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Different storage and freezing protocols for extracellular vesicles: a systematic review

Stem Cell Research & Therapy volume 15, Article number: 453 (2024) Cite this article

Abstract

Background

Extracellular vesicles (EVs) have been considered promising tools in regenerative medicine. However, the nanoscale properties of EVs make them sensitive to environmental conditions. Optimal storage protocols are crucial for maintaining EV structural, molecular, and functional integrity. This systematic review aimed to gather evidence on the effects of various storage protocols on EV characteristics and integrity.

Strategy

A comprehensive search was conducted for original studies investigating the impacts of storage temperature, freezing techniques, freeze-thaw cycles, and stabilizing strategies on EV concentration, size distribution, morphology, cargo content, and bioactivity. Results from 50 included studies were analyzed.

Results

Data indicated that rapid freezing procedures and constant subzero temperatures (optimally − 80 °C) resulted in appropriate EV quantity and cargo preservation. Subjecting EVs to multiple freeze-thaw cycles decreased particle concentrations, RNA content, impaired bioactivity, and increased EV size and aggregation. Electron microscopy revealed vesicle enlargement, and fusion, along with membrane deformation after being exposed to substandard storage protocols. The addition of stabilizers like trehalose helped EVs to maintain integrity. Of note, storage in native biofluids offered improved stability over purified EVs in buffers.

Conclusion

Data emphasize the critical need for precise storage protocols for EVs to ensure reproducible research outcomes and clinical applications. Further studies using reliable methods are necessary to create specific guidelines for improving the stability of EVs in various applications.

Introduction

Extracellular vesicles (EVs) have emerged as one of the most promising tools in regenerative medicine and drug delivery. These nano-sized and cell-derived particles play important roles in intercellular communication and also have potential applications in the field of diagnostics and therapeutics. However, the clinical translation of EV-based therapies faces significant challenges, especially in maintaining EV stability during storage, transfer, and administration.

EVs are heterogenous populations and consist of three main categories based on their biogenesis and size: exosomes (Exos, 30–150 nm), microvesicles (MVs, 50–1000 nm), and apoptotic bodies (several micrometers) [1, 2]. EVs are released during different physiological and pathological conditions and based on their distinct formation mechanisms, each class of EVs have a different cargo profile that resonates with their diverse biological functions [3,4,5]. These lipid bilayer-enclosed particles can transfer bioactive molecules, including proteins, nucleic acids, lipids, and metabolites, between cells and modulate recipient cell behavior as well as function [6, 7].

Exosomes are formed via the invagination of the endosomal membrane in endosomes and multivesicular bodies (MVBs), leading to the formation of numerous intraluminal vesicles (ILVs) with various cytoplasmic contents [8,9,10]. Exos generally can harbor certain tetraspanins (e.g., CD63, CD9, and CD81), heat shock proteins (HSP70), and endosome-associated proteins (i.e., TSG101, Alix) [11,12,13,14]. MVs are directly produced and released via the outward budding of the plasma membrane [9]. Apoptotic bodies originate from apoptotic cells containing various cellular components, including organelles and fragmented DNA. Unlike Exos and MVs, apoptotic bodies are a result of cell death and are typically engulfed and cleared through phagocytosis by local immune cells or native non-immune system cells [15]. It is thought that both Exos and MVs contain proteins, as well as nucleic acids (mRNAs, miRNAs, lncRNAs, and DNA), lipids, and metabolites [4, 11, 16]. Cargo sorting is regulated by complex molecular pathways and mechanisms inside the cells that sequester selectively of the contents [17]. Thus, the cargo of EVs is not a random sample of cellular content but it is a selective sorter that represents their parental cells’ cytoplasmic condition under physiological and pathological status [4, 16]. Since EVs can mimic the cytoplasmic state of their parental cells and carry functional biomolecules, this capacity makes them putative therapeutic tools for various pathological conditions [9]. However, there is a need to overcome challenges in EV production and preservation up to years, especially optimizing storage conditions for maintaining the integrity and functionality of EVs, to realize their therapeutic potential.

To date, EVs have been isolated from diverse biofluids including blood, urine, saliva, breast milk, semen, follicular and amniotic fluid, and synovia [12, 18,19,20,21,22,23]. EVs can also be obtained from tissue lysates or cultured [16, 24]. Recently the application of EVs has expanded in biomedical fields due to their suitable biodistribution and delivery platform eligibility [9, 16, 25]. It has been observed that EVs are powerful and minimally invasive agents with remarkable therapeutic potential in various diseases [8, 26], however, the clinical application of EVs requires standardized protocols to isolate, characterize, and store EVs to maintain their structural integrity and dynamic activity [9, 27, 28].

