Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles

Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles Ryosuke Inayama, Saki Horie, Risako Okada, Naoomi Tominaga This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7586495/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Extracellular vesicles (EVs) are crucial mediators of intercellular communication and have significant diagnostic and therapeutic potential. However, their preservation poses considerable challenges. This study examined the stability of EVs derived from PC3 cells when stored in PBS, HBSS, or HBSS with 5% glucose under long-term storage conditions (4 °C, -30 °C, and -80 °C) and subjected to up to six freeze-thaw cycles. The physicochemical properties of EVs, including their concentration, size, morphology, and protein content, were assessed over 90-day period. The findings indicate that storage at -30 °C significantly compromised EV integrity across all buffers, resulting in particle loss and morphological degradation. Although storage at -80 °C better preserved particle concentrations, morphological instability persisted at this temperature. In contrast, storage at 4 °C in PBS was most effective for maintaining morphology, albeit with a reduction in protein content over time. Repeated freeze-thaw cycles, particularly at -30 °C, caused substantial damage. These results show that storage temperature, duration, and suspension buffer affect EV integrity, providing useful information for developing improved preservation methods. Biological sciences/Biological techniques Biological sciences/Biophysics Biological sciences/Biotechnology Physical sciences/Materials science Extracellular vesicles Preservation Cryopreservation Freeze-Thaw Cycles Storage Temperature Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Extracellular vesicles (EVs) are nanometer-sized structures encased in lipid bilayers, released from cells, and play a vital role in intercellular communication through their encapsulated proteins, nucleic acids, and lipids 1–3 . EVs have been extensively researched for their potential as diagnostic biomarkers for various diseases, including cancer, neurodegenerative disorders, and cardiovascular conditions, as well as for their application as drug delivery systems (DDS) to transport encapsulated substances to target cells 4–6 . Notably, their biocompatibility, low immunogenicity, and ability to cross biological barriers have been recognized as advantages over conventional nanocarriers, and their potential as therapeutic agents has been explored . For the application of EVs in diagnosis and therapy, mass production, purification, and stable storage are essential. Particularly, when considering the use of EVs as pharmaceutical products, it is crucial to establish a technology that enables the long-term preservation of EVs while maintaining their functions and characteristics from manufacturing to administration to patients. However, several technical challenges exist in preserving EVs. Currently, refrigerated (4℃) storage, cryopreservation, and lyophilization are the primary methods considered 7,8 . These methods may affect the function and therapeutic efficacy of EVs, making them unsuitable for long-term storage. Conversely, cryopreservation is widely employed as a method suitable for long-term storage, but there is a risk that the membrane structure of EVs may be compromised or aggregation may occur due to ice crystal formation or osmotic pressure changes during the freezing and thawing processes 9,10 . In particular, the freezing speed and the type and concentration of cryoprotectant significantly affect the condition of EVs, necessitating the study of optimal conditions. Furthermore, it has been suggested that multiple freeze-thaw cycles of cryopreserved EVs significantly reduce their physical properties and biological activity 11 . This may lead to changes in the particle size distribution of EVs, fluctuations in the expression of surface markers, and even reduced efficiency of cellular uptake 12 , 13 . Thus, the stability of EVs varies greatly depending on the method of storage, and it is essential to evaluate in detail the effects of these storage conditions on the condition of EVs to ensure long-term storage, and particularly their quality as pharmaceutical products. To commercialize EVs as safe and effective pharmaceutical products, it is essential to establish the safety and efficacy of the manufactured EVs, which must be scientifically proven to maintain their physicochemical properties and biological activity for extended periods under appropriate storage conditions. The study investigates the difficulties of preserving EVs over extended durations at temperatures of 4℃, -30℃, and -80℃ in different solvents, as well as the impact of cryopreservation and repeated freeze-thaw cycles on their integrity. We present the findings of our research, which have contributed to the development of stable storage techniques for the future utilization of EVs, providing crucial insights for pharmaceutical applications. Results Initial characterization of PC3 cell-derived EVs reveals suspension buffer affects particle concentration without significantly altering particle size or zeta potential. To characterize the isolated EVs, dynamic light scattering (DLS), electrophoretic light scattering, Western blotting, and transmission electron microscopy (TEM) observations were performed. Isolated EVs were suspended in different suspension buffers (PBS, HBSS, and HBSS with 5% glucose) and stored under three different temperature conditions (4℃, -30℃, and-80℃) for up to 90 days. In addition, EVs suspended in each buffer (PBS, HBSS, and HBSS with 5% glucose) were also freeze-thawed 1, 2, 3, and 6 times after storage at -30℃ and -80℃. During storage, samples were periodically taken to measure the particle concentration, particle size, polydispersity index (PDI), zeta potential, electrical conductivity, and scattered light intensity using ELSZneo, and to observe morphology using electron microscopy (Fig.1A). Physicochemical properties of EVs were evaluated immediately after isolation (Day 1). The particle concentration of EVs in various suspension buffers was measured using ELSZneo (Fig. 1B). The results showed that the particle concentrations in PBS, HBSS, and HBSS with 5% glucose were 2.53 × 10¹⁰ particles/mL, 2.16 × 10¹⁰ particles/mL, and 3.26 × 10¹⁰ particles/mL, respectively. Particle size and zeta potential of EVs immediately after isolation (Day 1) were also measured using ELSZneo (Fig. 1B), noting that HBSS and HBSS with 5% glucose are more viscous than PBS, which may affect particle count measurements. Particle size was approximately 170 nm for all buffers, with no significant differences (PBS: 171.6 nm, HBSS: 171.9 nm, HBSS with 5% glucose: 170.7 nm). Zeta potentials were negative in all buffers, approximately -12.55 mV (PBS), -12.97 mV (HBSS), and -12.22 mV (HBSS w/ 5% glucose). Subsequently, the presence of EV marker proteins was assessed by western blotting. The expression of CD63, a marker protein specific to isolated EVs, was confirmed by western blotting, although it exhibited low intensity in the cell lysate (Fig. 1C). Additionally, Cytochrome-C was not detected in EVs compared to that in the cell lysate. Morphological analysis of the isolated EVs was conducted using TEM (Fig. 1D). The analysis revealed that EVs in PBS displayed typical cup-shaped or circular membrane structures. However, EVs in HBSS and HBSS with 5% glucose demonstrated particle degradation, aggregation, and emergence of spike-like structures from Day 1. -30℃ Storage Significantly Impairs EV Physicochemical Stability To investigate the effects of long-term storage at -30°C, we conducted further experiments. The particle counts of EVs varied under the tested conditions. In PBS suspension, the particle count decreased to 2.0 × 10 10 particles/mL by day 45 when stored at 4℃ and -80℃, followed by an increase by day 90 (Fig. 2A). Notably, storage at -30℃ resulted in a reduction of particle count to half by day 15, with levels remaining low thereafter, suggesting potential aggregation or destruction of EVs during a single freeze-thaw cycle at this temperature. Conversely, storage in HBSS suspension at -80℃ resulted in relatively stable particle concentrations, closely approximating the initial values, whereas particle concentrations gradually increased over time when stored at 4℃. In contrast, the particle concentrations remained relatively stable at low temperatures of -30℃ and -80℃. Additionally, an increase in the particle count was observed in HBSS (5% glucose) at 4℃. At -30℃ and -80℃, the particle concentrations were comparably stable. Subsequently, the particle sizes of EVs were analyzed (Fig. 2B). In the PBS suspension, the particle size remained relatively stable within the range of approximately 150-170 nm at 4℃ and -80℃, whereas at -30℃, the particle size increased from day 30 to day 60, followed by instability. In the HBSS or HBSS (5% glucose) suspension exhibited remarkable stability in the size range of approximately 160-180 nm across all temperature conditions. Regarding the PDI, which indicates particle size uniformity, a value of 0.1 or less was considered to be near monodisperse. Storage at 4℃ in PBS suspension increased PDI up to day 45, followed by a decrease after day 60 (Fig. 2C). At -30℃ and -80℃, the PDI exceeded 0.1 after day 15, with a marked increase in particle size heterogeneity, particularly at -30℃. In the HBSS suspension, the PDI remained relatively low during storage at 4℃. At -30℃ and -80℃, the PDI exceeded 0.1, indicating a slight increase in heterogeneity. In HBSS with 5% glucose, the PDI remained low (below 0.1) at 4℃. However, at -30℃ and -80℃, the PDI generally remained above 0.1. The zeta potential, an indicator of particle dispersion stability, remained relatively stable in PBS and HBSS (5% glucose) suspensions, ranging from -10 to -15 mV under all temperature conditions (Fig. 2D). However, when the HBSS suspension was stored at -30℃, the absolute value of zeta potential significantly decreased on day 75, approaching -20 mV, suggesting alterations in the particle surface state and potential aggregation. Among the electrical properties shown in Supplementary Figure 1, electrophoretic mobility is linked to the zeta potential. In PBS and HBSS (5% glucose) suspensions, electrophoretic mobility remained relatively stable across all temperature conditions (Supplementary Fig. 1A). In contrast, conductivity, which reflects the ion concentration in the suspension, remained stable at approximately 14-15 mS/cm in both PBS and HBSS suspensions throughout the 90-day period at all temperatures (Supplementary Fig. 1B). Collectively, these findings suggest that the polydisperse index tends to vary with storage duration, favoring storage at 4℃ or -80℃. In contrast, the physicochemical indices of EVs were significantly affected by storage at -30℃. PBS More Effectively Preserves EV Morphology at 4℃ The physical properties of EVs were evaluated by particle count and size measurements using ELSZneo, as shown in Figure 2. However, to thoroughly assess EV quality, it is essential to directly observe the morphological characteristics of individual vesicles, including membrane integrity and aggregation levels. To evaluate morphological stability over time, TEM observations were conducted at 15-day intervals from Day 1 to Day 90 (Fig.3). On Day 1, the EVs stored in PBS displayed a typical cup or spherical shape with diameters ranging from approximately 50 to 200 nm and a well-defined lipid bilayer structure (Fig. 3A). However, as the storage duration increased, morphological changes progressed, and by Day 30, some EVs began to show obscured membrane structures and partial vesicle collapse. This morphological deterioration became more pronounced after Day 45, and by Days 60 and 75, the number of EVs retaining their original shape was significantly