Table 1 Summary of Original Research Studies. This table outlines key information, including the EV source and isolation methods, the evaluation of storage conditions, durations, freeze-thaw cycles on EVs parameters and overall conclusions

[3]

human adipose stem cells (hASCs)

Isolation: tangential flow filtration (TFF)

Storage: encapsulated in microneedles (EV@MN) made of hyaluronic acid (HA) at -20, + 4, and + 25 °C for six months, and up to 10 freeze-thaw cycles.

• Significant decrease in EVs in PBS over time

• No significant decrease up to three months, negligible decrease after six months for EVs in EV@MN (> 85% remained)

-

• Protein activity lost in PBS at any temperature in 2 weeks, while preserved at over 99% in EV@MN at 4 °C or -20 °C storage.

EV@MN preserves EV functions (cell proliferation and fibroblast migration) for up to six months.

• HA in EV@MN preserved EV bioactivity after 6 months at 4 °C

• EVs in PBS lost activity quickly

• EV@MN retains over 99% activity at 4 °C or -20 °C during short-term storage

[53]

Mouse J774A.1 cells

Isolation: size exclusion chromatography (SEC)

Storage: Engineered EVs (E-EVs) in PBS at 37 °C (1 week), RT, and − 80 °C (1 year); E-EVs loaded into microneedles (MN-EVs) at RT (12 months) supplemented with trehalose and cellulose

E-EVs in PBS:

• 70 to 90% of E-EV’s cargo remained intact for 3 h, up to 45% by day 7

• RT and − 80 °C stored E-EVs had a larger mean diameter

MN-EVs:

• Maintained count and size for 12 months at RT.

• RT and − 80 °C E-EVs showed aggregation and some membrane disruption.

• MN-EVs maintained intact membranes, over 12 months at RT.

-

• Only a 3% loss in bioactivity of MN-EVs over 12 months at RT.

• Almost no biological activity of E-EVs in PBS at RT for 12 months • Improved EVs stability with trehalose and cellulose in MN-EVs.

• EVs encapsulated in microneedles remained stable at RT for at least one year with no impact on their bioactivities.

[33]

HEK293T cells conditioned media (CM)

Isolation: ultracentrifugation, TFF, SEC.

Storage: +4 °C, -20 °C, or -80 °C; multiple freeze/thaw cycles; resuspended in various buffers e.g. human albumin and trehalose (PBS-HAT); different tube types tested.

• Concentrations decreased at any temperature in PBS.

• Unaffected EVs at 4 °C for 8 days.

• Up to 26 weeks: 90% loss at -20 °C, less at -80 °C; better size preservation at -80 °C versus − 20 °C.

• 2 years Long-term storage: Highest count in PBS-HAT at -80 °C, with less size increase; additives improved freezing recovery.

TEM after 20 weeks in various conditions:

No consistent differences in shape, diameter, or intactness

• EV protein in PBS remained stable for 1 week at + 4 °C, declined at -20 °C after 8 weeks, and had better long-term stability at -80 °C.

RNA:

• 50% decline in 1 week at + 4 °C in PBS, significant loss in long-term at -80 °C, but stable for 2 years in PBS-HAT buffer.

• PBS with cryoprotectants kept EVs stability significantly.

• Long-term storage in PBS (2 years) reduced cellular uptake.

• Storage in PBS hurt EV; PBS-HAT represented a promising solution

• Human serum albumin reduces EV adsorption to tubes

• Buffers maintained EV stability

[11]

Human umbilical cord-MSCs (hUC-MSC) conditioned media

Isolation: Ultrafiltration and SEC

Storage: Immediate, RT, 4 °C, -20 °C (7 days), -80 °C (1 month); one freeze-thaw cycle

• No particle count differences, unaffected by storage.

• -80 °C storage had more uniform and smaller EVs.

• Size increased at -20 °C.

• Zeta potentials unaffected.