Cryopreservation is a commonly used technique to preserve original characteristics such as concentration, size, morphology, and functional properties of EVs after storage [4, 29]. However, the freezing process often causes vesicle rupture, cargo lost, aggregation, and precipitation (Fig. 1). However, there is no universally optimal protocol for varied EV types, and therefore attempts should be focused on the establishment of standard storage conditions based on nanovesicle source and intended future application [4, 29, 30]. Cryoprotectants such as dimethyl sulfoxide (DMSO), and glycerol have been used to minimize cryodamage, however, numerous experiments have indicated the possible cytotoxicity and inhibition of specific downstream processes [22].

Fig. 1
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EV cargo stability during optimal, suboptimal, and poor storage conditions.

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This systematic review aims to comprehensively evaluate various storage and cryopreservation methods for EVs, and assess their possible impact on physicochemical parameters, and functional qualities for potential clinical applications. This information will help identify optimal storage conditions and could advance EV-based therapies and establish standardized protocols for EV storage and cryopreservation. Moreover, the influencing factors such as temperature, freezing rate, cryoprotectant type and concentration, and post-thaw processing were highlighted. Eventually, this review highlights areas where further research is needed to support the translation of EV-based therapies from bench to bedside.

Methods

This systematic review aimed to find the appropriate protocol to store and freeze EVs without causing prominent damage in short- and long-term storage. The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [31] were implemented for data extraction and analysis.

Search strategy

A comprehensive search using PubMed and Scopus databases was conducted for the published literature up to August 2024. The search strategy was a combination of Boolean operators, keywords and Medical Terms (MeSH) related to “extracellular vesicles”, “storage”, “freezing”, “cryopreservation”, “stability”, “functionality” and “integrity”. The search was limited to articles published in English. In addition to a systematic search in PubMed and Scopus databases, a supplemented search was conducted to ensure comprehensive coverage of including papers. To this end, the reference lists of included studies were screened and a hand search on Google Scholar was conducted for additional relevant studies. These additional sources are represented as “other” in the flow chart of PRISMA (Fig. 2).

Fig. 2
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PRISMA flow diagram. The identified documents were carefully reviewed, and studies meeting the eligibility criteria were included in the systematic review as illustrated in this flow diagram

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Selection criteria

Studies were included if they reported on “the impact of storage and freezing conditions on the integrity of EVs”. All studies measuring the vascularization rate (EV release of blood cells during storage time) were excluded since those studies did not examine the stability capacity of EVs themselves. Reviews, editorials, commentaries, letters, and conference abstracts were also excluded. Two authors (SA and NJ) independently screened titles and abstracts and non-related articles were excluded. Full-text articles were obtained for eligible studies and were independently assessed by three authors (SA, NJ, and AG).

Data extraction

Data were extracted using a standardized data extraction table. The following information was extracted from each study: source of EVs and isolation methods, storage and freezing conditions, methods used to assess EVs integrity and stability (concentration, size, morphology, protein/DNA/RNA contents), and main findings. Any disagreements between authors during the data extraction process were resolved through discussion or consultation with a fourth author (MM).

Outcomes

Literature search

The initial systematic search resulted in 626 papers from PubMed, Scopus, and other databases out of which 170 duplicates were removed resulting in 456 total papers. Subsequently, title and abstract screening identified 58 potentially related papers, which were further assessed in full text. Studies assessing the influence of storage temperature, freezing method, free-thaw cycles, and stabilizing agents on EV parameters such as concentration, size, morphology, protein and DNA/RNA content, and functional bioactivity consisting of the final 50 papers were considered, while the excluded studies primarily focused on RBC vascularization (EV release during storage periods) or employed methodologies beyond the scope of our study. The study selection process, following PRISMA guidelines [31], is presented in the flow diagram (Fig. 2). The comprehensive summary of papers is presented in Table 1.

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
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Storage temperature and duration effects

Studies revealed that storage temperature significantly influences EV recovery and integrity depending on the specific temperature range. The effect of various storage temperatures on EVs as reported in multiple included studies will be summarized in the following.

Impact of lower temperatures

Most of the studies indicated that storing EVs at -80 °C yielded better results in terms of particle concentration, RNA content, morphology, and biological functionality for long-term preservation compared to higher temperatures [4, 11, 12, 16, 20, 21, 25, 27, 29, 30, 32,33,34,35,36,37,38,39,40]. Additionally, it was also stated that ultra-low storage at -80 °C preserved EVs’ integrity and function in short-term storage (more than 1 week) [41]. These findings were consistent across various EV sources, including conditioned media, biofluids, and tissue extracts. For instance, one-month storage of EVs from human umbilical cord mesenchymal stem cells (hUC-MSCs) at -80 °C did not significantly affect their uniform size, integrity, and bioactivity, while EVs stored at -20 °C showed a significant particle aggregation and size increase [11]. Similarly, HEK293T and MSC-derived EVs preserved their size, concentration, morphology, and RNA/protein content better when stored at -80 °C versus − 20 °C up to 26 weeks [33].