reduced, with many forming aggregates or becoming fragmented. By Day 90, few EVs with clear vesicular structures were observed. In contrast, EVs preserved in HBSS exhibited morphology on Days 1 and 15 similar to those preserved in PBS (Fig. 3B). However, the initial morphology of EVs tended to be somewhat unstable compared to those preserved in PBS, with aggregation and morphological heterogeneity becoming apparent at approximately Day 30. By Day 90, only a few EVs maintained a stable morphology, similar to that in the PBS-preserved condition. Conversely, EVs stored in HBSS containing 5% glucose showed morphological instability as early as Day 1, with collapsed vesicle structures observed in some instances (Fig. 3C). The instability of the morphology increased with storage time, and in addition to the formation of string-like protrusions, further collapse and aggregation of the vesicles were observed. Based on the TEM observations, PBS alone is more effective than HBSS alone or HBSS with 5% glucose in maintaining the highest morphological stability of vesicles during long-term storage of EVs at 4℃. -30℃ Storage Results in Significant EV Morphological Degradation The morphology of extracellular vesicles preserved at -30°C was subsequently analyzed using TEM. EVs stored in PBS at -30℃ began to show compromised membrane integrity on the Day 15 of freezing (Fig. 4A). By the Day 30, the vesicular structure had deteriorated, leading to the formation of substantial aggregates. By the Day 45, morphological anomalies such as protruding structures and vesicle collapse were evident. On Day 60, vesicle crushing, persistent protrusions, and extensive aggregation were observed. EVs stored in HBSS at -30℃ exhibited scattered structural instability from an early stage (Fig. 4B). By the 15th day, some aggregation and minor membrane damage were apparent, although the morphology was somewhat better than that observed with PBS storage. By the 30th day, aggregation and morphological diversity became pronounced, and by the 45th day, there was an increase in aggregates and collapsed vesicles. By the Day 90, most particles showed structural instability, including protruding structures and vesicle collapse. In contrast, EVs in HBSS with 5% glucose stored at -30℃ consistently displayed morphological deterioration, such as protruding structures, vesicle collapse, and aggregation, throughout the observation period from the Day 15 to the Day 90 (Fig. 4C). TEM revealed that long-term cryopreservation of EVs at -30℃ resulted in significant morphological degradation over time across all three tested conditions. Although the initial morphology during PBS storage was satisfactory, the effects of one-time freeze-thaw cycles and prolonged storage were unavoidable. This suggests that storage at -30℃ may not be effective in maintaining morphological stability. Significant EV Morphological Degradation Despite -80 ℃ Cryopreservation The morphology of extracellular vesicles preserved at -80°C was subsequently analyzed using TEM. EVs preserved in PBS at -80℃ retained a relatively stable particle structure for up to 15 days after freezing (Fig. 5A). However, by Day 30, the vesicle structure began to show signs of collapse, with protruding formations and notable aggregation becoming apparent. This morphological degradation continued over time, and by Day 60, only a few clear and intact particle structures remained visible. EVs stored in HBSS at -80℃ exhibited significant morphological degradation, including protruding formations, vesicle collapse, and aggregation, starting from Day 15 post-freezing, with structural instability persisting throughout the storage period (Fig. 5B). Similarly, EVs in HBSS with 5% glucose, also stored at -80℃, showed comparable morphological degradations from Day 15 post-freezing, with the particle structure remaining unstable throughout the observation period (Fig. 5C). TEM revealed that, despite long-term cryopreservation at -80℃, all three solution conditions tested resulted in significant morphological degradation over time. Morphological instability was observed earlier in HBSS alone and in HBSS with 5% glucose, particularly in the latter, which demonstrated no morpho‐protective effect under -80℃ storage conditions. Solution and Temperature Determine EV Response to Freeze-Thaw Stress Freezing and thawing can negatively impact the membrane structure of EVs due to ice crystal formation and osmotic pressure changes, potentially leading to aggregation and collapse, which may subsequently affect particle size, particle count, and the quality of their contents. To evaluate the changes in particle size and particle count of EVs during multiple freeze-thaw cycles, we tested varying freezing temperatures (-30℃ and -80℃) and three distinct storage solutions: PBS, HBSS, and HBSS with 5% glucose. The experimental procedure involved suspending the isolated EVs in their respective solutions, freezing them at either -30℃ or -80℃, and thawing them at room temperature, repeated 1, 2, 3 times, and 6 times. Notably, after six cycles, there was a significant increase in the particle size compared to the pre-freeze state (Fig.6A). When EVs suspended in HBSS were stored at -30℃, the particle size remained consistent, regardless of the number of cycles (Fig.6B). EVs suspended in HBSS with 5% glucose exhibited a notable increase in particle size after 3 and 6 freeze-thaw cycles at -80℃ storage conditions compared to storage at -30℃ (Fig.6C). For EVs suspended in PBS, a single freeze-thaw cycle led to a significant reduction in particle numbers, dropping to approximately 50% of the initial count at both -30℃ and -80℃ storage temperatures (Fig.6D). Regardless of the temperature, the particle numbers continued to decline with additional freeze-thaw cycles, although they remained significantly higher at -80℃ than at -30℃. When EVs were stored in HBSS with 5% glucose at -30℃, the particle count remained relatively high at approximately 85% of the initial number after one cycle and decreased to approximately 40% after six cycles (Fig.6E). This trend of maintaining a relatively high level persisted through two and three cycles (Fig.6E). Regardless of the temperature, the particle numbers continued to decline with additional freeze-thaw cycles, although they remained significantly higher at -80℃ than at -30℃ (Fig.6D). When EVs were stored in HBSS at -30℃ or -80℃, the particle count remained relatively high at approximately 50% of the initial number after one cycle and decreased to approximately 40% after six cycles (Fig.6E). Conversely, when EVs were stored in HBSS with 5% glucose at -30℃ or -80℃, the particle count remained relatively high, approximately 70% to 80% of the initial count after one cycle. However, after six cycles, the particle count decreased to approximately 120% when preserved at -30℃ and 80% when preserved at -80℃ (Fig. 6F). These findings suggest that freeze-thaw cycles affect both the size and number of EV particles, with the extent of the effect influenced by the freezing temperature and preservation solution used. Specifically, storage at -80℃ in PBS was more effective in maintaining particle numbers, although the particle size increased after six cycles at -30℃. These insights could aid in developmentping of optimal cryopreservation protocols to preserve EV quality. Freeze-Thaw Cycles Alter EV Polydispersity and Zeta Potential Depending on Solution and Temperature EVs underwent several freeze-thaw cycles at -30℃ and -80℃, with their Polydispersity Index (PDI) and zeta potential assessed in three different storage solutions: PBS, HBSS, and HBSS with 5% glucose. (Electrophoretic mobility and Electrical conductivity are shown in Supplementary Figures 2A-2F). For EVs in PBS, the initial PDI was approximately 0.08 (Fig. 6G). A single freeze-thaw cycle at both -30℃ and -80℃ significantly increased the PDI to ~0.18-0.20, indicating a loss of particle size homogeneity. In subsequent cycles, PDI values remained stable between 0.20 and 0.24. For EVs in HBSS, the pre-freezing PDI was also ~0.08 (Fig. 6H). Following one cycle, the PDI similarly increased to ~0.18-0.20 at both temperatures. At -30℃, a slight temporary decrease to ~0.16 occurred in the third cycle, but it returned to ~0.18 after six cycles. At -80℃, the PDI remained stable at ~0.20 throughout all cycles. For EVs in HBSS with 5% glucose, the pre-freezing PDI was ~0.09 (Fig. 6I). At -30℃, the PDI increased to ~0.16 after the first cycle, fluctuated, and settled at ~0.15 after six cycles. Conversely, at -80℃, the PDI surged to ~0.21 after one cycle and remained elevated. Notably, after 1, 3, and 6 cycles, the PDI values at -80℃ were significantly higher than those at -30℃. Regarding surface charge, EVs in PBS initially had a zeta potential of approximately -12 mV (Fig. 6J). At -30℃, the absolute zeta potential increased to ~ -15 mV after one cycle before decreasing to ~ -9 mV after two cycles. At -80℃, it fluctuated more, reaching ~ -16 mV after two cycles but ending at ~ -13 mV. After six cycles, the absolute zeta potential at -80℃ was significantly greater than at -30℃. The EVs in HBSS initially had a zeta potential of ~ -13 mV (Fig. 6K). At -30℃, the absolute value increased to ~ -15 mV after one cycle and rose further to ~ -20 mV after six cycles. At -80℃, the zeta potential remained relatively stable between -12 and -15 mV. Before freezing, EVs in HBSS with 5% glucose exhibited a zeta potential of ~ -12 mV (Fig. 6L). At -30℃, the absolute zeta potential significantly increased to ~ -18 mV after one cycle and remained high. At -80℃, the zeta potential varied between -13 and -16 mV, with absolute values generally lower than those at -30℃. These findings suggest freeze-thaw cycling affects EV PDI and zeta potential, reducing particle size uniformity and altering surface charge. Zeta potential variations were influenced by both the storage solution and freezing temperature, indicating a complex impact on the EV surface. The significant PDI increase for HBSS with 5% glucose at -80℃, and the rise in absolute zeta potential at -30℃, represent substantial changes. These physicochemical alterations influence EV aggregation and dispersion, providing valuable insights for optimizing storage conditions. Freeze-Thaw Cycling Inflicts Progressive Morphological Damage on EVs TEM was employed to examine the morphological changes in EVs subjected to multiple freeze-thaw cycles (1, 2, 3, and 6 cycles) at varying freezing temperatures (-30℃ and -80℃) and in three different storage solutions: PBS, HBSS, and HBSS with 5% glucose. For EVs in HBSS treated at -30℃, vesicle collapse and aggregation were noticeable after 1, 2, 3, and 6 freeze-thaw cycles (Fig. 7B). Similarly, in EVs suspended in HBSS with 5% glucose and treated at -30℃, morphological degradation, such as vesicle collapse, aggregation, and the emergence of protruding structures, was observed (Fig. 7C). In EVs suspended in PBS and subjected to freeze-thawing at -80℃, many vesicles maintained a relatively intact structure after one or two freeze-thaw cycles (Fig. 8A). However, as the number of cycles increased, morphological degradation progressed, with some vesicles collapsing after three cycles of expansion and contraction. After six cycles, the number of vesicles retaining their original shape was significantly reduced, as vesicle collapse, membrane fragmentation, and substantial aggregate formation were observed. For EVs in HBSS treated at -80℃, vesicle collapse was evident after one freeze-thaw cycle (Fig. 8B). With more cycles, vesicle disintegration, protruding structures, and aggregation intensified, and after 3 and 6 cycles, many vesicles lost their structure, becoming primarily fragmented membrane components and aggregates. In EVs in HBSS with 5% glucose treated at -80℃, vesicle disintegration, membrane heterogeneity, and aggregation were clearly visible after one freeze-thaw cycle, with these morphological degradations becoming more