• Size differences between − 20 °C storage and RT

• TEM shows a distinct bilayer at -80 °C (more homogeneous EVs)

• Better protein markers preservation at -80 °C

• No significant miRNA differences, slight reduction at 4 °C

• No impact on the effect of EVs on sarcoma cell growth with storage

• Storing at -80 °C maintained EV size uniformity and integrity

[54]

Leaf-Derived EVs (LEVs)

Isolation: centrifugation

Storage: -20 °C, 4 °C, 25 °C, 45 °C (4 weeks); freeze-thaw (0, 1, or 3 cycles); storage media: 1,3-butylene glycol, Saliguard TMO

• LEVs stable at -20 °C

• Larger sizes at higher temperatures

• Freeze/thaw cycles made LEVs larger and aggregated

• Similar zeta potentials, except LEVs-TMO at 45 °C turned negative after 4 weeks

Freeze-thaw cycles caused LEV:

• Increase and vary in size.

• Aggregation and disruption.

• Protein levels in LEVs and LEVs-TMO decreased over time, especially at higher temperatures.

• LEVs-TMO at 4 °C had the highest protein levels.

• No significant impact on protein content with freeze-thaw cycles.

• LEV uptake decreases with freeze/thaw cycles

• LEVs-TMO uptake stable after freeze/thaw cycles

• LEVs at 25 °C uptake was better than − 20 °C and 4 °C

• Low LEV uptake at 45 °C (aggregated)

• LEVs in TMO: Stable at 4 °C for 4 weeks

• Freeze-thaw cycles altered LEV size

[9]

Cell culture supernatant

Isolation: ultracentrifugation, freeze-drying at -80ºC.

Examined after Lyophilization

Storage: PBS, 20 mM HEPES buffer, 8.5% sucrose for lyophilization

• EV size, surface charge, and PDI consistent after freeze-drying with cryoprotectant.

• Lyophilization did not reduce EV counts.

• Morphology changed with more debris and aggregates without 8.5% sucrose in the lyophilization buffer.

• No significant differences in total protein content

• Cellular uptake confirmed EV functionality post-lyophilization

• Lyophilization preserved EVs’ physicochemical properties and functionality

[62]

U937 and CT26 cells.

Isolation: centrifugation

Storage: Lyophilized and stored at 4 °C, 25 °C, 3 months, and 6 months.

Storage media: Sucrose or trehalose, with/without polysorbate 80.

• No significant size changes • Trehalose or sucrose plus polysorbate 80 could maintain EV size after lyophilization

Well-preserved

• Protein concentration, structure, and activity declined during storage.

• Trehalose, sucrose, and polysorbate 80 maintained protein levels before and after lyophilization.

• EVs in cryoprotectant had a similar bioactivity, antioxidant enzyme activity, and reduced heart infarct size like fresh EVs

• Trehalose provides higher storage stability than sucrose.

• Trehalose with polysorbate 80 maintained EV bioactivity.

[30]

Human BDMSCs

Isolation: ultracentrifugation, freeze-drying

Storage: RT, 4 °C, -20 °C, -80 °C for 1 week, 4 weeks, 6 weeks; up to five freeze-thaw cycles

• EV size increased after 28 days at -20 °C

• Mode size increased after 5 freeze-thaw cycles at -80 °C

• EV morphology and size retained after lyophilization

• Protein content stable at RT and 4 °C.

• CD63 and TSG101 are stable across temperatures.

• Stable upon five freeze-thaw cycles.

• Bioactivity decreased after 5 freeze-thaw cycles.

• Frozen EVs led to lower IL-6 secretion.

• Lyophilization preserved EV bioactivity.

• 4 °C and lyophilization were best for long-term bioactivity.

• EV proteins were stable in storage conditions.

• Freeze-thaw cycles and long-term storage harmfully affected bioactivity.

[32]

Murine melanoma B16BL6 cells

Isolation: ultracentrifugation, lyophilization

Storage: -80 °C and RT (1 week); cryoprotectant: Trehalose

• Lyophilization without trehalose led to aggregation

• Wider PDI compared to -80 °C storage

• Not aggregated Exos at -80 °C and lyophilized with trehalose.

• No significant changes compared to -80 °C storage.

• Trehalose preserved protein and RNA integrity in lyophilization Exos.

• Luciferase activity and cytokine release stimulation potential of EVs remained stable at RT

• Lyophilization with trehalose preserves Exos.

• RT storage did not affect exosome content or function.

• RT maintained protein, RNA, pharmacokinetic, and physicochemical properties.