Although storage at -80 °C has been shown to provide promising preservation potential, -196 °C (liquid nitrogen) is less commonly used in the studies and there is very limited data on the comparison of these two ultra-low temperatures. The available data seems to report a better outcome from storing EVs at -80 °C compared to liquid nitrogen. For example, unlike − 80 °C, both microvesicles and exosomes from mice bone marrow MSCs stored in liquid nitrogen for one month exhibited a size reduction [4]. Another study showed less EV concentration loss when stored at -80 °C compared to liquid nitrogen [34]. One study demonstrated membrane disruption in EVs freezing in liquid nitrogen followed by storing at -80 [40]. Therefore, based on the evidence − 80 °C remains the most practical and commonly recommended option for long-term EV preservation since it effectively maintains particle integrity without the need for ultra-low temperatures (Fig. 3).

Fig. 3
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Temperature and time effects on EV stability.

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Freezing at -20 °C exhibited moderate preservation, better than 4 °C but not as reliable as -80 °C. For instance, one study recommended the superiority of -70 °C over − 20 °C for EV maintenance and storage [42]. Another study demonstrated good preservation of EV protein, RNA content, and cellular uptake at -20 °C compared to 4 °C after 28 days [34]. In another study, it is mentioned that short-term storage at − 20 °C is suitable for up to 3 months to preserve EV miRNAs [43]. Oppositely, a study reported amorphous, deformation and shrink Exos after 1 month storage at − 20 °C [44]. Therefore, − 20 °C could serve as an alternative for mid-term storage when − 80 °C facilities are unavailable. However, further studies could clarify the exact effectiveness of − 20 °C for EV preservation.

Impact of moderate temperatures (4 °C)

Moderate temperatures like 4 °C are often used for short-term storage, especially when EVs are needed within a few days. In some studies, storing at 4 °C was thought to yield appropriate conditions for preserving EV size, quantity, cargo, and bioactivity over short-term durations of up to 1 week [22, 25, 39, 42, 44,45,46]. For example, eight days of storage of HEK293T-EVs at 4 °C did not adversely affect their count, structure, and RNA and protein content [33]. Similarly, no significant changes in morphology, RNA content, or biological function were observed in hUM-MSCs-derived EVs after two weeks of storage at 4 °C [45]. In a study associated with postmortem serum and exosomal miRNA expression profiling, it was found that Exos may be stable at 4 °C for 3 days [47]. Another study claimed that a week of refrigeration of bovine milk samples at 4 °C before EV isolation did not adversely affect their quality [41]. Additionally, bronchoalveolar lavage fluid EVs retained their biological functionality and membrane integrity when stored at 4 °C for 4 days of storage, indicating that moderate temperatures without freezing are a safer choice for short storage durations to lessen the damage to macromolecules by ice crystal formation [48]. These findings suggest that 4 °C is a viable short-term storage option (Fig. 3).

However, as the storage period exceeds one week, the stability of the EV decreases. For instance, some studies have reported size increase, membrane disruption, aggregation, and protein degradation in EVs stored at 4 °C for longer periods [22, 34, 36, 44, 46, 49,50,51,52]. This indicates that 4 °C may be suitable for short-term storage, however, its effectiveness diminishes significantly with time and it is generally not recommended for long-term preservation.

Nevertheless, in a study, long-time EV storage at 4 °C for 20 months preserved their membrane integrity [19]. One study reported no significant differences in EV number in different storage temperatures at 4 °C or -80 °C for up to one month [25]. Another study reported better preservative results for erythrocyte EVs when stored at 4 °C compared to -80 °C up to 6 months [49].

Impact of moderate temperatures ( room temperature and above)

Storage at room temperature (RT, ~ 25 °C) or higher leads to rapid and substantial degradation of EVs. Most studies show that EVs stored at room temperature exhibit significant decreases in particle concentration and integrity within just a few days. Storing EVs above 4 °C reduces stability even at room temperature (RT) and decreases EV quality [13, 30, 36, 37, 40, 51, 53]. For instance, mouse J774A.1 cells-derived EVs stored at RT for 12 months showed almost no biological activity [53]. Additionally, RT storage of HEK293T-EVs displayed significant size enlargement and membrane disruption [51]. Even over shorter periods, higher temperatures of > + 20–30 °C in other studies caused a reduction in EV markers, proteins, and RNA cargo within days [40, 43, 46]. The EV degradation process accelerates at higher temperatures, such as 37 °C within hours to days [37, 40, 42,43,44, 54]. Therefore, EVs should kept away from higher temperatures, otherwise, they might lose all their bioactivity after just four days of storage.