pronounced as the number of cycles increased (Fig. 8C). After three and six cycles, few vesicles remained in their original form, and extensive aggregation and disintegration of membrane structures were observed. These TEM observations visually confirmed the severe damage inflicted on EV morphology by the freeze-thaw cycles. At both -30℃ and -80℃, vesicle disintegration, aggregation, and fragmentation advanced with more cycles. The type of storage solution also played a role, suggesting that PBS frozen at -80℃ might preserve some morphology, although instability from the initial state was noted at -30℃. These morphological evaluations should be considered alongside particle size and number, PDI, and zeta potential measurements by ELSZneo, highlighting the importance of TEM observations in assessing EV quality. Storage and Freeze-Thaw in PBS Reduce EV Protein Content The protein content of EVs is a crucial marker for evaluating their functionality and quality. Protein levels were measured using the microBCA method, with changes in protein concentration compared to baseline values (Day 1 or pre-freeze samples), set as a relative value of 1.0. For EVs stored in PBS at 4℃, the protein concentration dropped to 25% of the Day 1 level by Day 15 (Supplementary Fig. 3A). Although fluctuations occurred during the storage period, the concentration remained lower than the initial level. When EVs were stored in PBS at -30℃, the protein concentration fell to approximately 40% of the initial value by Day 15 and further decreased to approximately 15% by Day 45. Despite variations throughout storage, the concentration consistently remained lower than the initial level. For EVs stored in PBS at -80℃, the protein concentration was reduced to approximately 30% of the initial value by Day 15. Although it varied during storage, it remained lower than the initial level. For EVs suspended in PBS and subjected to freeze-thaw cycles at -30℃, the protein concentration decreased to approximately 50% of the initial value after one cycle (Supplementary Fig. 3B). In EVs at -80℃, the concentration decreased to approximately 60% after one cycle and then stabilized at around 50% after six cycles. HBSS and HBSS with 5% glucose could not be measured using microBCA due to solvent inhibitors. These findings suggest that during prolonged storage of EVs in PBS, the protein concentration significantly decreased from the initial value under all temperature conditions. In the freeze-thaw cycle treatment, the concentration was reduced by approximately half in the first cycle, with no significant further decrease as cycles increased. These fluctuations may result from factors such as EV aggregation, disintegration, or effects on the measurement system, and should be interpreted in conjunction with other physicochemical and morphological observations. Discussion The study reveals that storing extracellular vesicles (EVs) in phosphate-buffered saline (PBS) at 4°C is optimal for maintaining their morphology for up to 15 days. During this period, vesicles remain intact with minimal aggregation. However, by day 45, a mix of intact and disintegrated particles is observed, with EV aggregation becoming more pronounced over 75 days post-purification. This finding highlights a balance between structural preservation and potential biochemical alterations that may occur during storage at 4°C in PBS 14,15 16 17 . Compared to storage at -30°C and -80°C, the 4°C PBS condition proves superior. At -30°C, significant deterioration is observed across all buffers, including reduced particle concentration, increased polydispersity index (PDI), and severe morphological damage. While storage at -80°C better preserves particle numbers, it fails to prevent structural degradation over time or during freeze-thaw cycles 18 19 , 20 . Despite the 4°C PBS condition being the most effective in preserving vesicle morphology, changes in particle count and protein content indicate ongoing degradation processes. This suggests that even under optimal storage conditions, EVs undergo gradual deterioration. These findings have important implications for EV research and potential therapeutic applications. The choice of storage conditions can significantly impact the integrity and functionality of EVs, potentially affecting experimental outcomes or therapeutic efficacy. Researchers and clinicians working with EVs should carefully consider these storage effects when designing experiments or developing EV-based therapies. Further research is needed to fully understand the biochemical changes occurring during storage and to develop improved preservation methods. This may include investigating alternative storage buffers, exploring the use of cryoprotectants, or developing novel preservation techniques to better maintain EV integrity over extended periods. Dynamic light scattering (DLS) was used to characterize extracellular vesicle (EV) samples in accordance with the MISEV2023 guidelines 21 . DLS is a high-throughput technique that is effective for the qualitative identification of submicron particles and aggregates within EV samples. The data obtained from DLS underscore its utility in quality control, specifically in confirming the presence or absence of aggregation during EV preparation. However, as noted by MISEV2023, DLS has limitations in quantitatively determining characteristics such as the mean hydrodynamic diameter of polydisperse EV samples. This limitation arises because the DLS analysis algorithm assumes monodispersity, which can lead to size overestimation in polydisperse EV. Compared to nanoparticle tracking analysis (NTA) and imaging methods, the establishment of the limit of detection (LOD) for particle detection in DLS remains underdeveloped and is acknowledged as a future challenge. Therefore, DLS data should be regarded as a guiding tool, illuminating general trends in size distribution rather than providing precise measurements of individual EV dimensions. To effectively utilize EVs in medical applications, they must be stored properly to preserve their morphology, quantity, and functionality. Our study shows that no single optimal storage method exists; various factors must be balanced based on specific needs. Short-term storage at 4℃ in PBS preserves morphology but requires protein levels monitoring. Because freezing and thawing affect EVs, single-use portions are recommended. Future research should focus on developing methods to protect EVs during freezing or drying and assess their post-storage functionality. Standardized methods are crucial for ensuring EV safety in both research and medical applications. This study examined PC3-derived EVs under various storage conditions, showing that temperature, duration, buffer, and freezing affect EVs. Storage at 4℃ in PBS is optimal for morphology, while -80℃ in PBS better maintains particle numbers during freezing compared to -30℃, despite morphological and protein challenges. These findings contribute to the development of enhanced storage methods for medical applications. Material and Methods Cell culture The human prostate cancer cell line, PC-3 (RCB2145), was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan on 2022~2024. The cell lines authentication was performed by the RIKEN BRC. PC-3 was cultured in RPMI1640 medium (Nacalai Tesque, Japan, No.30264-85) supplemented with 10% heat-inactivated FBS (Corning, No.35-079-CV) and antibiotic–antimycotic agents (Nacalai Tesque, Japan, No.09366-44) at 37℃ in 5% CO2. EVs isolation Cells were seeded onto 15cm dishes at 70-80% confluency and cultured overnight in a 5% CO2 incubator. The medium was then replaced with Advanced RPMI-1640 medium (Gibco, Massachusetts, No.12633020) supplemented with 2 mM L-glutamine (Nacalai Tesque, Japan, No.16948-04) and 1% antibiotic-antimycotic solution (Nacalai Tesque, Japan, No. 09367-34) after twice washes with 10 mL Dulbecco's Phosphate Buffered Saline (PBS) (-) (Nacalai Tesque, Japan, No. 14249-24). Following a 48-hour incubation period, the culture supernatant was collected by centrifugation at 10,000 xg for 10 min at 4℃. To ensure the complete removal of cellular debris, the supernatant was subsequently filtered through a 0.22 μm PVDF filter (Millipore Merck, Massachusetts, No.17461-05). The resulting filtered supernatants were used for EV isolation. EVs were isolated by ultracentrifugation at 110,000 xg for 70 min at 4℃ using a SW41Ti swinging-bucket rotor (Beckman Coulter). The pellets were then washed with PBS (-), HBSS, or HBSS containing 5% glucose by repeat ultracentrifugation at 110,000 xg for 70 min at 4℃ and resuspended in PBS (-). The isolated EVs were stored at 4℃ until further experimentation. Regents and preservation, freeze-thaw processes PBS, Hank's Balanced Salt Solution (HBSS), and HBSS with 5% glucose were used for resuspension regent of EVs. HBSS with 5% glucose was prepared by dissolving glucose (Fujifilme-wako, no.049-31165) at a concentration of 5% in standard HBSS solution. In the experiments of “Long-term preservation”, EVs in each regent were measured Particle concentration, Particle size distribution, Scattered light intensity, Polydispersity Index (PDI), Zeta potential, Electrophoretic mobility, and Electrical conductivity using ELSZneo on Day 1, 15, 30, 45, 60, 75, and 90. In addition, EVs were observed morphology using transmission electron microscope on each day. Protein concentration was measured using microBCA Protein Assay Kit (Thermofisher Scientific, no. 23235). For the “Repeated freeze-thaw processes” experiments, the same examinations were conducted after 1, 2, 3, and 6 cycles. Dynamic Light Scattering (DLS) The concentrated EVs samples were measured using a dynamic light scattering (DLS) instrument (ELSZneo, Otsuka Electronics, Co., Ltd., Osaka, Japan) 22 . The instrument is equipped with a 40 mW and 638 nm Semiconductor laser diode. The laser is detected by the APD module. The scattering angle is 165◦, at a constant temperature of 25 ℃. The optimal light scattering intensity is between 84,000 cps and 114,000 cps (photon count rate per second). This system is equipped with an attenuation filter that automatically regulates the incident light if the scattering intensity is above the range. Each measurement consisted of 30 runs. The measurements were performed three times. Transmission electron microscopy (TEM) We followed the protocol established in our previous study 4 . EVs were confirmed morphologically using a transmission electron microscopy. Formvar/carbon-coated copper grid (Nisshin EM, Tokyo, Japan) was hydrophilized with JFC-1600 Auto Fine Coater (JEOL, Tokyo, Japan). 