From: Different storage and freezing protocols for extracellular vesicles: a systematic review
Ref. | Source/ Isolation/ Storage | Characterization | Morphology | Protein, RNA, and DNA evaluations | Biological function | Highlights |
---|---|---|---|---|---|---|
[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 |