EV Storage Stability: Freezing, Lyophilization and Trehalose

By Benn Bluestein-Veyra, M.Sc. Organic Chemistry · Updated 20 October 2026

Extracellular vesicle storage is a measured experimental variable. Temperature, freeze-thaw history, buffer composition and drying each change the particle count, size, cargo and activity that a later assay reports.^[1][2] Jeyaram and Jay concluded in an early review that −80 °C was the most promising condition and that storage effects varied with the vesicle source.^[3]

This page summarizes what storage studies measure, how temperature, freeze-thaw cycles and buffers compare, how trehalose protects membranes, what lyophilization studies report, and how MISEV2023 asks authors to document storage.

Extracellular vesicle storage studies measure particles, cargo and activity

A storage study divides one vesicle preparation into aliquots, holds each under a defined condition, and compares it with fresh material. Gelibter and colleagues, for example, measured concentration, size and zeta potential by tunable resistive pulse sensing, together with contaminating protein.^[4]

The readouts fall into a few groups, each sensitive to a different kind of change (Fig. 1). Most studies combine at least three of them, because particle number can stay constant while size, cargo or activity shifts.^[5] Electron microscopy then shows whether the remaining particles are single, aggregated or multilamellar.^[6]

• Particle concentration, by nanoparticle tracking analysis, resistive pulse sensing or calibrated flow cytometry.^[7][8]
• Size distribution and zeta potential, which reveal aggregation, fusion and surface changes.^[4][6]
• Protein markers such as ALIX, HSP70 and TSG101 by western blot, and whole proteomes by mass spectrometry.^[6][9]
• RNA content and morphology by transmission or cryo-electron microscopy.^[10][11]
• Cellular uptake, biodistribution in mice, or a defined biological readout in cell culture.^[9][12]

Storage temperature shapes vesicle recovery and structure

Temperature comparisons usually test 4 °C, −20 °C and −80 °C against fresh material. A systematic review of 50 studies by Ahmadian and colleagues found that rapid freezing and a constant temperature, optimally −80 °C, best preserved vesicle quantity and cargo.^[2]

Short holds and refrigeration

Cheng and colleagues held HEK293T exosomes for 24 h at five temperatures. Material kept at 4 °C retained the highest particle concentration and the highest ALIX, HSP70 and TSG101 signal.^[9]

Mean size stayed similar across conditions, so the authors attributed the losses to degradation of whole vesicles. Over about a month, the same markers declined at every temperature, most slowly at −80 °C.^[9] Sokolova and colleagues reported that exosome size from three human cell types decreased during storage at 4 °C and 37 °C.^[13]

Cheng and colleagues also varied pH. Concentration and marker levels fell most at pH 4, less at pH 10 and least at pH 7, which the authors took to mean that acidic conditions are the most disruptive for stored exosomes.^[9]

Freezing at −20 °C and −80 °C

Lőrincz and colleagues stored vesicles from human neutrophils at four temperatures. At −20 °C the vesicle count held for 4 weeks while size shifted and antibacterial activity was almost entirely lost, and at −80 °C count and size were stable with partial retention of activity.^[5]

Wu and colleagues compared 4 °C, −20 °C and −80 °C over 4 weeks. Each condition changed size distribution, quantity, cargo, cellular uptake and biodistribution relative to fresh vesicles, and the authors favored −80 °C for long-term storage.^[12]

Maroto and colleagues found that mouse airway exosomes grew about 10 percent in diameter at 4 °C and about 25 percent at −80 °C, with multilamellar structures after freezing. Distinct protein groups appeared in the supernatant at each temperature, consistent with leakage or loss of loosely bound proteins.^[6]

Gelibter and colleagues reported that −80 °C storage of plasma and microglial vesicles reduced concentration and purity over time and increased size and its variability. These effects appeared under all eight strategies they tested, and the authors advised processing fresh samples in most cases.^[4]

Freeze-thaw cycles reduce particle counts and enlarge vesicles

Repeated freezing and thawing is the most consistently reported source of change. In the Gelibter study, concentration fell after the first cycle and particle size rose with each further cycle.^[4] Cheng and colleagues saw concentration and marker protein levels decline across five cycles, while uptake per remaining vesicle changed little in their cell assay.^[9]

Gelibter and colleagues also mixed GFP-labeled and mCherry-labeled vesicles before storage. After storage a significant population carried both labels, which the authors interpreted as evidence of vesicle fusion during storage. Two-color designs of this kind separate fusion from aggregation, which bulk size measurements report together.^[4]

Responses vary by source and buffer. Sokolova and colleagues found exosome size unchanged through repeated freezing to −20 °C,^[13] while Bosch and colleagues saw concentration and size spread increase after freeze-thaw in PBS and remain steady in PBS with trehalose.^[10]

Storage buffers and excipients govern vesicle recovery

Görgens and colleagues compared storage buffers at several temperatures for up to 2 years, using vesicles from stromal and HEK293T cells. Plain PBS led to sharply reduced recovery over time, and PBS used as a diluent reduced recovery within minutes.^[7]

Their lead formulation, PBS-HAT, adds 25 mM HEPES, 0.2 percent human albumin and 25 mM trehalose to PBS. It improved short-term and long-term recovery at −80 °C, preserved vesicles through several freeze-thaw cycles, and improved recovery when used as a diluent.^[7]

Van de Wakker and colleagues found higher recovery with HEPES-buffered saline than PBS, and with polypropylene tubes than glass. Bovine albumin and Tween 20 improved preservation without changing activity in an endothelial migration assay, and both 4 °C and −80 °C were suitable for short-term storage.^[14]