[22]

Human umbilical cord-MSCs conditioned medium

Isolation: centrifugation, lyophilization

Storage: 4 °C, -20 °C, -70 °C, -196 °C (liquid nitrogen) for 2–3 weeks; storage media: DMSO (2.5 to 10%).

• Microvesicle concentration stable at 4 °C for 1 and 2 weeks (95.0% and 64.8% of initial levels), followed by over 50% after the third week.

• Lyophilization extended shelf life

-

-

-

• Microvesicles sensitive to freezing

• Lower DMSO (< 5%) preserved 90–95% of microvesicles and outperforms higher DMSO (7.5–10%)

[29]

Purified plasma (pPFP) and BV2 microglia cell line

Isolation: qEV SEC columns, ultracentrifugation.

Storage: -80 °C with/without preservatives (trehalose), lyophilization; various freeze-thaw cycles; storage media: Trehalose, DMSO, Glycerol, Protease Inhibitor, Sodium Azide

• Storage at -80 °C reduces EV yield and counts after 6 months.

• Freeze-thaw cycles decreased EV yield and increased size.

• Stored EVs became more positively charged after 6 months.

• Reduced EVs yield, and increased particle size after freeze-thaw cycles.

• Increased contaminant protein concentration during storage

-

• Storage at − 80 °C was best for EVs

• −80 °C preservation of EVs in their biofluids is preferable over isolated EV

• Freeze-thaw damaged EVs’ membrane

• Time affects protein and size

[55]

Human umbilical cord-MSCs

Isolation: centrifugation for apoptotic vesicles, ultracentrifugation for exosomes

Storage: lyophilized or encapsulated in hyaluronic acid hydrogel (EV- HA) and stored at -80 °C or RT for 2 months; storage media: Trehalose, polyvinylpyrrolidone

• No change in size and slight changes in total numbers and zeta potentials after lyophilization.

• Cryo-EM showed intact membrane structures of lyophilized apoptotic vesicles (apoVs).

• Minimal decrease in tissue factors expression in lyophilized apoVs.

• Lyophilized apoVs and EV- HA, maintained their procoagulant ability at both RT and − 80 °C.

• Lyophilization or encapsulation in hydrogels fulfilled storage challenges, kept bioactivity, and facilitated the transportation of EVs.

[64]

RO cells (ACC452)

Isolation: ultracentrifugation, SEC

Storage: Lyophilized and stored up to 6 months; stabilizers: sucrose, poloxamer 188, polysorbate 20, polyvinylpyrrolidone

• PBS-EVs lost intact vesicles.

• Sucrose reduced freeze-thaw particle growth.

• P188 and sucrose preserved the highest number of intact vesicles (size, zeta value, and concentration)

-

-

-

• EV stability is affected by freezing and drying

• P188 and especially sucrose preserves EV stability for 6 months

• Storage at 2–8 °C suitable for at least 1 month

[34]

bEnd.3 endothelial cells

Isolation: ultracentrifugation.

Storage: 4 °C, -20 °C, and − 80 °C (up to 28 days); freeze-thaw: 1–5 times (to 4 °C)

• Widened size range for all storage conditions

• Most significant enlargement at -20 °C

• TEM observations: aggregations, membrane destruction, fusion.

• Protein and CD63 levels decreased post-storage.

• Decreased protein levels after one week at 4 °C but stable at -80 °C.

• No significant total RNA decreases at 4 °C within a week

• Storage reduced EV’s autologous uptake in vivo and ex vivo

• Storage affects the size, quantity, content, cellular uptake, and biodistribution

[12]

Human semen

Isolation: ExoQuick (EQ) precipitation

Storage: -80 °C in biofluid (30 years)

• No impact from storage duration

• Morphology unchanged after freezing and storage

• No aggregation, fusion, or membrane damage

• Stable RNA and protein in Exos when frozen.

• Some cargo components, like AChE activity, decreased

-

• Freezing duration did not affect semen Exos and their protein content

[35]

MIN6 supernatants

Isolation: ultracentrifugation, ultrafiltration.

Storage: 4 °C (1 day) or -80 °C (up to 1 year); four freeze-thaw cycles; storage media: PBS or trehalose

Trehalose:

• Increased EV count and yield, maintained zeta potential, uniform size distribution, and reduced mean size.