3µL of purified EVs in PBS were placed on the hydrophilized grid and absorbed for 3 min. We washed them using 500 µL double-distilled H2O successive four drops, then negatively stained using 30 µL of 2.0% uranyl acetate successive four drops. The grid was air-dried after absorbing 2.0% uranyl acetate on the grid with filter paper. We imaged the grid with Tecnai G2 Spirit BioTWIN electron microscopy (FEI, OR, USA) operating 120 kV equipped with a EMSIS Phurona CMOS Camera. After exporting the raw data images to TIFF format, we used Fiji (ImageJ 1.53t) for data analysis. Western blotting We followed the protocol established in our previous study 23 . Proteins were isolated from cells using M-PER (Thermo Scientific, MA, USA) separated in Mini-PROTEAN TGX Gel (4–12%, Bio-Rad) and electro transferred onto a PVDF membrane (Bio-Rad, No.1704274). After blocking in Block Ace (KAC, Japan, No.UKB80), the membranes were incubated for 1-hr at room temperature with primary antibodies, which included anti-CD63 (purified mouse anti-human CD63, H5C6, 1:200, BD), anti-Cytochrome C (Rabbit polyclonal, 4272S, 1;1000, CST). Secondary antibodies (HRP-linked anti-mouse IgG, NA931V or HRP-linked anti-rabbit IgG, NA934V, GE Healthcare) were used at a dilution of 1:10000. The membrane was then exposed to ImmunoStar LD (Fujifilm-Wako, Japan, No.292-69903). Quantification and statistical analysis We conducted statistical analyses and visualized the data utilizing R software (version 4.2.1). To compare differences between two groups, we employed the Student's t-test. Graphical representations display the mean ± standard deviation (S.D.). Declarations Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Acknowledgements We extend our sincere gratitude to Mr. Yuuta Miyagi, Mr. Tsubasa Yamanaka (an employee of Otsuka Electronics Co. Ltd.), and Mr. Taizo Hasegawa (an employee of Otsuka Electronics Co. Ltd.) for generously donating his time. We thank the Science Research Center of Yamaguchi University for Institute of Gene Research, Institute for Biomedical Research and Institute of Life Science and Medicine. This work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting construction of core facilities), Grant Number JPMXS0440400024. Fundings This work was supported by Otsuka Electronics Co. This work was supported in part by a Grant-in-Aid for Research Activity Start-up (No. 21K21219), Early-Career Scientists (No.22K17826, No. 25K21103) from Japan Society of the Promotion of Science (JSPS). This work was supported by Japan Science and Technology Agency (JST), ACT-X Grant Number JPMJAX222B, Japan. This work was supported in part by a Grant-in-Aid for Yamaguchi University Fund, Start-up Research Fund, New frontier project-2021, and FOCS 2023 (Yamaguchi University Original-University Fund). This work was supported in part by a Grant-in-Aid for the Ube City Next-Generation Researchers Project. The funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Author contribution R.I. and N.T. conceived and designed this study. R.I. performed the experiments using ELSZneo. S.H. and R.O. performed the experiments of the western blotting, transmission electron microscopy analysis. R.I. and N.T. performed data analysis and interpretation. All authors have reviewed and edited the manuscript. The manuscript was finalized by N.T. with the assistance of all authors. All authors have approved the final manuscript. Declaration of Interest The author N.T. is a founder and director of ADDVEMO Inc. The ADDVEMO Inc. was not involved in the study design, data collection, analysis, interpretation of data, the writing of the report, or the decision to submit the article for publication. Author N.T. receives research funding from Otsuka Electronics Co. for this study. Author R.I. is an employee of Otsuka Electronics Co. Ltd., and ELSZneo evaluated in this study was developed by Otsuka Electronics Co. Ltd. The authors S.H. and R.O. declare no competing interests. References Théry, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3 , Unit 3.22 (2006). Yáñez-Mó, M. et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4 , 27066 (2015). Tominaga, N. Anti-Cancer Role and Therapeutic Potential of Extracellular Vesicles. Cancers (Basel) 13 , 6303 (2021). Yamada, N. et al. Glycosylation changes of vWF in circulating extracellular vesicles to predict depression. Sci Rep 14 , 29066 (2024). Kalluri, R. The biology and function of exosomes in cancer. J Clin Invest 126 , 1208–1215 (2016). Luan, X. et al. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 38 , 754–763 (2017). Yuan, F., Li, Y.-M. & Wang, Z. Preserving extracellular vesicles for biomedical applications: consideration of storage stability before and after isolation. Drug Deliv 28 , 1501–1509 (2021). Jeyaram, A. & Jay, S. M. Preservation and Storage Stability of Extracellular Vesicles for Therapeutic Applications. AAPS J 20 , 1 (2017). Baxter, A. A. et al. Analysis of extracellular vesicles generated from monocytes under conditions of lytic cell death. Sci Rep 9 , 7538 (2019). Larssen, P. et al. Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assays. Mol Cell Proteomics 16 , 502–511 (2017). Gelibter, S. et al. The impact of storage on extracellular vesicles: A systematic study. J Extracell Vesicles 11 , e12162 (2022). Park, S. J., Jeon, H., Yoo, S.-M. & Lee, M.-S. The effect of storage temperature on the biological activity of extracellular vesicles for the complement system. In Vitro Cell Dev Biol Anim 54 , 423–429 (2018). Gelibter, S. et al. The impact of storage on extracellular vesicles: A systematic study. J Extracell Vesicles 11 , e12162 (2022). Weldon, K. C. et al. Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives. Int Urogynecol J 36 , 839–847 (2025). Rubin, B. E. R. et al. Investigating the impact of storage conditions on microbial community composition in soil samples. PLoS One 8 , e70460 (2013). Görgens, A. et al. Identification of storage conditions stabilizing extracellular vesicles preparations. J of Extracellular Vesicle 11 , e12238 (2022). Wu, J.-Y., Li, Y.-J., Hu, X.-B., Huang, S. & Xiang, D.-X. Preservation of small extracellular vesicles for functional analysis and therapeutic applications: a comparative evaluation of storage conditions. Drug Delivery 28 , 162–170 (2021). Liu, B., Zhang, Q., Zhao, Y., Ren, L. & Yuan, X. Trehalose-functional glycopeptide enhances glycerol-free cryopreservation of red blood cells. J Mater Chem B 7 , 5695–5703 (2019). Fry, L. J. et al. Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts. Transfusion 53 , 1834–1842 (2013). Zaloga, J. et al. Different storage conditions influence biocompatibility and physicochemical properties of iron oxide nanoparticles. Int J Mol Sci 16 , 9368–9384 (2015). Welsh, J. A. et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J of Extracellular Vesicle 13 , e12404 (2024). Tanaka, S., Naruse, Y., Terasaka, K. & Fujioka, S. Concentration and Dilution of Ultrafine Bubbles in Water. Colloids and Interfaces 4 , 50 (2020). Tominaga, N. et al. Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood–brain barrier. Nat Commun 6 , 6716 (2015). Additional Declarations Competing interest reported. The author N.T. is a founder and director of ADDVEMO Inc. The ADDVEMO Inc. was not involved in the study design, data collection, analysis, interpretation of data, the writing of the report, or the decision to submit the article for publication. Author N.T. receives research funding from Otsuka Electronics Co. for this study. Author R.I. is an employee of Otsuka Electronics Co. Ltd., and ELSZneo evaluated in this study was developed by Otsuka Electronics Co. Ltd. The authors S.H. and R.O. declare no competing interests. Supplementary Files ManuscriptOtsukav7SRv3UncroppedWB.pdf ManuscriptOtsukav7SRv3Supplimentaly.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7586495","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":521418439,"identity":"ca48b2ad-2cba-4e05-9c65-67e264f70d44","order_by":0,"name":"Ryosuke Inayama","email":"","orcid":"","institution":"Otsuka Electronics Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Inayama","suffix":""},{"id":521418440,"identity":"a84c91e0-698d-4bf2-9a09-d0d03a083f67","order_by":1,"name":"Saki Horie","email":"","orcid":"","institution":"Yamaguchi University","correspondingAuthor":false,"prefix":"","firstName":"Saki","middleName":"","lastName":"Horie","suffix":""},{"id":521418441,"identity":"a46734ee-c1b5-498d-88e6-3fe9193ecc53","order_by":2,"name":"Risako Okada","email":"","orcid":"","institution":"Yamaguchi University","correspondingAuthor":false,"prefix":"","firstName":"Risako","middleName":"","lastName":"Okada","suffix":""},{"id":521418442,"identity":"15046987-9f17-46e3-bd59-ef29e02e9561","order_by":3,"name":"Naoomi Tominaga","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYLCCBCDmh3EMmKEMxgYcytmgWiRhCojTAlZ5AMYg5CZz+eajGx7usLPbfCP56YYfvw7Lm7MzsEkw1NgxMM/Gbo1lG1vajcQzycnbbqSZ3eztO2y4sxmk5VgyA+OcA1i1GBzjMbuR2MacbHYjwewGb8/hBIPD/N8kGNgOMDDOSMCnpT7ZeEb6t5t/wVpAtvwjqOWwnYFEjtltnh9QLYxt+LSkAf3SdjxB4sybstuyDemGGw4zMFsk9iXz4PTL4cPHbv5sq7bnb0/fdvPNH2t5g/MHGG98+GYnZ4gjxGAgsUEA6AzGNigXyOYxnIFXB4M9Az/IGX+QhOQl8GsZBaNgFIyCEQMAaMJk8d6Xr8YAAAAASUVORK5CYII=","orcid":"","institution":"Yamaguchi University","correspondingAuthor":true,"prefix":"","firstName":"Naoomi","middleName":"","lastName":"Tominaga","suffix":""}],"badges":[],"createdAt":"2025-09-11 00:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7586495/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7586495/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92411502,"identity":"c3c2f649-217d-4a69-93ef-221d17cc8242","added_by":"auto","created_at":"2025-09-29 12:36:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":324853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design and baseline characterization of PC3-derived EVs in different suspension solutions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the experimental plan for long-term EV storage. EVs were stored in PBS, HBSS, or HBSS with 5% glucose at 4℃, −30℃, and −80℃. Analyses included particle characteristics (concentration, size, polydispersity, zeta potential, electrophoretic mobility, conductivity), morphology (electron microscopy), protein content, and Western blotting. (B) Western blotting of CD63 and Cytocrom-C. (C) EV particle concentration (×10\u003csup\u003e10\u003c/sup\u003e particles/mL), particle size (nm), zeta potential (mV) in PBS, HBSS, or HBSS (5%Gluc.) on Day 1. Data are presented as mean ± SD. (D) Representative transmission electron micrographs of EVs in PBS (left), HBSS (center), and HBSS (5%Gluc.) (right). White arrowheads point to vesicles which unstable structure. Scale bar, 0.3 µm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/a6ab3cb6117dfe76c3d0fca1.png"},{"id":92411500,"identity":"0bb8adc4-ce12-4b2c-9110-16887717dddd","added_by":"auto","created_at":"2025-09-29 12:36:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStability of PC3-derived EVs under various storage conditions over 90 days.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTime-course analysis of (A) particle concentration (×10\u003csup\u003e10\u003c/sup\u003e particles/mL), (B) particle size (nm), (C) Polydispersity Index (PDI), and (D) zeta potential (mV) of EVs. EVs were suspended in PBS (left column), HBSS (middle column), or HBSS with 5% glucose (HBSS (5%Gluc.), right column) and stored at 4 ℃(light blue line with circles), −30 ℃ (purple line with triangles), or −80 ℃ (dark blue line with squares) for up to 90 days. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/5cb4cc8c54f97647a4ff2788.png"},{"id":92412222,"identity":"6557dd92-54d8-4d60-a4d5-735458a8f87a","added_by":"auto","created_at":"2025-09-29 12:44:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of PC3-derived extracellular vesicles (EVs) stored at 4 \u003c/strong\u003e℃\u003cstrong\u003e over 90 days.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative TEM images of EVs. EVs were suspended in (A) PBS, (B) HBSS, or (C) HBSS with 5% glucose and stored at 4 ℃. Images were taken on Day 1 and at various time points (Day 15, 30, 45, 60, 75, and 90) after storage. Scale bars, 0.3 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/5063a92157d9f7517fde44ee.png"},{"id":92411303,"identity":"19d7aac1-6ee8-4d8b-bfaa-efe872bc313f","added_by":"auto","created_at":"2025-09-29 12:28:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":403008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of PC3-derived EVs stored at −30 ℃ over 90 days.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative TEM images of EVs. EVs were suspended in (A) PBS, (B) HBSS, or (C) HBSS with 5% glucose and stored at −30 ℃. Images were taken at various time points (Day 15, 30, 45, 60, 75, and 90) after storage. Scale bars, 0.3 µm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/8b0b57a1132c10cf1a2443cc.png"},{"id":92411298,"identity":"48bbdcf0-2a55-4a1a-83b0-14609516d620","added_by":"auto","created_at":"2025-09-29 12:28:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":393644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of PC3-derived EVs stored at −80 \u003c/strong\u003e℃\u003cstrong\u003eover 90 days.