Storage duration effects

The period of storage influenced EV integrity independent of temperature, although the results are not consistent. For instance, in urine and plasma EVs, particle concentration and RNA content were decreased after being stored for more than 6 months at -80 °C [21, 29]. However, another study reported no significant change in the count and size of Exos after freezing milk for 6 months at -80 °C [38]. There are very limited studies that investigate the long-term sustainability of EVs (more than one year), which is a critical limitation in the field of studying the sustainability of EVs. Most of the studies do not extend beyond 6–12 months with sufficient focus on the impact of ultra-low temperature, storage medium, and cryoprotectants on EV stability during extended periods. Although relevant papers suggest that storage at -80 °C generally preserves EV up to several months, whether this remains effective over the years has yet to be determined in multiple comprehensive studies.

Zeta potential and polydispersity index

Zeta potential and polydispersity index are critical physical properties that play a significant role in understanding the stability and uniformity of EVs during storage. Zeta potential measures the surface charge of EVs, which influences their colloidal stability and aggregation tendencies under different storage conditions. A high zeta potential indicates better stability, reducing the risk of aggregation over time. Changes in zeta potential during storage may reflect alterations in EV surface properties or aggregation. The polydispersity index reflects the size distribution of EVs, with lower values indicating a more uniform population. Monitoring these properties during storage can provide valuable insights into EV stability and potential functional changes. However, few studies evaluated the impact of parameters like temperature, freeze-thaw cycles, and storage period on the zeta potential of EVs. One investigation found that the zeta potential remained consistent during short-term storage at room temperature or refrigeration [36]. In contrast, significant declines in zeta potential values were observed after freezing at -80 °C and thawing, indicating possible structural disruption [48]. Another study noted that the zeta potential of EVs was unchanged after 3 months of -80 °C storage, but became more positive after 6 months, suggesting alterations to surface charge over time [29]. Another trial reported slight changes in zeta potential values with EV lyophilization [55]. Changes to zeta potential could reflect cargo leakage, membrane instability, and vesicle aggregation during suboptimal storage.

Freezing methodology

Rapid freezing protocols implementing direct liquid nitrogen vitrification or snap freezing exhibited comparatively better maintained EV particle numbers compared to slow freezing, without cryoprotectants in both methods [11, 16]. It should be noted that freezing in both − 80 °C and liquid nitrogen can lead to membrane disruption of EVs to some extent, however, lesser damage was reported in one study at -80 °C than in liquid nitrogen [16]. Controlling the rate of reducing temperature (-1 °C/minute) partially recovered RNA yield versus unfrozen controls [56]. One investigation found EV diameter increased after storage at -20 °C but not at 4 °C [34], whereas another study found similar results at -80 °C versus 4 °C [48]. Hypothetically, there is the possibility to implement cryoprotectants in rapid freezing protocols to enhance the efficiency and protective effects. Since cryoprotectants like DMSO, glycerol, or trehalose can prevent ice crystal formation, there might be less membrane disruption in EVs. Further research is needed to develop optimum EV-specific freezing protocols to enhance the stability and functionality of EVs.

Freeze-thaw effects

Multiple studies revealed that repeated freeze-thaw cycles reduced EV number and RNA content coincided with increased mean size, aggregation rate, and weakened bioactivity [13, 29, 30, 34, 35, 43, 54, 56,57,58,59,60]. Additionally, studies consistently report that multiple freeze-thaw cycles can lead to membrane disruption that negatively affects the structural integrity of EVs. For instance, one study reported a 23–36% loss of EVs derived from various blood cells after a single freeze-thaw cycle [58]. Another found a 37–43% reduction in EVs quantity after 3 freeze-thaw cycles [57]. An approximately 70% EV miRNA degradation was seen followed by a single freeze-thaw cycle in a report [43]. In plasma-derived EVs, RNA yield declined with each additional freeze-thaw procedure [56]. Another study reported that three cycles of freezing to -80 °C and thawing at RT increased vesicle size and polydispersity index (PDI) [35]. Two investigations reported minimal effects on EV number, RNA, or miRNA content after one freeze-thaw cycle, while significant declines occurred after two cycles [59, 61]. Most of the published reports focused on the effect of freeze-thaw cycles at -80 °C, and there is a gap in the comparison of the effect of freeze-thaw cycles between − 20 and − 80 °C. However, since − 80 °C storage generally shows better EV preservation post-storage thawing, it is possible that freeze-thaw cycles at -20 °C might result in greater losses. In only one study, it was reported that freeze-thaw cycles resulted in decreased biological activity in the endothelial gap closure assessment in -20 °C EVs compared to the − 80 °C [30]. These findings emphasize the critical importance of minimizing freeze-thaw cycles in EV transport and storage. Designing experiments or protocols that implement only a single-time freeze-thaw cycle or alternative storage methods such as lyophilization may help to reduce the harmful effects of repeated freezing and thawing.

