, lowering the pH. Murphy and colleagues described this in 2013, along with two remedies: briefly uncapping the tube while it is still frozen to vent the gas, or holding the sample at -80 °C for several days so that the CO₂ diffuses out.⁹ Cheng and colleagues found that pH alone changed both EV counts and cellular uptake,¹⁸ so venting is worth building into any receiving protocol.

Chilled shipments carry their own risk. Purified EVs sent at 4 °C are exposed to wall losses for the full transit time, and dilute samples lose the most.³ Send them at the highest practical concentration, in low-binding tubes, and in a stabilising buffer if the downstream application tolerates one. Do not dilute in advance to the range the instrument needs, since diluting in PBS reduced EV recovery within minutes in the Görgens study.¹³

For samples coming to us for NTA, please include:

• The exact buffer composition, including any albumin, sugars, glycerol, DMSO or detergent.
• A small volume of the same buffer on its own, so background particles can be measured.
• The isolation method and the approximate EV source.
• Storage temperature and duration since isolation, and the number of freeze-thaw cycles.
• Whether the sample has been diluted, and in what.

Planning Storage for Your Samples

The planner below suggests a starting point from sample type, time to analysis and intended use. It reflects the published evidence summarised above, and individual preparations may behave differently.

Summary

Purified EVs are less stable in a tube than most protocols assume, and plain PBS is one of the main reasons. Where a sample has to be kept, -80 °C in single-use aliquots is the best supported option, and a buffer containing trehalose and HEPES, with albumin where the downstream work allows it, protects against most of the losses seen in PBS. Storing the cleared biofluid rather than the isolate helps further when it is practical. Storage at -20 °C can preserve counts while function is lost, and 4 °C is suitable only for short holds.

For shipping, the main risks are partial thawing, carbon dioxide from dry ice, and wall losses in chilled dilute samples, and each can be managed. Freeze-drying is an option for formulations that have been validated.

Several questions remain open. It is not clear how far any one buffer transfers between EV sources, whether stability at -80 °C holds beyond two years, or how much of the disagreement between studies reflects biology rather than method. Until those are answered, the most practical step is to record exactly how each sample was stored and to avoid treating stored and fresh material as interchangeable.

References

1. Jeyaram A, Jay SM. Preservation and storage stability of extracellular vesicles for therapeutic applications. AAPS J. 2017;20(1):1.
2. Ahmadian S, Jafari N, Tamadon A, et al. Different storage and freezing protocols for extracellular vesicles: a systematic review. Stem Cell Res Ther. 2024;15:453.
3. Evtushenko EG, Bagrov DV, Lazarev VN, et al. Adsorption of extracellular vesicles onto the tube walls during storage in solution. PLoS One. 2020;15(12):e0243738.
4. Gelibter S, Marostica G, Mandelli A, et al. The impact of storage on extracellular vesicles: A systematic study. J Extracell Vesicles. 2022;11(2):e12162.
5. Maroto R, Zhao Y, Jamaluddin M, et al. Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. J Extracell Vesicles. 2017;6(1):1359478.
6. Bosch S, de Beaurepaire L, Allard M, et al. Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep. 2016;6:36162.
7. Gómez G, Pikal MJ, Rodríguez-Hornedo N. Effect of initial buffer composition on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions. Pharm Res. 2001;18(1):90-97.
8. Pikal-Cleland KA, Cleland JL, Anchordoquy TJ, Carpenter JF. Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems. J Pharm Sci. 2002;91(9):1969-1979.
9. Murphy BM, Swarts S, Mueller B, et al. Protein instability following transport or storage on dry ice. Nat Methods. 2013;10(4):278-279.
10. LÅ‘rincz ÁM, Timár CI, Marosvári KA, et al. Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes. J Extracell Vesicles. 2014;3:25465.
11. Wu JY, Li YJ, Hu XB, et al. Preservation of small extracellular vesicles for functional analysis and therapeutic applications: a comparative evaluation of storage conditions. Drug Deliv. 2021;28(1):162-170.
12. van de Wakker SI, van Oudheusden J, Mol EA, et al. Influence of short term storage conditions, concentration methods and excipients on extracellular vesicle recovery and function. Eur J Pharm Biopharm. 2022;170:59-69.
13. Görgens A, Corso G, Hagey DW, et al. Identification of storage conditions stabilizing extracellular vesicles preparations. J Extracell Vesicles. 2022;11(6):e12238.
14. Charoenviriyakul C, Takahashi Y, Nishikawa M, Takakura Y. Preservation of exosomes at room temperature using lyophilization. Int J Pharm. 2018;553(1-2):1-7.
15. Kawai-Harada Y, El Itawi H, Komuro H, Harada M. Evaluation of EV storage buffer for efficient preservation of engineered extracellular vesicles. Int J Mol Sci. 2023;24(16):12841.
16. Ge Q, Zhou Y, Lu J, et al. miRNA in plasma exosome is stable under different storage conditions. Molecules. 2014;19(2):1568-1575.
17. Welsh JA, Goberdhan DCI, O'Driscoll L, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024;13(2):e12404.
18. Cheng Y, Zeng Q, Han Q, Xia W. Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Protein Cell. 2019;10(4):295-299.

Cell Guidance Systems offers an NTA size profiling service for EV concentration and size distribution, alongside wider EV and exosome services. For samples that need to travel without a cold chain, our freeze drying service and custom freeze dried EVs may help. To discuss a project or send samples, use the EV/exosome service enquiry or the service requisition form, and further background is available in our exosome resources.

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