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative TEM images of EVs. EVs were suspended in (A) PBS, (B) HBSS, or (C) HBSS with 5% glucose and stored at −80 ℃. Images were taken at various time points (Day 15, 30, 45, 60, 75, and 90) after storage. Scale bars, 0.3 µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/cb3dbc16382f699971bf77d3.png"},{"id":92411300,"identity":"a498e5af-9eeb-4227-b981-8ee6152e38ad","added_by":"auto","created_at":"2025-09-29 12:28:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":322310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStability of PC3-derived EVs against repeated freeze-thaw cycles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs were suspended in PBS (A, D, G, J), HBSS (B, E, H, K), or HBSS with 5% glucose (C, F, I, L) and subjected to 0, 1, 2, 3, or 6 freeze-thaw (FT) cycles. Freeze-thaw cycles were performed by freezing at either −30 ℃(light blue bars) or −80 ∘C (dark blue bars) and thawing at room temperature. (A-C) Particle size relative to the non-frozen control (0 FT cycles). (D-F) Particle number relative to the non-frozen control (0 FT cycles). (G-I) Polydispersity Index (PDI). The dotted line at PDI = 0.1 indicates a threshold for acceptable homogeneity. (J-L) ζ-potential (mV). Data are presented as mean ± SD (n=5-6 per group). Statistical significance was determined by student's test. p \u0026lt; 0.05, p \u0026lt; 0.01, p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/9d0af3f6abad2143b27faeb2.png"},{"id":92411305,"identity":"af160084-3b33-469d-8cac-75b63aa1bca9","added_by":"auto","created_at":"2025-09-29 12:28:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":441639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of PC3-derived EVs after repeated freeze-thaw cycles at −30 ℃.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative TEM images of EVs. EVs were suspended in (A) PBS, (B) HBSS, or (C) HBSS with 5% glucose and subjected to 1, 2, 3, or 6 freeze-thaw cycles at −30 ℃. Scale bars, 0.3 µm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/ca782ce39dcfc4f5bbdb1b90.png"},{"id":92411503,"identity":"d2f51eb0-b77f-4686-adf3-b44068a15633","added_by":"auto","created_at":"2025-09-29 12:36:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":557595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis of PC3-derived EVs after repeated freeze-thaw cycles at −80 ℃.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative TEM images of EVs. EVs were suspended in (A) PBS, (B) HBSS, or (C) HBSS with 5% glucose and subjected to 1, 2, 3, or 6 freeze-thaw cycles at −80 ∘C. Scale bars, 0.3 µm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/f043b6d2644511b6e5c31fa0.png"},{"id":96244414,"identity":"64fad8a3-8616-4bef-bcdc-71fa99509e8c","added_by":"auto","created_at":"2025-11-19 07:18:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4442889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/c136ff90-417b-4fe0-a07e-151a2d3ab6c0.pdf"},{"id":92411297,"identity":"bd0d1bed-6571-4333-aa91-41d4d667fee8","added_by":"auto","created_at":"2025-09-29 12:28:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":191124,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptOtsukav7SRv3UncroppedWB.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/48dcf634f591e57450c8083c.pdf"},{"id":92411504,"identity":"d4aa2d1d-2ed6-4d51-b65e-01f791596667","added_by":"auto","created_at":"2025-09-29 12:36:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":329570,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptOtsukav7SRv3Supplimentaly.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586495/v1/ed5a7e266f5b67a26fa9c6ca.pdf"}],"financialInterests":"Competing interest reported. The author N.T. is a founder and director of ADDVEMO Inc. The ADDVEMO Inc. was not involved in the study design, data collection, analysis, interpretation of data, the writing of the report, or the decision to submit the article for publication. Author N.T. receives research funding from Otsuka Electronics Co. for this study. Author R.I. is an employee of Otsuka Electronics Co. Ltd., and ELSZneo evaluated in this study was developed by Otsuka Electronics Co. Ltd. The authors S.H. and R.O. declare no competing interests.","formattedTitle":"Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtracellular vesicles (EVs) are nanometer-sized structures encased in lipid bilayers, released from cells, and play a vital role in intercellular communication through their encapsulated proteins, nucleic acids, and lipids \u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. EVs have been extensively researched for their potential as diagnostic biomarkers for various diseases, including cancer, neurodegenerative disorders, and cardiovascular conditions, as well as for their application as drug delivery systems (DDS) to transport encapsulated substances to target cells \u003csup\u003e4\u0026ndash;6\u003c/sup\u003e. Notably, their biocompatibility, low immunogenicity, and ability to cross biological barriers have been recognized as advantages over conventional nanocarriers, and their potential as therapeutic agents has been explored . For the application of EVs in diagnosis and therapy, mass production, purification, and stable storage are essential. Particularly, when considering the use of EVs as pharmaceutical products, it is crucial to establish a technology that enables the long-term preservation of EVs while maintaining their functions and characteristics from manufacturing to administration to patients. However, several technical challenges exist in preserving EVs. Currently, refrigerated (4℃) storage, cryopreservation, and lyophilization are the primary methods considered \u003csup\u003e7,8\u003c/sup\u003e. These methods may affect the function and therapeutic efficacy of EVs, making them unsuitable for long-term storage. Conversely, cryopreservation is widely employed as a method suitable for long-term storage, but there is a risk that the membrane structure of EVs may be compromised or aggregation may occur due to ice crystal formation or osmotic pressure changes during the freezing and thawing processes \u003csup\u003e9,10\u003c/sup\u003e. In particular, the freezing speed and the type and concentration of cryoprotectant significantly affect the condition of EVs, necessitating the study of optimal conditions. Furthermore, it has been suggested that multiple freeze-thaw cycles of cryopreserved EVs significantly reduce their physical properties and biological activity \u003csup\u003e11\u003c/sup\u003e. This may lead to changes in the particle size distribution of EVs, fluctuations in the expression of surface markers, and even reduced efficiency of cellular uptake \u003csup\u003e12\u003c/sup\u003e\u003csup\u003e,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e13\u003c/sup\u003e. Thus, the stability of EVs varies greatly depending on the method of storage, and it is essential to evaluate in detail the effects of these storage conditions on the condition of EVs to ensure long-term storage, and particularly their quality as pharmaceutical products. To commercialize EVs as safe and effective pharmaceutical products, it is essential to establish the safety and efficacy of the manufactured EVs, which must be scientifically proven to maintain their physicochemical properties and biological activity for extended periods under appropriate storage conditions. The study investigates the difficulties of preserving EVs over extended durations at temperatures of 4℃, -30℃, and -80℃ in different solvents, as well as the impact of cryopreservation and repeated freeze-thaw cycles on their integrity. We present the findings of our research, which have contributed to the development of stable storage techniques for the future utilization of EVs, providing crucial insights for pharmaceutical applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInitial characterization of PC3 cell-derived EVs reveals suspension buffer affects particle concentration without significantly altering particle size or zeta potential.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the isolated EVs, dynamic light scattering (DLS), electrophoretic light scattering, Western blotting, and transmission electron microscopy (TEM) observations were performed. Isolated EVs were suspended in different suspension buffers (PBS, HBSS, and HBSS with 5% glucose) and stored under three different temperature conditions (4℃, -30℃, and-80℃) for up to 90 days. In addition, EVs suspended in each buffer (PBS, HBSS, and HBSS with 5% glucose) were also freeze-thawed 1, 2, 3, and 6 times after storage at -30℃ and -80℃. During storage, samples were periodically taken to measure the particle concentration, particle size, polydispersity index (PDI), zeta potential, electrical conductivity, and scattered light intensity using ELSZneo, and to observe morphology using electron microscopy (Fig.1A). Physicochemical properties of EVs were evaluated immediately after isolation (Day 1). The particle concentration of EVs in various suspension buffers was measured using ELSZneo (Fig. 1B). The results showed that the particle concentrations in PBS, HBSS, and HBSS with 5% glucose were 2.53 \u0026times; 10\u0026sup1;⁰ particles/mL, 2.16 \u0026times; 10\u0026sup1;⁰ particles/mL, and 3.26 \u0026times; 10\u0026sup1;⁰ particles/mL, respectively. Particle size and zeta potential of EVs immediately after isolation (Day 1) were also measured using ELSZneo (Fig. 1B), noting that HBSS and HBSS with 5% glucose are more viscous than PBS, which may affect particle count measurements. Particle size was approximately 170 nm for all buffers, with no significant differences (PBS: 171.6 nm, HBSS: 171.9 nm, HBSS with 5% glucose: 170.7 nm). Zeta potentials were negative in all buffers, approximately -12.55 mV (PBS), -12.97 mV (HBSS), and -12.22 mV (HBSS w/ 5% glucose). Subsequently, the presence of EV marker proteins was assessed by western blotting. The expression of CD63, a marker protein specific to isolated EVs, was confirmed by western blotting, although it exhibited low intensity in the cell lysate (Fig. 1C). Additionally, Cytochrome-C was not detected in EVs compared to that in the cell lysate. Morphological analysis of the isolated EVs was conducted using TEM (Fig. 1D). The analysis revealed that EVs in PBS displayed typical cup-shaped or circular membrane structures. However, EVs in HBSS and HBSS with 5% glucose demonstrated particle degradation, aggregation, and emergence of spike-like structures from Day 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-30℃ Storage Significantly Impairs EV Physicochemical Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of long-term storage at -30\u0026deg;C, we conducted further experiments. The particle counts of EVs varied under the tested conditions. In PBS suspension, the particle count decreased to 2.0 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e particles/mL by day 45 when stored at 4℃ and -80℃, followed by an increase by day 90 (Fig. 2A). Notably, storage at -30℃ resulted in a reduction of particle count to half by day 15, with levels remaining low thereafter, suggesting potential aggregation or destruction of EVs during a single freeze-thaw cycle at this temperature. Conversely, storage in HBSS suspension at -80℃ resulted in relatively stable particle concentrations, closely approximating the initial values, whereas particle concentrations gradually increased over time when stored at 4℃. In contrast, the particle concentrations remained relatively stable at low temperatures of -30℃ and -80℃. Additionally, an increase in the particle count was observed in HBSS (5% glucose) at 4℃. At -30℃ and -80℃, the particle concentrations were comparably stable.