Morphological effects

By using transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryogenic electron microscopy (cryo-EM), and flow cytometry, it became clear that suboptimal storage temperatures and repeated freeze-thaw cycles cause EV size enlargement, aggregation, membrane disruption, and structural deformation [4, 20, 29, 34, 37, 48, 49, 54]. In one study, storing at 4 °C for 6 weeks yielded empty and degraded EVs [49]. In another study, -80 °C led to multi-lamellar membrane formation [48]. Only one study found minimal morphological differences after storage for up to one month at -80 °C versus 4 °C [25]. The observed morphological changes highlight the sensitivity of EVs to suboptimum storage conditions and also emphasize the need for careful optimization of storage protocols to maintain EV structural integrity for their biological function preservation and therapeutic potential maintain.

Protein and RNA content

Western blotting analysis revealed that storing samples at -20 and − 80 °C can result in favorable outcomes for the preservation of EV tetraspanins (CD63, CD9, CD81, TSG101, and HSP70) compared to higher temperatures such as 4 °C and RT. The most favorable results were detected at -80 °C however longer storage periods led to greater protein degradation [11, 12, 33,34,35,36,37, 48]. In one study, these markers became hard to detect with the Dot Blotting technique within 7 days in EVs stored at RT [11]. In another study, significant decreases in protein content were noticed within 8 weeks for EVs stored at both − 20 and − 80 °C [33]. Similarly, multiple studies reported that storing at lower temperatures, closer to -80 °C led to appropriate EV-associated RNA compared to storing at higher temperatures [21, 27, 33, 46]. In contrast to RNA content, EV-related DNA seems to be fairly stable at different temperatures, since it was not affected by storage temperature and period [13, 45]. For instance, the DNA content of serum EVs remained relatively unaffected under different storing conditions, including 4 °C and RT [13]. Freeze-thaw cycles, however, can cause a significant loss of DNA content [13]. In biofluids like milk and urine, storage at -80 °C caused better EV protein and RNA preservation compared to -20 °C or maintenance at RT [21, 50, 57]. In this case, one study noticed no significant differences in RNA yield between EVs stored in their physiological biofluids at RT for 7 days compared to -80 °C [57].

Functional effects

In addition to physical and molecular changes, few studies evaluated the functional potential of stored EVs on recipient cells. One study found that EVs stored at 4 °C exhibited reduced endothelial cell gap closure compared to fresh EVs [30]. Another study reported reduced bioactivity after 5 freeze-thaw cycles despite preserved cytokine secretion from DC2.4 cells [32]. Two studies observed impaired EV uptake and activation of target cells following inappropriate storage [34, 54]. Remarkably, in two studies, freeze-dried EVs preserved their function when lyophilized with trehalose [52, 62]. These studies emphasize the importance of validating storage protocols both for structural and molecular preservation and the desired biological activity maintaining of EVs for their following intended applications.

Stabilizing agents

Phosphate-buffered saline (PBS) as the most commonly used buffer to resuspend isolated EVs, may result from EV aggregation, decline in count, and loss of cargo and bioactivity [3, 4, 33, 35, 36, 43, 53, 60, 63, 64]. Several studies disclosed that using chemical stabilizers and cryoprotectants helped maintain EV integrity during handling and storage. Accordingly, the application of protease inhibitors, trehalose, and human serum albumin inhibited the EV number loss enriched from various sources [4, 29, 32, 33, 35, 36, 52, 62, 63]. For instance, one study reported no variations were found in EV concentration following storage for up to one year at -80 °C supplemented with trehalose [35]. Two years of storage at -80 °C of EVs in PBS supplemented with human serum albumin and trehalose minimally degraded their RNA and protein content compared with EVs in PBS with no supplements [33]. Other cryoprotectants like DMSO, glycerol, and sucrose helped maintain particle quantity, RNA content, and bioactivity during freezing and thawing procedures [4, 9, 22, 29, 33, 48, 50, 56, 62]. One study observed aggregation occurred when trehalose was not used as a cryoprotectant for lyophilized EVs [32]. Another study observed that freeze-thaw cycles and lyophilization with stabilizers like sucrose and poloxamer 188 (P188) can protect EVs from swelling. This strategy also maintained EVs’ morphology, protein content, and bioactivity for 6 months at both RT and − 80 °C [64]. Utilizing supplementing buffers and stabilizers such as trehalose, Tween-20, or bovine serum albumin (BSA), it is possible to preserve EV particle count, cargo, and function for at least one week of storage [39, 63]. In contrast, only in one study it was claimed that DMSO did not exert any protective effects after a week of storage at 25 °C, 4 °C, -20 °C, -80 °C, and − 196 °C [60]. By preserving EVs inside hydrogel-forming microneedles, EV parameters were stable for up to 12 months at 4 °C or RT [3, 53]. Based on evidence, the use of appropriate stabilizing agents can lead to the preservation of EV integrity during storage and transportation. However, the choice of stabilizer should be based on various factors. For example, the source and type of EV, storage conditions, and intended downstream applications must be carefully considered to ensure optimal preservation of EV stability while avoiding toxicity.