Additive effects depend on the vesicle type. Lőrincz and colleagues reported that snap-freezing gave no added benefit for neutrophil vesicles, and that the widely used cryoprotectants they tested caused vesicle lysis.^[5]

Surface adsorption explains part of the buffer and vessel effects. MISEV2023 asks authors to report storage vessels and their materials, because vesicles can be lost by attaching to surfaces,^[1] and the higher recovery from polypropylene over glass fits that picture.^[14]

Trehalose protects membranes by hydrogen bonding and glass formation

Trehalose is a nonreducing disaccharide of two glucose units joined through their anomeric carbons, C12H22O11 (Fig. 2).^[15] It accumulates at high concentrations in organisms that survive near-complete drying, and Crowe and colleagues studied it as a stabilizer of dry membranes in such organisms.^[16]

Crowe and colleagues showed in 1984 that dry dipalmitoylphosphatidylcholine with trehalose kept a phase transition temperature close to that of the hydrated lipid, about 30 K lower than lipid dried alone. Infrared spectra indicated hydrogen bonds between trehalose hydroxyl groups and the phospholipid head groups.^[16]

In freeze-dried liposomes, trehalose inhibited fusion between vesicles and allowed almost all trapped solute to be retained after rehydration. Retention needed more than ten times the trehalose required to block fusion, which tied it to keeping the lipids in their fluid phase.^[17]

A later review by Crowe, Carpenter and Crowe concluded that both direct hydrogen bonding and vitrification, the formation of an amorphous glass, are required (Fig. 3). Trehalose has a high glass transition temperature and forms a dihydrate crystal that shields the remaining glass from absorbed water.^[18]

Bosch and colleagues brought trehalose to vesicle work in 2016. Adding 25 mM trehalose to the isolation and storage buffer for pancreatic beta-cell vesicles narrowed the size distribution and raised the particle count per microgram of protein, and cryo-electron tomography showed intact vesicles.^[10]

Lyophilization holds vesicles in a dry sugar matrix

Lyophilization, or freeze-drying, removes water from a frozen sample by sublimation under vacuum (Fig. 4). Reviews of vesicle preservation describe it as an alternative to −80 °C storage and as a reliable method for preserving thermolabile materials.^[3][19]

Charoenviriyakul and colleagues found that exosomes lyophilized without a cryoprotectant aggregated, and that trehalose prevented the aggregation. Protein and RNA profiles were retained, and luciferase-labeled exosomes kept their reporter activity for about 4 weeks at 25 °C.^[11]

Trenkenschuh and colleagues screened formulations by freeze-thawing first. Sucrose or potassium phosphate buffer and neutral pH limited aggregation and fusion, a low level of poloxamer 188 helped further, and polyvinylpyrrolidone gave little protection on freeze-drying.^[20]

Their lyophilizates containing 10 mM phosphate buffer, 0.02 percent poloxamer 188 and 5 percent sucrose retained particle size and concentration for 6 months at 40 °C. The activity of a vesicle-associated enzyme, beta-glucuronidase, held for 1 month and declined by 6 months.^[20]

Bari and colleagues described a pilot process for freeze-dried stromal cell secretome under good manufacturing practice conditions. Nanoparticle tracking found a non-aggregated vesicle population in the powder, infrared spectra showed both protein and lipid, and calorimetry confirmed the drying process.^[21]

Biofluid handling before isolation sets the starting population

Many vesicle studies begin with stored plasma, urine or saliva, so pre-isolation storage is a variable of its own. Witwer and colleagues called for standardized specimen handling and well-controlled reporting in an early ISEV workshop paper.^[22]

Yuana and colleagues measured vesicles in plasma, urine and saliva before and after a single freeze-thaw cycle and after storage for up to 1 year. These steps had no significant effect on measured concentration or diameter, and they recommended freezing and storing all samples identically.^[8]

Kim and colleagues found that freezing plasma with residual platelets generated new vesicle subpopulations and cell-free mRNA ex vivo, changes that later processing could not remove.^[23] Kalra and colleagues reported a stable TSG101 signal for plasma exosomes over about three months of storage.^[24]

Ahmadian and colleagues noted across the literature that vesicles kept in their native biofluid were more stable than purified vesicles in buffer.^[2] MISEV2023 advises pre-processing source material before storage to remove cells and other interfering material.^[1]

MISEV2023 asks studies to report every storage parameter

The MISEV2023 guidelines from Welsh and colleagues give storage its own section. They ask authors to report all storage parameters before and after separation, including preservatives or cryoprotectants, temperature, time, freezing procedure and storage vessel.^[1]

The guidelines list the freezing method, suspension buffer, temperature, duration, thawing method and number of freeze-thaw cycles as conditions that may need optimizing for each vesicle source. They also ask that the effects of storage and formulation on activity be assessed in functional studies.^[1]

• Freezing method, such as snap-freezing in liquid nitrogen or gradual freezing.^[1]
• Suspension buffer, cryoprotectants and other additives.^[1][7]
• Temperature, storage duration and vessel material.^[1][14]
• Thawing method, and the number of freeze-thaw cycles each aliquot has seen.^[1]

Reporting these details lets readers compare studies whose storage designs differ. The cold-chain validation reference covers the separate question of how temperature is documented during shipping.

Where to go from here

The isolation methods reference explains how the vesicle preparations described here are separated, and the nomenclature and protein marker references define what is being counted. The quality references on particle counting and MISEV2023 compliance show how these measurements are documented.

The cold-chain validation reference covers storage and shipping documentation. Verified practices can request lot documentation, and our team is glad to discuss the methods described on this page.