• Protected from freeze-thaw cycles compared to PBS.

More particles in trehalose -EVs, with less aggregation and fusion

• No differences in total protein and RNA concentrations.

• Enhanced bioactivity in trehalose EVs more than PBS (stimulation of TNF-alpha).

• Freeze-thaw: trehalose preserved EV bioactivity better than PBS.

• Trehalose preserved count, purity, size, charge, and cargo and inhibted aggregation.

• Exos in trehalose were more bioactive than in PBS.

[48]

Bronchoalveolar lavage fluid

Isolation: ultracentrifugation.

Storage: +4 °C (4 days), -80 °C (4 days); one freeze-thaw cycle

• +4 °C increased exosome size slightly.

• -80 °C significantly increased size and PDI.

• Thawing from − 80 °C disrupted ζ values and structure.

• Fresh and + 4 °C: separated, membrane-encapsulated.

• -80 °C: nanovesicle Aggregation, diminished ζ values, and multi-lamellar membranes.

• +4 °C storage: Lost 457 proteins.

• -80 °C storage: Lost 315 proteins.

• Altered biological function.

• Freezing made Exos enlarged and formed multilamellar vesicles.

• Storage led to leakage of non-membrane-integrated proteins.

[36]

A549 cell line

Isolation: ultracentrifugation

Storage: short-term at RT (PBS, PBS with trehalose or DMSO, 14 days); long-term at 4 °C, -20 °C, -80 °C (PBS, PBS with trehalose or DMSO, 8 weeks)

• Appropriate short-term stability in all preservatives

• Best long-term stability at -80 °C with trehalose

• No change in size distributions in short or long-term storage

• Reduced concentration at -20 °C and 4 °C

• No significant aggregation in 2-month-storage

• Stable exosome protein for 2 weeks at RT and 2 months at low temperatures

• 20% decrease in PBS at RT

-

• Exos stored in PBS with trehalose had the best stability in terms of concentration, zeta potential at RT and low temperatures

[45]

Placental cell culture media

Isolation: ultracentrifugation

Storage: RT, 4 °C, -20 °C (14 days); one freeze-thaw cycle

• No concentration differences at RT or 4 °C

• Storage at -20 °C reduced concentration with no significant size changes on days 7 and 14

-

• Stable protein levels at RT or 4 °C.

• Unaffected DNA by storage conditions.

• EVs at RT or 4 °C retained endothelial cell activation prevention ability

• Reduction observed for EVs at different durations and temperatures

• Placental EVs stable at RT or 4 °C for 14 days

• Storage at -20 °C reduces EVs concentration

• Functional activity unaffected at -20 °C

[42]

KSHV-infected HUVECs

Isolation: ultracentrifugation

Storage: -70 °C, -20 °C, 4 °C, 37 °C (25 days)

• Higher temperatures caused higher decreases over time.

• On day 16, -70 °C had more particles.

• Counts decreased at -20 °C and − 70 °C by day 25

• Size decreased over time at all temperatures, especially at -70 °C.

-

• No notable alterations in Surface protein stability of EVs up to day 8.

• A decline in protein levels at 37 °C by day 16

• Protein levels remained stable at other temperatures by day 25.

• Activity maintained only at 4 °C and − 70 °C by day 25.

• -20 °C less effective by day 16.

• 4 °C slightly better than − 70 °C, possibly due to freeze-thaw.

• 37 °C lost activity in 4 days.

• EV number decreased during storage

• 4 °C offers good stability

• -20 °C storage reduced activity

• Storage at -70 °C preserved EV activity

• EVs at 4 °C more active than − 70 °C

[57]

CSF from glioblastoma patients

Isolation: ultracentrifugation

Storage: RT (1 and 7 days), -80 °C (7 days); freeze-thaw (1, 2, or 3 cycles)

• 37–43% reduction after 3 freeze-thaw cycles in EV count.

TEM:

• No membrane damage.

• No morphology change.