\u003c/p\u003e\n\u003cp\u003eSubsequently, the particle sizes of EVs were analyzed (Fig. 2B). In the PBS suspension, the particle size remained relatively stable within the range of approximately 150-170 nm at 4℃ and -80℃, whereas at -30℃, the particle size increased from day 30 to day 60, followed by instability. In the HBSS or HBSS (5% glucose) suspension exhibited remarkable stability in the size range of approximately 160-180 nm across all temperature conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the PDI, which indicates particle size uniformity, a value of 0.1 or less was considered to be near monodisperse. Storage at 4℃ in PBS suspension increased PDI up to day 45, followed by a decrease after day 60 (Fig. 2C). At -30℃ and -80℃, the PDI exceeded 0.1 after day 15, with a marked increase in particle size heterogeneity, particularly at -30℃. In the HBSS suspension, the PDI remained relatively low during storage at 4℃. At -30℃ and -80℃, the PDI exceeded 0.1, indicating a slight increase in heterogeneity. In HBSS with 5% glucose, the PDI remained low (below 0.1) at 4℃. However, at -30℃ and -80℃, the PDI generally remained above 0.1.\u003c/p\u003e\n\u003cp\u003eThe zeta potential, an indicator of particle dispersion stability, remained relatively stable in PBS and HBSS (5% glucose) suspensions, ranging from -10 to -15 mV under all temperature conditions (Fig. 2D). However, when the HBSS suspension was stored at -30℃, the absolute value of zeta potential significantly decreased on day 75, approaching -20 mV, suggesting alterations in the particle surface state and potential aggregation.\u003c/p\u003e\n\u003cp\u003eAmong the electrical properties shown in Supplementary Figure 1, electrophoretic mobility is linked to the zeta potential. In PBS and HBSS (5% glucose) suspensions, electrophoretic mobility remained relatively stable across all temperature conditions (Supplementary Fig. 1A). In contrast, conductivity, which reflects the ion concentration in the suspension, remained stable at approximately 14-15 mS/cm in both PBS and HBSS suspensions throughout the 90-day period at all temperatures (Supplementary Fig. 1B). Collectively, these findings suggest that the polydisperse index tends to vary with storage duration, favoring storage at 4℃ or -80℃. In contrast, the physicochemical indices of EVs were significantly affected by storage at -30℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS More Effectively Preserves EV Morphology at 4℃\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical properties of EVs were evaluated by particle count and size measurements using ELSZneo, as shown in Figure 2. However, to thoroughly assess EV quality, it is essential to directly observe the morphological characteristics of individual vesicles, including membrane integrity and aggregation levels. To evaluate morphological stability over time, TEM observations were conducted at 15-day intervals from Day 1 to Day 90 (Fig.3). On Day 1, the EVs stored in PBS displayed a typical cup or spherical shape with diameters ranging from approximately 50 to 200 nm and a well-defined lipid bilayer structure (Fig. 3A). However, as the storage duration increased, morphological changes progressed, and by Day 30, some EVs began to show obscured membrane structures and partial vesicle collapse. This morphological deterioration became more pronounced after Day 45, and by Days 60 and 75, the number of EVs retaining their original shape was significantly reduced, with many forming aggregates or becoming fragmented. By Day 90, few EVs with clear vesicular structures were observed. In contrast, EVs preserved in HBSS exhibited morphology on Days 1 and 15 similar to those preserved in PBS (Fig. 3B). However, the initial morphology of EVs tended to be somewhat unstable compared to those preserved in PBS, with aggregation and morphological heterogeneity becoming apparent at approximately Day 30. By Day 90, only a few EVs maintained a stable morphology, similar to that in the PBS-preserved condition. Conversely, EVs stored in HBSS containing 5% glucose showed morphological instability as early as Day 1, with collapsed vesicle structures observed in some instances (Fig. 3C). The instability of the morphology increased with storage time, and in addition to the formation of string-like protrusions, further collapse and aggregation of the vesicles were observed. Based on the TEM observations, PBS alone is more effective than HBSS alone or HBSS with 5% glucose in maintaining the highest morphological stability of vesicles during long-term storage of EVs at 4℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-30℃ Storage Results in Significant EV Morphological Degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of extracellular vesicles preserved at -30\u0026deg;C was subsequently analyzed using TEM. EVs stored in PBS at -30℃ began to show compromised membrane integrity on the Day 15 of freezing (Fig. 4A). By the Day 30, the vesicular structure had deteriorated, leading to the formation of substantial aggregates. By the Day 45, morphological anomalies such as protruding structures and vesicle collapse were evident. On Day 60, vesicle crushing, persistent protrusions, and extensive aggregation were observed. EVs stored in HBSS at -30℃ exhibited scattered structural instability from an early stage (Fig. 4B). By the 15th day, some aggregation and minor membrane damage were apparent, although the morphology was somewhat better than that observed with PBS storage. By the 30th day, aggregation and morphological diversity became pronounced, and by the 45th day, there was an increase in aggregates and collapsed vesicles. By the Day 90, most particles showed structural instability, including protruding structures and vesicle collapse. In contrast, EVs in HBSS with 5% glucose stored at -30℃ consistently displayed morphological deterioration, such as protruding structures, vesicle collapse, and aggregation, throughout the observation period from the Day 15 to the Day 90 (Fig. 4C). TEM revealed that long-term cryopreservation of EVs at -30℃ resulted in significant morphological degradation over time across all three tested conditions. Although the initial morphology during PBS storage was satisfactory, the effects of one-time freeze-thaw cycles and prolonged storage were unavoidable. This suggests that storage at -30℃ may not be effective in maintaining morphological stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignificant EV Morphological Degradation Despite -80\u003c/strong\u003e℃\u003cstrong\u003e\u0026nbsp;Cryopreservation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of extracellular vesicles preserved at -80\u0026deg;C was subsequently analyzed using TEM. EVs preserved in PBS at -80℃ retained a relatively stable particle structure for up to 15 days after freezing (Fig. 5A). However, by Day 30, the vesicle structure began to show signs of collapse, with protruding formations and notable aggregation becoming apparent. This morphological degradation continued over time, and by Day 60, only a few clear and intact particle structures remained visible. EVs stored in HBSS at -80℃ exhibited significant morphological degradation, including protruding formations, vesicle collapse, and aggregation, starting from Day 15 post-freezing, with structural instability persisting throughout the storage period (Fig. 5B). Similarly, EVs in HBSS with 5% glucose, also stored at -80℃, showed comparable morphological degradations from Day 15 post-freezing, with the particle structure remaining unstable throughout the observation period (Fig. 5C). TEM revealed that, despite long-term cryopreservation at -80℃, all three solution conditions tested resulted in significant morphological degradation over time. Morphological instability was observed earlier in HBSS alone and in HBSS with 5% glucose, particularly in the latter, which demonstrated no morpho‐protective effect under -80℃ storage conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolution and Temperature Determine EV Response to Freeze-Thaw Stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFreezing and thawing can negatively impact the membrane structure of EVs due to ice crystal formation and osmotic pressure changes, potentially leading to aggregation and collapse, which may subsequently affect particle size, particle count, and the quality of their contents. To evaluate the changes in particle size and particle count of EVs during multiple freeze-thaw cycles, we tested varying freezing temperatures (-30℃ and -80℃) and three distinct storage solutions: PBS, HBSS, and HBSS with 5% glucose. The experimental procedure involved suspending the isolated EVs in their respective solutions, freezing them at either -30℃ or -80℃, and thawing them at room temperature, repeated 1, 2, 3 times, and 6 times. Notably, after six cycles, there was a significant increase in the particle size compared to the pre-freeze state (Fig.6A). When EVs suspended in HBSS were stored at -30℃, the particle size remained consistent, regardless of the number of cycles (Fig.6B). EVs suspended in HBSS with 5% glucose exhibited a notable increase in particle size after 3 and 6 freeze-thaw cycles at -80℃ storage conditions compared to storage at -30℃ (Fig.6C).\u003c/p\u003e\n\u003cp\u003eFor EVs suspended in PBS, a single freeze-thaw cycle led to a significant reduction in particle numbers, dropping to approximately 50% of the initial count at both -30℃ and -80℃ storage temperatures (Fig.6D). Regardless of the temperature, the particle numbers continued to decline with additional freeze-thaw cycles, although they remained significantly higher at -80℃ than at -30℃. When EVs were stored in HBSS with 5% glucose at -30℃, the particle count remained relatively high at approximately 85% of the initial number after one cycle and decreased to approximately 40% after six cycles (Fig.6E). This trend of maintaining a relatively high level persisted through two and three cycles (Fig.6E). Regardless of the temperature, the particle numbers continued to decline with additional freeze-thaw cycles, although they remained significantly higher at -80℃ than at -30℃ (Fig.6D). When EVs were stored in HBSS at -30℃ or -80℃, the particle count remained relatively high at approximately 50% of the initial number after one cycle and decreased to approximately 40% after six cycles (Fig.6E). Conversely, when EVs were stored in HBSS with 5% glucose at -30℃ or -80℃, the particle count remained relatively high, approximately 70% to 80% of the initial count after one cycle. However, after six cycles, the particle count decreased to approximately 120% when preserved at -30℃ and 80% when preserved at -80℃ (Fig. 6F).\u003c/p\u003e\n\u003cp\u003eThese findings suggest that freeze-thaw cycles affect both the size and number of EV particles, with the extent of the effect influenced by the freezing temperature and preservation solution used. Specifically, storage at -80℃ in PBS was more effective in maintaining particle numbers, although the particle size increased after six cycles at -30℃. These insights could aid in developmentping of optimal cryopreservation protocols to preserve EV quality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFreeze-Thaw Cycles Alter EV Polydispersity and Zeta Potential Depending on Solution and Temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs underwent several freeze-thaw cycles at -30℃ and -80℃, with their Polydispersity Index (PDI) and zeta potential assessed in three different storage solutions: PBS, HBSS, and HBSS with 5% glucose. (Electrophoretic mobility and Electrical conductivity are shown in Supplementary Figures 2A-2F). For EVs in PBS, the initial PDI was approximately 0.08 (Fig. 6G). A single freeze-thaw cycle at both -30℃ and -80℃ significantly increased the PDI to ~0.18-0.20, indicating a loss of particle size homogeneity. In subsequent cycles, PDI values remained stable between 0.20 and 0.24. For EVs in HBSS, the pre-freezing PDI was also ~0.08 (Fig. 6H). Following one cycle, the PDI similarly increased to ~0.18-0.20 at both temperatures. At -30℃, a slight temporary decrease to ~0.16 occurred in the third cycle, but it returned to ~0.18 after six cycles. At -80℃, the PDI remained stable at ~0.20 throughout all cycles. For EVs in HBSS with 5% glucose, the pre-freezing PDI was ~0.09 (Fig. 6I). At -30℃, the PDI increased to ~0.16 after the first cycle, fluctuated, and settled at ~0.15 after six cycles. Conversely, at -80℃, the PDI surged to ~0.21 after one cycle and remained elevated. Notably, after 1, 3, and 6 cycles, the PDI values at -80℃ were significantly higher than those at -30℃. Regarding surface charge, EVs in PBS initially had a zeta potential of approximately -12 mV (Fig. 6J). At -30℃, the absolute zeta potential increased to ~ -15 mV after one cycle before decreasing to ~ -9 mV after two cycles. At -80℃, it fluctuated more, reaching ~ -16 mV after two cycles but ending at ~ -13 mV. After six cycles, the absolute zeta potential at -80℃ was significantly greater than at -30℃. The EVs in HBSS initially had a zeta potential of ~ -13 mV (Fig. 6K). At -30℃, the absolute value increased to ~ -15 mV after one cycle and rose further to ~ -20 mV after six cycles. At -80℃, the zeta potential remained relatively stable between -12 and -15 mV. Before freezing, EVs in HBSS with 5% glucose exhibited a zeta potential of ~ -12 mV (Fig. 6L). At -30℃, the absolute zeta potential significantly increased to ~ -18 mV after one cycle and remained high. At -80℃, the zeta potential varied between -13 and -16 mV, with absolute values generally lower than those at -30℃. These findings suggest freeze-thaw cycling affects EV PDI and zeta potential, reducing particle size uniformity and altering surface charge. Zeta potential variations were influenced by both the storage solution and freezing temperature, indicating a complex impact on the EV surface.