Storage in biofluids

Several studies revealed that storage of EVs in native biofluids like urine, milk, but also cell culture media can lead to better-preserved particle concentration, RNA content, and surface protein expression compared to purified EVs stored in buffer solutions [11, 16, 19,20,21, 29, 44, 57]. The observation that EVs are better preserved in their native biofluids compared to purified EVs in buffer solutions may be attributed to several factors. Firstly, native biofluids provide a complex, physiological environment that may help maintain EV integrity. This environment includes proteins, lipids, and other molecules that could act as natural stabilizers to reduce the likelihood of EV aggregation or degradation. Secondly, native biofluids likely provide a more suitable osmotic environment for EVs compared to artificial buffers, thereby preserving their cargo, such as RNA, and surface proteins more effectively than in artificial buffer solutions. This hypothesis suggests that storing EVs in their natural context may offer a more supportive environment with enhanced long-term stability. Further research is needed to uncover the mechanisms behind biofluid preservation and develop synthetic media that mimic these protective effects.

Lyophilization, hydrogels, and biomaterials for conserving EV stability

Recently, emerging methods such as lyophilization (freeze-drying) and the use of hydrogels have been considered as potential solutions to overcome the limitations of traditional liquid phase storage. In lyophilization, EVs are stored in a dry and solid state that maintains their structural integrity and biological functionality even at room temperature. Similarly, hydrogels provide an encapsulation environment that captures and stabilizes EVs. This approach not only can enhance EV stability but also facilitate controlled release in therapeutic applications. Several studies investigated the potential of lyophilization techniques and biomaterials like hydrogels for preserving EV stability during the storage period (Fig. 4). Lyophilization was found to maintain EV size, morphology, protein content, and bioactivity when appropriate cryoprotectants were simultaneously used [3, 9, 22, 30, 32, 55, 62]. According to several reports trehalose and sucrose with polysorbate 80 provided effective lyophilization and prevented aggregation [9, 32, 52, 62]. In one study, authors reported well-preserved, spherical vesicles after lyophilization with trehalose or sucrose and polysorbate 80 [62]. Lyophilized EVs showed similar cellular uptake and functional effects related to freshly isolated EVs [9, 62].

Fig. 4
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Storage methods and additives for EV preservation. The figure was created with BioRender.com

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A study demonstrated that RT storage of lyophilized EVs with trehalose did not affect their protein, RNA contents, or functional properties [32]. Similar results were obtained without using cryoprotectant [30]. Hydrogels have also shown promising platforms for stabilizing and storing EVs. The encapsulation of EVs in hydrogel microneedles composed of hyaluronic acid preserved EV integrity [3]. This approach protected the EVs even after 6 months of storage, maintaining cargo integrity and biological activity. Overall, lyophilization and incorporation into stabilizing hydrogel matrices emerge as an effective approach for long-term EV preservation. Nevertheless, a study reported that lyophilization had limited ability to mitigate the effects of storage on samples and both lyophilization with and without cryoprotectant failed to outperform the effectiveness of storing samples at -80 °C [29]. Therefore, additional studies are urgently needed to address the critical issues related to the application of hydrogel for EV protection and storage.

Storage tube material effects on EVs

The adsorption of EVs onto storage tube walls during refrigeration can result in significant vesicle loss [28]. It is reported that about a 32% reduction occurred for total EV count after 48 h of storage at 4 °C. This effect would be related to EVs binding on ordinary polypropylene tubes. However, the use of specialized low protein-binding tubes prevented over 50% of these adsorption-mediated losses. These features indicate that the tube material significantly impacts the recovery of EVs after low-temperature storage [28]. Of note, use of glass tubes resulted in lower particle recovery versus plastic tubes after 1 week at 4 °C (Fig. 4) [39].