• No significant changes in miRNA levels

-

• EV miRNAs stable at RT for 7 days

• Single freeze-thaw cycle did not affect EVs or miRNAs

• Decreased parameters after two freeze-thaw cycles

[19]

Human whole saliva primary culture

Isolation: gel filtration chromatography, ultracentrifugation

Storage: 4 °C (up to 20 months); slow freeze-thaw cycles; storage media: Tris-buffered saline

• Stable size after 20 months at 4 °C

• Intact in its biofluid (saliva) for 28 days at 4 °C

• Resistant to NP-40 and Triton X-100 detergents

• Morphology unchanged during storage

• Membrane integrity preserved

• Stored Exos showed some protein degradation.

• Key exosomal marker proteins remained intact.

-

• Salivary Exos were stable at 4 °C

• Storing in whole saliva-preserved Exos for at least 28 days at 4 °C

[20]

Human plasma

Isolation: ultracentrifugation, Immunoaffinity pull-down, OptiPrep density gradient separation.

Storage: 4 °C, -20 °C, -80 °C (3 months)

-

• Exos were detected in all conditions in their biofluid

• Aggregation in plasma samples stored at 4 °C after both 30 and 90 days.

• TSG101 detected in Exos from plasma-stored samples

• Exosome uptake remained stable and active by cells after 30 days at -20 °C

• Exos were stable for 90 days in plasma

[4]

Mice BM-conditioned media

Isolation: ultracentrifugation

Storage: 1 month (4 °C, -20 °C, -80 °C, -196 °C); one freeze-thaw cycle; storage media: PBS, PBS + trehalose + DMSO (TRE)

• Cryopreserved Exos increased in size at -20 °C in PBS, not in TRE.

• At 4 °C, exosome size decreased in PBS and increased in TRE.

• Morphology stable at -80 °C (PBS, TRE)

• Membrane disruption and fusion at 4 °C

• Less aggregation in TRE

-

• Trehalose avoided biological functionality lost during storage (EV uptake and migration potential)

• Storing at -80 °C with trehalose preserved structure, integrity, and bioactivity of EVs

[21]

Urine samples (healthy, diabetic, normal/micro/macroalbuminuria)

Isolation: ultracentrifugation

Storage: -20 °C vs. -80 °C, up to 4 years

• Particle concentration decreased after 14 days

• Stable particle size and size distribution

• Stored at -80 °C up to 24 months, maintained particle size, concentration, structure, and EV protein markers

• TSG101, CD9, CD63 levels decrease after 4 months at -20 °C.

• Healthy control EVs show detectable EV markers at -20 °C for 1.5 months, comparable to -80 °C.

• Lower RNA yield at -20 °C compared to -80 °C storage.

-

• Temperature affects EV protein markers

- Urine (biofluid) storage resulted in EV-enriched protein markers

[16]

Epithelial ovarian cancer (EOC) tissue lysates

Isolation: ultracentrifugation, centrifugation

Storage: lysates or tissues at -80 °C for 15 days

-

• Membrane structure and morphological diversity maintained after − 80 °C storage

• CD81 slightly reduced in frozen tissue lysate EVs

• TSG101, ALIX, and Flotillin-1 levels stable

• No significant difference between fresh and frozen tissue-derived EVs

• Freezing did not affect cellular uptake

• Freezing did not affect EV’s uptake or structure

• Cryopreservation is suitable for EV membrane structure and size maintenance

[28]

HT-29 human colorectal adenocarcinoma cell line

Isolation: ultracentrifugation

Storage: -80 °C (8 weeks), then thawed at + 4 °C and stored (up to 48 h) in various tubes; one freeze-thaw cycle

• Significant concentration loss in ordinary tubes at 48 h resulted from EV adsorption on tube walls.

• Particle counts reduced in all samples in PBS at + 4 °C, particularly in ordinary tubes.

• No aggregation, fusion, or membrane disruption during storage or freeze/thaw cycles

-

-

• Adsorption of EVs onto tube walls causes concentration losses.

• Surface block with excess protein or BSA or use of Eppendorf Protein LoBind tubes can alleviate EV adsorption.

[56]

Plasma samples and BM1 cells

Isolation: ultracentrifugation, qEV columns

Storage: -80 °C (10–12 days); slow freezing; one freeze-thaw cycle; cryoprotectant: 10% DMSO

• Lower concentrations in stored EVs.

• Similar size distribution for fresh and stored EVs.