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe significant PDI increase for HBSS with 5% glucose at -80℃, and the rise in absolute zeta potential at -30℃, represent substantial changes. These physicochemical alterations influence EV aggregation and dispersion, providing valuable insights for optimizing storage conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFreeze-Thaw Cycling Inflicts Progressive Morphological Damage on EVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEM was employed to examine the morphological changes in EVs subjected to multiple freeze-thaw cycles (1, 2, 3, and 6 cycles) at varying freezing temperatures (-30℃ and -80℃) and in three different storage solutions: PBS, HBSS, and HBSS with 5% glucose. For EVs in HBSS treated at -30℃, vesicle collapse and aggregation were noticeable after 1, 2, 3, and 6 freeze-thaw cycles (Fig. 7B). Similarly, in EVs suspended in HBSS with 5% glucose and treated at -30℃, morphological degradation, such as vesicle collapse, aggregation, and the emergence of protruding structures, was observed (Fig. 7C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn EVs suspended in PBS and subjected to freeze-thawing at -80℃, many vesicles maintained a relatively intact structure after one or two freeze-thaw cycles (Fig. 8A). However, as the number of cycles increased, morphological degradation progressed, with some vesicles collapsing after three cycles of expansion and contraction. After six cycles, the number of vesicles retaining their original shape was significantly reduced, as vesicle collapse, membrane fragmentation, and substantial aggregate formation were observed. For EVs in HBSS treated at -80℃, vesicle collapse was evident after one freeze-thaw cycle (Fig. 8B). With more cycles, vesicle disintegration, protruding structures, and aggregation intensified, and after 3 and 6 cycles, many vesicles lost their structure, becoming primarily fragmented membrane components and aggregates. In EVs in HBSS with 5% glucose treated at -80℃, vesicle disintegration, membrane heterogeneity, and aggregation were clearly visible after one freeze-thaw cycle, with these morphological degradations becoming more pronounced as the number of cycles increased (Fig. 8C). After three and six cycles, few vesicles remained in their original form, and extensive aggregation and disintegration of membrane structures were observed. These TEM observations visually confirmed the severe damage inflicted on EV morphology by the freeze-thaw cycles. At both -30℃ and -80℃, vesicle disintegration, aggregation, and fragmentation advanced with more cycles. The type of storage solution also played a role, suggesting that PBS frozen at -80℃ might preserve some morphology, although instability from the initial state was noted at -30℃. These morphological evaluations should be considered alongside particle size and number, PDI, and zeta potential measurements by ELSZneo, highlighting the importance of TEM observations in assessing EV quality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStorage and Freeze-Thaw in PBS Reduce EV Protein Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein content of EVs is a crucial marker for evaluating their functionality and quality. Protein levels were measured using the microBCA method, with changes in protein concentration compared to baseline values (Day 1 or pre-freeze samples), set as a relative value of 1.0. For EVs stored in PBS at 4℃, the protein concentration dropped to 25% of the Day 1 level by Day 15 (Supplementary Fig. 3A). Although fluctuations occurred during the storage period, the concentration remained lower than the initial level. When EVs were stored in PBS at -30℃, the protein concentration fell to approximately 40% of the initial value by Day 15 and further decreased to approximately 15% by Day 45. Despite variations throughout storage, the concentration consistently remained lower than the initial level. For EVs stored in PBS at -80℃, the protein concentration was reduced to approximately 30% of the initial value by Day 15. Although it varied during storage, it remained lower than the initial level. For EVs suspended in PBS and subjected to freeze-thaw cycles at -30℃, the protein concentration decreased to approximately 50% of the initial value after one cycle (Supplementary Fig. 3B). In EVs at -80℃, the concentration decreased to approximately 60% after one cycle and then stabilized at around 50% after six cycles. HBSS and HBSS with 5% glucose could not be measured using microBCA due to solvent inhibitors. These findings suggest that during prolonged storage of EVs in PBS, the protein concentration significantly decreased from the initial value under all temperature conditions. In the freeze-thaw cycle treatment, the concentration was reduced by approximately half in the first cycle, with no significant further decrease as cycles increased. These fluctuations may result from factors such as EV aggregation, disintegration, or effects on the measurement system, and should be interpreted in conjunction with other physicochemical and morphological observations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study reveals that storing extracellular vesicles (EVs) in phosphate-buffered saline (PBS) at 4\u0026deg;C is optimal for maintaining their morphology for up to 15 days. During this period, vesicles remain intact with minimal aggregation. However, by day 45, a mix of intact and disintegrated particles is observed, with EV aggregation becoming more pronounced over 75 days post-purification. This finding highlights a balance between structural preservation and potential biochemical alterations that may occur during storage at 4\u0026deg;C in PBS \u003csup\u003e14,15\u003c/sup\u003e\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e. Compared to storage at -30\u0026deg;C and -80\u0026deg;C, the 4\u0026deg;C PBS condition proves superior. At -30\u0026deg;C, significant deterioration is observed across all buffers, including reduced particle concentration, increased polydispersity index (PDI), and severe morphological damage. While storage at -80\u0026deg;C better preserves particle numbers, it fails to prevent structural degradation over time or during freeze-thaw cycles\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e19\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e20\u003c/sup\u003e. Despite the 4\u0026deg;C PBS condition being the most effective in preserving vesicle morphology, changes in particle count and protein content indicate ongoing degradation processes. This suggests that even under optimal storage conditions, EVs undergo gradual deterioration. These findings have important implications for EV research and potential therapeutic applications. The choice of storage conditions can significantly impact the integrity and functionality of EVs, potentially affecting experimental outcomes or therapeutic efficacy. Researchers and clinicians working with EVs should carefully consider these storage effects when designing experiments or developing EV-based therapies. Further research is needed to fully understand the biochemical changes occurring during storage and to develop improved preservation methods. This may include investigating alternative storage buffers, exploring the use of cryoprotectants, or developing novel preservation techniques to better maintain EV integrity over extended periods.\u003c/p\u003e\n\u003cp\u003eDynamic light scattering (DLS) was used to characterize extracellular vesicle (EV) samples in accordance with the MISEV2023 guidelines \u003csup\u003e21\u003c/sup\u003e. DLS is a high-throughput technique that is effective for the qualitative identification of submicron particles and aggregates within EV samples. The data obtained from DLS underscore its utility in quality control, specifically in confirming the presence or absence of aggregation during EV preparation. However, as noted by MISEV2023, DLS has limitations in quantitatively determining characteristics such as the mean hydrodynamic diameter of polydisperse EV samples. This limitation arises because the DLS analysis algorithm assumes monodispersity, which can lead to size overestimation in polydisperse EV. Compared to nanoparticle tracking analysis (NTA) and imaging methods, the establishment of the limit of detection (LOD) for particle detection in DLS remains underdeveloped and is acknowledged as a future challenge. Therefore, DLS data should be regarded as a guiding tool, illuminating general trends in size distribution rather than providing precise measurements of individual EV dimensions.\u003c/p\u003e\n\u003cp\u003eTo effectively utilize EVs in medical applications, they must be stored properly to preserve their morphology, quantity, and functionality. Our study shows that no single optimal storage method exists; various factors must be balanced based on specific needs. Short-term storage at 4℃ in PBS preserves morphology but requires protein levels monitoring. Because freezing and thawing affect EVs, single-use portions are recommended. Future research should focus on developing methods to protect EVs during freezing or drying and assess their post-storage functionality. Standardized methods are crucial for ensuring EV safety in both research and medical applications. This study examined PC3-derived EVs under various storage conditions, showing that temperature, duration, buffer, and freezing affect EVs. Storage at 4℃ in PBS is optimal for morphology, while -80℃ in PBS better maintains particle numbers during freezing compared to -30℃, despite morphological and protein challenges. These findings contribute to the development of enhanced storage methods for medical applications.