Discrepancies

There are several controversies regarding the effects of -20 °C versus 4 °C storage on the physicochemical properties of EVs. In one study, it was suggested that the preservation capacity of -20 °C was better in terms of EV size distribution, concentration, and RNA content compared to 4 °C [34], while another reported reduced EV concentration at -20 °C, but not at 4 °C [42]. Also as mentioned before, in a study abnormal and shrink Exos were observed after 1 month storage at − 20 °C [44]. Similarly, there were various results for the impacts of short storage at RT. One study observed no significant differences in EV number after 14 days at RT [45], while several other studies noted decreased EV concentrations at RT starting from 48 h [13, 30, 36, 37, 51]. Another study concluded that RT is suitable for short-term storage (less than a month), however, longer storing durations at RT will affect EVs protein and RNA contents [65]. These inconsistencies in the tolerance of EVs to RT might be due to different isolation and assay methods.

Discussion

In this systematic review, 50 original studies were included. These studies examined various storage conditions and how they can affect the physicochemical features of EVs, such as their initial number, size distribution, shape, content, and functional activity. EVs have a nano-scale physical structure, which makes them very sensitive to surrounding microenvironmental conditions. Optimized protocols are required to maintain EV structural, molecular, and functional integrity during handling, freezing, long-term storage, and thawing.

EVs help cells communicate with each other by exchanging important substances such as nucleic acids, proteins, lipids, and metabolites [9, 16, 25]. The potential clinical application of EVs can be directly affected by alterations in their cargo content resulting from inappropriate storage durations, temperatures, and freeze-thaw cycles. Multiple studies in the current systematic review mentioned that storing EVs resulted in a loss of their ability to affect recipient cells irrespective of storage temperature. All biological properties like cell uptake, migration, and cytokine secretion stimulation were affected by different degrees [30, 32, 33, 39]. Since EVs are gaining continuous attraction as drug delivery platforms, cell-free therapeutics, and clinical biomarkers, maintaining their functional properties during processing and storage using methods is vital. However, the wide range of methods of isolation, optimal temperature, freeze-thaw limits, effective stabilizing substances, various sources of EVs, and functional assessments make it extremely complex to establish consistent and broadly applicable procedures.

Despite the existence of conflicting data and lack of complete convergence, some consistent findings became apparent. In general, storage at -80 °C outperformed higher temperatures for preserving EV quantity, cargo, and morphology, especially for longer than 1 week (Fig. 3). Rapid freezing helped better maintain EV parameters, emphasizing the importance of quickly transitioning samples to ultra-low temperatures to face minimum freezing damage [11, 16]. Subjecting EVs to repeated freeze-thaw cycles caused consistent damage which emphasizes the need for protocols with the least need for or without thawing and refreezing [13, 29, 30, 34, 35, 54, 57, 58]. Storing EVs in their natural biofluids, rather than resuspending them in standard buffers, makes them more stable which indicates the benefit of keeping their physiological conditions in the storage process [11, 16, 19,20,21, 29, 44, 57].

In addition to conventional methods, emerging storage techniques such as Lyophilization and biomaterial encapsulation helped to reduce the detrimental effects of storage on EVs. Lyophilization, when coupled with cryoprotectants, has been shown to preserve EV morphology and cargo content over long periods at room temperature. Likewise, hydrogels provide a protective matrix for EVs which enhance stability and functionality while offering the potential for controlled therapeutic release. These methods prevent vesicle clumping and leakage, addressing challenges in preserving EVs in liquid form. Optimizing lyophilization conditions can maintain EV structure and function, even at RT. However, scalability for clinical-grade EVs needs more research. Using hydrogel matrices to stabilize EVs is another option, extending their lifespan and enabling controlled drug release. Exploring the synergy between EV encapsulation and their release could uncover ideal materials and methods. Developing integrated processes for EV isolation, lyophilization, and encapsulation to meet GMP standards will advance clinical use (Fig. 4). The development of standardized protocols for the use of these innovative approaches will be key to utilizing their maximum potential in EV-based therapies. This technique offers advantages for ease of storage and transportation, particularly for clinical use where refrigeration might not always be feasible.

Storage tubes could also alter EV parameters [28, 39]. Standard tubes can cause EVs to adhere to the wall, leading to inaccurate measurements and potential research and therapy problems [28, 39]. Using anti-adhesion coated tubes can be a simple solution to improve the reliability of EV studies and production. Exploring alternative approaches like lyophilization and bio scaffolds can eliminate plastic tube-related issues (Fig. 3).

This review revealed several key controversies on the best storage temperatures and durations to preserve EV integrity across different studies. These discrepancies likely come from differences in EV isolation method, storage, and analyze, emphasizing once again, the importance of standardized protocols. Additionally, since various isolation methods can unintentionally contain contaminants that affect EV stability, further research using reliable techniques is needed to establish the ideal isolation methods, temperature ranges, and appropriate buffers.