-

• Freezing reduced RNA yield in EVs

• Slow freezing and rapid thawing did not fully recover RNA levels

• Freeze-thaw cycles caused RNA loss

• DMSO-protected RNA yield

-

• Cryopreservation resulted in EV and RNA loss.

• 10% DMSO improved RNA yield in cryopreserved samples.

[49]

Human erythrocytes

Isolation: ultracentrifugation

Storage: 4 °C (up to 7 months); resuspended in PBS-citrate

• Light scattering intensity decreased in the first week and remained constant afterward (may be due to vesicle adhesion)

• After 6 weeks at 4 °C: empty and degraded vesicles seen.

• EVs stability at 15–60 °C: morphology almost preserved.

-

-

• EVs were stable at various pH levels, osmolarities, and temperatures

• Minimal changes observed when stored at 4 °C

[50]

Human Milk & Infant Formulas

Isolation: ultracentrifugation

Storage: 4 °C, -80 °C with/without glycerol and DMSO

• 4 °C caused loss of Exos in human milk after 4 weeks

• No significant loss at 4 weeks in frozen samples or with preservatives

• No significant size changes.

-

• Stored milk at -80 °C or > 24 h resulted in low RNA yield.

-

• Exosome-sized vesicles Lost in human milk at 4 °C after 4 weeks

- No significant loss in frozen samples

[37]

HEK 293 conditioned medium

Isolation: Exo-Quick kit.

Storage: Short-term (4 °C to 90 °C, 30 min), long-term (-70 °C to RT, 10 days)

-

• Altered exosome morphology during storage

• Increased dispersion in 10-day RT

• maintained morphology at -70 °C storage

Short-term storage:

• Stable exosome markers at 4 °C, 37 °C, and RT.

• Slight loss at 60 °C.

• All proteins degraded at 90 °C

Long-term storage:

• Stable markers below − 20 °C; lost in higher temperatures.

-

• High temperatures degraded exosomal proteins

• Freezing conditions were best for long-term storage

• Above − 20 °C was not ideal for exosome preservation

• Cold storage is recommended for long-term preservation.

[13]

Peripheral blood from metastatic colorectal cancer patients

Isolation: ExoQuick or PureExo^® kit

Storage: serum at 4 °C (24, 72, 168 h); RT (6, 12, 24, 48 h); up to 5 freeze-thaw cycles

-

-

• EV markers were consistent in different conditions.

• High DNA stability, especially at 4 °C, while decreased over 48 h at RT.

• Freeze-thaw cycles caused significant DNA decline.

-

• Serum EVs and their DNA contents were stable under different storage conditions.

- Freeze-thaw cycles had the most significant impact on EV stability.

[25]

LPS-stimulated THP-1 cells

Isolation: ultracentrifugation, ExoEasy purification.

Storage: 4 °C, -80 °C (up to 1 month); thawed at RT

• No difference in EV number at 4 °C or -80 °C over time

-

-

-

• 4 °C or -80 °C were suitable for EV storage up to one month

• Best preserved at -80 °C

[38]

Human milk

Isolation: Ultracentrifugation.

Storage: -80 °C (up to 6 months)

• No significant trends in count and size

-

-

-

• Storage did not affect Exos

[27]

Endothelial progenitor cells (EPC)

Isolation: centrifugation with PEG

Storage: -80 °C (2 months); storage media: shear-thinning gel (STG)

-

-

• Frozen EVs maintained RNA and miRNA purity.

• Minimal degradation over 8 weeks at -80 °C.

• -80 °C maintained EV’s bio-functionality during storage (antibacterial, angiogenesis)

• Fresh and frozen EVs stored at -80 °C maintained function for over 2 months

[39]

Cardiac progenitor cells conditioned medium

Isolation: TFF, HiScreen Capto Core 700 column

Storage: 4 °C–− 80 °C for 7 days in various tubes; buffers: PBS with Tween 20 or BSA

• EVs stored at 4 °C or -80 °C decreased particle counts over time.

• Storage in glass tubes led to EV loss compared to other tube materials.

• Better count and size preservation in Tween or BSA.

-

• No differences in protein content between storage temperatures.

• -80 °C had stronger cell migration effects in-vitro compared to 4 °C after 7 days

• Tween 20 and BSA assisted EVs to preserve their function

• Polypropylene tubes enhanced EV recovery.

• BSA and Tween 20 protected EVs.

• 4 °C or -80 °C suitable for short-term EV storage.

• -80 °C is better for longer preservation

[58]

Blood samples

Isolation: centrifugation

Storage: Various tubes; plasma: -80 °C; one freeze-thaw cycle

• EVs decreased after a single freeze-thaw cycle.

-

-

-

• EV concentration decreased after a freeze-thaw cycle

[65]

First-morning urine sample

Isolation: ultracentrifugation, immunoaffinity, chemical precipitation

Storage: -80 °C (6 months, long-term), RT (1 month, short-term); with or without preservative; one freeze-thaw cycle

• Slight decrease in EVs after 1 month at RT.

• Size distribution of EVs unaffected by 1-month RT storage.

• EVs aggregate during long-term storage at RT.

• Stable protein and RNA contents at RT for 1 month

• Higher protein and RNA content at -80 °C for 6 months

• ALIX and TSG101 levels remained unchanged

• Small non-coding RNAs unaffected by storage conditions

- EVs’ biological function was not affected by storage conditions.

• Urine EV RNA was stable at RT for short- and long-term storage

• RT is not suitable for protein analysis, but acceptable for short-term (1 month).

• Cryoprotectants maintained EV stability.

• Freeze/thaw harmed EVs and RNA integrity.

[59]

Plasma

Isolation: ExoQuick kit

Storage: Short-term: 4 °C (2 weeks), -20 °C, -80 °C (up to 2 months); long-term: -20 °C (3 or 5 years); up to 2 freeze-thaw cycles

• No significant size and concentration differences were observed after storage

-

• Exosomal miRNAs were stable in different storage conditions.

-

• Exosome miRNAs stable across storage conditions.

[51]

HEK293T-palmGFP cell line conditioned medium.

Isolation: ultracentrifugation

Storage: 4 °C or RT (up to 12 weeks); 3–4 freeze-thaw cycles; storage media: NaCl-HEPES, PVP nanofibers

• Particle count decreased after 2 weeks at 4 °C and RT

-

• EV marker (CD81) decreased after 4 °C and RT storage.

-

• Polymer base preservation improved EV stability.

• Electrospinning offers practical vesicle storage stability.

[61]

Mouse fibroblast transfected cell culture media

Isolation: ultracentrifugation

Storage: 4 °C or -80 °C for 21 days; up to 3 freeze-thaw: cycles

• Count and size remained consistent after storage

• Count increased with the first freeze-thaw, but decreased with subsequent cycle

-

• A slight decrease in protein content with 2 freeze-thaw cycles.

-

• EVs were stable in storage, freeze-thaw, and high salt.

[63]

Genetically engineered HEK293T cells

Isolation: ultracentrifugation

Storage: 4 °C, -80 °C (up to 7 days); storage media: PBS, culture media, trehalose, BSA-HEPES

• Significant particle reductions and size increase in PBS over time

• Storage in culture media (before isolation) resulted in significant particle loss compared to isolated EV storage

• EVs in PBS may fuse or aggregate immediately after resuspension

• EVs in cryoprotectant buffer had higher DNA copies than EVs in cultured media or PBS at 4–− 80 °C storage over 7 days.

• EVs in buffer showed higher binding capacity to target cells

• Storing EVs in PBS or buffer for 24 h at 4 °C did not influence EV targeting capacity.

• Storage in PBS negatively affected EV integrity and functions.

• Cryoprotectants prevented EV loss and maintained EVs’ capacity.

[60]

Mouse brain tissue homogenate

Isolation: ultracentrifugation

Storage: RT, 4 °C, -20 °C, -80 °C, -196 °C (0.5 to 7 days); storage media: PBS, DMSO

• Significant concentration difference across temperatures in the PBS group, but not in the DMSO group

• Damaged microparticles (MPs) during storage

• DMSO had no protective effect on MPs

• Freezing caused agglomeration, lysis, or fusion.

-

• Procoagulant ability decreased at all temperatures.

• No protective effect from DMSO on cryopreserved MPs.

• Size, morphology, and biological function of MPs were downgraded by cryopreservation.

[41]

Dairy cow foremilk samples

Isolation: ultracentrifugation

Storage: milk samples at 4 °C and − 20 °C (7 days) before EV isolation; EVs at -80 °C up to 1 month

• No significant differences in particle size and concentration over 7 days of storage.