Another issue is that many studies only assessed EV stability for short durations, usually just days to weeks. Long-term monitoring over months or even years could provide a more accurate understanding of their stability. Based on the increasing interest in EV-based therapies, especially in clinical settings, it is crucial to develop practical and reliable protocols that can maintain EV function for long periods. Multiple studies suggest that storage at very low temperatures, such as -80 °C, can effectively preserve EV integrity over months, but further research is needed to determine whether these conditions are sufficient for long-term storage over one or two years. In addition, innovative preservation techniques such as lyophilization and encapsulation in hydrogels may demonstrate a remarkable potential for long-term EV stability, however, these approaches also require further validation for periods longer than one year. Only a few studies explored how storage affects surface charge (zeta potential) and aggregation mechanisms, which should be a focus of future research. Probably storage conditions like freezing, thawing, and long-term storage can alter zeta potential, leading to aggregation [29, 48]. A more comprehensive investigation of EV physical properties is necessary to better grasp the factors causing their instability. Several inconsistencies were observed among studies regarding the effects of storage temperature and the use of cryoprotectants on EV integrity. This might be due to variations in experimental conditions, including differences in EV isolation methods, the source of EVs (e.g., biofluids vs. tissue extracts), and the specific experimental design used in the studies. For example, different results under similar storage conditions could be explained that studies using centrifuge-based separation techniques may yield EVs that are more prone to aggregation due to the applied high gravity force compared to those obtained through size-exclusion chromatography. Additionally, variations in EV cargo (e.g., RNA, protein) may also contribute to inconsistent findings, since EVs from different sources may represent different cargo profiles that may cause different outcomes from the same storage protocols in various studies. Future studies should focus on standardizing isolation and experimental conditions to reduce variability and improve reproducibility in EV storage research.

Conclusions

This systematic review highlighted the significant impact of storage conditions and stabilizing strategies on key EV parameters such as concentration, size, structure, cargo content, and functionality. The findings emphasized the importance of maintaining physiological conditions, implementing rapid or immediate freezing, minimizing freeze-thaw cycles, and using stabilizing additives to preserve EV quality. These insights are crucial for developing standardized procedures to ensure consistent EV stability for both research and clinical applications. However, further high-quality studies are needed to optimize storage conditions, freeze-thaw limits, and cryoprotectants for different types of EVs. In addition, lyophilization and biomaterial-based strategies offer promising alternatives for preserving EVs, enabling solid-phase storage and sustained release. Developing specific lyophilization and encapsulation protocols for EVs can address key challenges in storage and delivery, making EVs more accessible for therapeutic applications. Finally, the review highlighted the importance of storage tube surface properties, emphasizing the need for optimizing tube coatings, understanding EV-surface interactions, and exploring tube-free storage methods like lyophilization to enhance EV storage consistency and analysis.

Data availability

All data generated or analyzed during this study are included in this published article.

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Acknowledgements

The authors would like to thank the Faculty of Advanced Medical Sciences for their support and help. Also, we thank Bernard Roelen, associate professor at Utrecht University, for his valuable comments and suggestions during the preparation of this manuscript that greatly contributed to improving the quality of this work. Figures 1, 3, and 4 were created with BioRender.com.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    Shahin Ahmadian, Negin Jafari, Alireza Ghaffarzadeh, Reza Rahbarghazi & Mahdi Mahdipour

  2. Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

    Shahin Ahmadian, Reza Rahbarghazi & Mahdi Mahdipour

  3. Department of Research and Development, PerciaVista R&D Co, Shiraz, Iran

    Amin Tamadon

  4. Department of Natural Sciences, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan

    Amin Tamadon

  5. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

    Mahdi Mahdipour

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Contributions

S.A., N.J., and A.G. collected data, performed a literature review, and wrote the initial draft of the manuscript. A.T. and R.R. reviewed and revised the initial draft of the manuscript. M. M. designed and conceptualized.

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Correspondence to Mahdi Mahdipour.

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This systematic review was published as a partial fulfillment of the PhD student project “Studying the regenerative effects of engineered exosomes loaded with Nrf2 activator in the rat model of azoospermia,” which was reviewed and approved on February 13, 2023, by the Research Ethics Committees of Laboratory Animals - Tabriz University of Medical Sciences (ethical code: IR.TBZMED.AEC.1401.084).

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Amin Tamadon was employed by PerciaVista R&D Co. The other authors declare that their research was conducted without any commercial or financial relationships that could be perceived as potential conflicts of interest.

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Ahmadian, S., Jafari, N., Tamadon, A. et al. Different storage and freezing protocols for extracellular vesicles: a systematic review. Stem Cell Res Ther 15, 453 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04005-7

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Stem Cell Research & Therapy

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