• No differences in morphology, aggregates, or contaminations over 7 days.

• No significant changes in protein, EV markers, RNA, and miRNA levels over storage time

-

• Storage of milk EVs at 4 °C for one week did not affect its protein concentration and markers

[47]

Human serum from autopsy cases

Isolation: ultracentrifugation

Storage: 4 °C, 20 °C, 30 °C (3 days)

• Size distributions not significantly changed by storage temperatures or periods

• Samples stored at 20 °C and 30 °C showed increased smaller-sized (< 33 nm) particles

-

• Protein and miRNA levels unchanged at 4 °C and 20 °C, but reduced at 30 °C

-

• Exos were stable up to 3 days at 4 °C and 20 °C, and 1 day at 30 °C.

[52]

Human corneal stromal stem cells (CSSC) conditioned medium

Isolation: Total Exosome Isolation Reagent

Storage: 4℃ and − 80℃ up to 4 weeks for EVs; RT for EVs lyophilized with trehalose

• Similar concentration of EVs after storage at 4 °C and − 80 °C.

• Better preservation of particle concentration and size with the addition of trehalose during lyophilization.

• Trehalose protected lyophilized EVs from aggregation.

• Trehalose avoided EV markers depletion after lyophilization

• Total RNA and miRNA levels of EVs stable up to 7 days after lyophilization.

• Trehalose-lyophilized EVs had the best anti-inflammatory and anti-fibrotic effects.

• EV integrity and function were better preserved at -80 °C than at 4 °C.

• Short-term storage (4 weeks) did not significantly alter EV integrity and function.

• Storage at -80 °C is optimal for EVs preserving

• Lyophilization with trehalose is effective in preserving EVs.

[44]

Human serum exosome

Isolation: ultracentrifugation

Storage: Pooled plasma stored at -80℃ before exosome isolation for up to 6 months; Isolated Exos stored at 4 °C (7 days), − 20 °C (1 month), or − 80 °C (up to 6 months)

• The highest concentration in one-week storage was at 4 °C and for longer storage was at − 80 °C

• Freeze-thaw cycle in short-term damaged Exos

• Storage at 4 °C–− 20 °C caused amorphous, deformation and shrink Exos

• No difference in exosome protein markers at 4 °C for 1 week

• In long-term storage higher temperatures had lower protein markers

-

• 4 °C is better than − 80 °C for 1 week storage

• −80 °C for long-term storage is better

• Plasma storage was better than isolated Exos in PBS

[46]

Urine samples derived Exos

Isolation: ExoLution kit

Storage: +4 °C (2, 7 or 14 days); +20 °C (2 days); +40 °C (2 days), -80 °C (2, 4 or 30 days)

Freeze-thaw: whole urine up to 2 cycles

-

-

• +20 °C and + 40 °C caused gradual mRNA degradation.

• No significant changes up to 7 days at + 4 °C

-

• High-temperature storage of urine samples causes mRNA content to lose

• +4 °C is better for less than 7 days of storage, longer storage should be at -80 °C

[43]

Serum samples derived EVs

Isolation: centrifugation

Storage: 25 °C, 37 °C, 4 °C, and − 20 °C up to 3 months

-

-

• EV miRNAs Stable at − 20 °C up to 3 months

• Higher temperature caused EV miRNA degradation faster

• A single freeze-thaw cycle caused EV miRNA degradation of up to 70%

-

• Short-term storage at − 20 °C is suitable for up to 3 months

• Adding protectant significantly slowed

down the degradation of EV miRNAs

[40]

Lymphocyte-derived EVs

Isolation: ultracentrifugation

Storage: -80 °C, − 20 °C, 4 °C, RT, and 37 °C up to 1 month and freeze-drying

Various cryoprotectants were used. Fast and slow freezing were tested.

• Stable EVs concentration at -80 °C for 30 days, and reduced EVs number at RT

• EVs strongly damaged at 37 °C

-

• Total protein increased, and EV marker decreased at RT, 4 °C, and − 80 °C after freezing in liquid nitrogen due to EV membrane damage

-

• Lyoprotectant maintained EV integrity upon lyophilization