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human prostate cancer cell line, PC-3 (RCB2145), was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan on 2022~2024. The cell lines authentication was performed by the RIKEN BRC. PC-3 was cultured in RPMI1640 medium (Nacalai Tesque, Japan, No.30264-85) supplemented with 10% heat-inactivated FBS (Corning, No.35-079-CV) and antibiotic\u0026ndash;antimycotic agents (Nacalai Tesque, Japan, No.09366-44) at 37℃ in 5% CO2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEVs isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded onto 15cm dishes at 70-80% confluency and cultured overnight in a 5% CO2 incubator. The medium was then replaced with Advanced RPMI-1640 medium (Gibco, Massachusetts, No.12633020) supplemented with 2 mM L-glutamine (Nacalai Tesque, Japan, No.16948-04) and 1% antibiotic-antimycotic solution (Nacalai Tesque, Japan, No. 09367-34) after twice washes with 10 mL Dulbecco\u0026apos;s Phosphate Buffered Saline (PBS) (-) (Nacalai Tesque, Japan, No. 14249-24). Following a 48-hour incubation period, the culture supernatant was collected by centrifugation at 10,000 xg for 10 min at 4℃. To ensure the complete removal of cellular debris, the supernatant was subsequently filtered through a 0.22 \u0026mu;m PVDF filter (Millipore Merck, Massachusetts, No.17461-05). The resulting filtered supernatants were used for EV isolation. EVs were isolated by ultracentrifugation at 110,000 xg for 70 min at 4℃ using a SW41Ti swinging-bucket rotor (Beckman Coulter). The pellets were then washed with PBS (-), HBSS, or HBSS containing 5% glucose by repeat ultracentrifugation at 110,000 xg for 70 min at 4℃ and resuspended in PBS (-). The isolated EVs were stored at 4℃ until further experimentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegents and preservation, freeze-thaw processes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBS, Hank\u0026apos;s Balanced Salt Solution (HBSS), and HBSS with 5% glucose were used for resuspension regent of EVs. HBSS with 5% glucose was prepared by dissolving glucose (Fujifilme-wako, no.049-31165) at a concentration of 5% in standard HBSS solution. In the experiments of \u0026ldquo;Long-term preservation\u0026rdquo;, EVs in each regent were measured Particle concentration, Particle size distribution, Scattered light intensity, Polydispersity Index (PDI), Zeta potential, Electrophoretic mobility, and Electrical conductivity using ELSZneo on Day 1, 15, 30, 45, 60, 75, and 90. In addition, EVs were observed morphology using transmission electron microscope on each day. Protein concentration was measured using microBCA Protein Assay Kit (Thermofisher Scientific, no.\u0026nbsp;23235). For the \u0026ldquo;Repeated freeze-thaw processes\u0026rdquo; experiments, the same examinations were conducted after 1, 2, 3, and 6 cycles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamic Light Scattering (DLS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrated EVs samples were measured using a dynamic light scattering (DLS) instrument (ELSZneo, Otsuka Electronics, Co., Ltd., Osaka, Japan)\u003csup\u003e22\u003c/sup\u003e. The instrument is equipped with a 40 mW and 638 nm Semiconductor laser diode. The laser is detected by the APD module. The scattering angle is 165◦, at a constant temperature of 25 ℃. The optimal light scattering intensity is between 84,000 cps and 114,000 cps (photon count rate per second). This system is equipped with an attenuation filter that automatically regulates the incident light if the scattering intensity is above the range. Each measurement consisted of 30 runs. The measurements were performed three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe followed the protocol established in our previous study\u003csup\u003e4\u003c/sup\u003e. EVs were confirmed morphologically using a transmission electron microscopy. Formvar/carbon-coated copper grid (Nisshin EM, Tokyo, Japan) was hydrophilized with JFC-1600 Auto Fine Coater (JEOL, Tokyo, Japan). 3\u0026micro;L of purified EVs in PBS were placed on the hydrophilized grid and absorbed for 3 min. We washed them using 500 \u0026micro;L double-distilled H2O successive four drops, then negatively stained using 30 \u0026micro;L of 2.0% uranyl acetate successive four drops. The grid was air-dried after absorbing 2.0% uranyl acetate on the grid with filter paper. We imaged the grid with Tecnai G2 Spirit BioTWIN electron microscopy (FEI, OR, USA) operating 120 kV equipped with a EMSIS Phurona CMOS Camera. After exporting the raw data images to TIFF format, we used Fiji (ImageJ 1.53t) for data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe followed the protocol established in our previous study\u003csup\u003e23\u003c/sup\u003e. Proteins were isolated from cells using M-PER (Thermo Scientific, MA, USA) separated in Mini-PROTEAN TGX Gel (4\u0026ndash;12%, Bio-Rad) and electro transferred onto a PVDF membrane (Bio-Rad, No.1704274). After blocking in Block Ace (KAC, Japan, No.UKB80), the membranes were incubated for 1-hr at room temperature with primary antibodies, which included anti-CD63 (purified mouse anti-human CD63, H5C6, 1:200, BD), anti-Cytochrome C (Rabbit polyclonal, 4272S, 1;1000, CST). Secondary antibodies (HRP-linked anti-mouse IgG, NA931V or HRP-linked anti-rabbit IgG, NA934V, GE Healthcare) were used at a dilution of 1:10000. The membrane was then exposed to ImmunoStar LD (Fujifilm-Wako, Japan, No.292-69903).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted statistical analyses and visualized the data utilizing R software (version 4.2.1). To compare differences between two groups, we employed the Student\u0026apos;s t-test. Graphical representations display the mean \u0026plusmn; standard deviation (S.D.).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere gratitude to Mr. Yuuta Miyagi, Mr.\u0026nbsp;Tsubasa Yamanaka (an employee of Otsuka Electronics Co. Ltd.), and Mr. Taizo Hasegawa (an employee of Otsuka Electronics Co. Ltd.) for generously donating his time. We thank the Science Research Center of Yamaguchi University for Institute of Gene Research, Institute for Biomedical Research and Institute of Life Science and Medicine. This work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting construction of core facilities), Grant Number JPMXS0440400024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Otsuka Electronics Co. This work was supported in part by a Grant-in-Aid for Research Activity Start-up (No. 21K21219), Early-Career Scientists (No.22K17826, No. 25K21103) from Japan Society of the Promotion of Science (JSPS). This work was supported by Japan Science and Technology Agency (JST), ACT-X Grant Number JPMJAX222B, Japan. This work was supported in part by a Grant-in-Aid for Yamaguchi University Fund, Start-up Research Fund, New frontier project-2021, and FOCS 2023 (Yamaguchi University Original-University Fund). This work was supported in part by a Grant-in-Aid for the Ube City Next-Generation Researchers Project. The funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.I. and N.T. conceived and designed this study. R.I. performed the experiments using ELSZneo. S.H. and R.O. performed the experiments of the western blotting, transmission electron microscopy analysis. R.I. and N.T. performed data analysis and interpretation. All authors have reviewed and edited the manuscript. The manuscript was finalized by N.T. with the assistance of all authors. All authors have approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author N.T. is a founder and director of ADDVEMO Inc. The ADDVEMO Inc. was not involved in the study design, data collection, analysis, interpretation of data, the writing of the report, or the decision to submit the article for publication. Author N.T. receives research funding from Otsuka Electronics Co. for this study. Author R.I. is an employee of Otsuka Electronics Co. Ltd., and ELSZneo evaluated in this study was developed by Otsuka Electronics Co. Ltd. The authors S.H. and R.O. declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTh\u0026eacute;ry, C., Amigorena, S., Raposo, G. \u0026amp; Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. \u003cem\u003eCurr Protoc Cell Biol\u003c/em\u003e \u003cstrong\u003eChapter 3\u003c/strong\u003e, Unit 3.22 (2006).\u003c/li\u003e\n\u003cli\u003eY\u0026aacute;\u0026ntilde;ez-M\u0026oacute;, M. \u003cem\u003eet al.\u003c/em\u003e Biological properties of extracellular vesicles and their physiological functions. \u003cem\u003eJ Extracell Vesicles\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 27066 (2015).\u003c/li\u003e\n\u003cli\u003eTominaga, N. Anti-Cancer Role and Therapeutic Potential of Extracellular Vesicles. \u003cem\u003eCancers (Basel)\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 6303 (2021).\u003c/li\u003e\n\u003cli\u003eYamada, N. \u003cem\u003eet al.\u003c/em\u003e Glycosylation changes of vWF in circulating extracellular vesicles to predict depression. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 29066 (2024).\u003c/li\u003e\n\u003cli\u003eKalluri, R. The biology and function of exosomes in cancer. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 1208\u0026ndash;1215 (2016).\u003c/li\u003e\n\u003cli\u003eLuan, X. \u003cem\u003eet al.\u003c/em\u003e Engineering exosomes as refined biological nanoplatforms for drug delivery. \u003cem\u003eActa Pharmacol Sin\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 754\u0026ndash;763 (2017).\u003c/li\u003e\n\u003cli\u003eYuan, F., Li, Y.-M. \u0026amp; Wang, Z. Preserving extracellular vesicles for biomedical applications: consideration of storage stability before and after isolation. \u003cem\u003eDrug Deliv\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 1501\u0026ndash;1509 (2021).\u003c/li\u003e\n\u003cli\u003eJeyaram, A. \u0026amp; Jay, S. M. Preservation and Storage Stability of Extracellular Vesicles for Therapeutic Applications. \u003cem\u003eAAPS J\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1 (2017).\u003c/li\u003e\n\u003cli\u003eBaxter, A. A. \u003cem\u003eet al.\u003c/em\u003e Analysis of extracellular vesicles generated from monocytes under conditions of lytic cell death. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 7538 (2019).\u003c/li\u003e\n\u003cli\u003eLarssen, P. \u003cem\u003eet al.\u003c/em\u003e Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assays. \u003cem\u003eMol Cell Proteomics\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 502\u0026ndash;511 (2017).\u003c/li\u003e\n\u003cli\u003eGelibter, S. \u003cem\u003eet al.\u003c/em\u003e The impact of storage on extracellular vesicles: A systematic study. \u003cem\u003eJ Extracell Vesicles\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e12162 (2022).\u003c/li\u003e\n\u003cli\u003ePark, S. J., Jeon, H., Yoo, S.-M. \u0026amp; Lee, M.-S. The effect of storage temperature on the biological activity of extracellular vesicles for the complement system. \u003cem\u003eIn Vitro Cell Dev Biol Anim\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 423\u0026ndash;429 (2018).\u003c/li\u003e\n\u003cli\u003eGelibter, S. \u003cem\u003eet al.\u003c/em\u003e The impact of storage on extracellular vesicles: A systematic study. \u003cem\u003eJ Extracell Vesicles\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e12162 (2022).\u003c/li\u003e\n\u003cli\u003eWeldon, K. C. \u003cem\u003eet al.\u003c/em\u003e Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives. \u003cem\u003eInt Urogynecol J\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 839\u0026ndash;847 (2025).\u003c/li\u003e\n\u003cli\u003eRubin, B. E. R. \u003cem\u003eet al.\u003c/em\u003e Investigating the impact of storage conditions on microbial community composition in soil samples. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e70460 (2013).\u003c/li\u003e\n\u003cli\u003eG\u0026ouml;rgens, A. \u003cem\u003eet al.\u003c/em\u003e Identification of storage conditions stabilizing extracellular vesicles preparations. \u003cem\u003eJ of Extracellular Vesicle\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e12238 (2022).\u003c/li\u003e\n\u003cli\u003eWu, J.-Y., Li, Y.-J., Hu, X.-B., Huang, S. \u0026amp; Xiang, D.-X. Preservation of small extracellular vesicles for functional analysis and therapeutic applications: a comparative evaluation of storage conditions. \u003cem\u003eDrug Delivery\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 162\u0026ndash;170 (2021).\u003c/li\u003e\n\u003cli\u003eLiu, B., Zhang, Q., Zhao, Y., Ren, L. \u0026amp; Yuan, X. Trehalose-functional glycopeptide enhances glycerol-free cryopreservation of red blood cells. \u003cem\u003eJ Mater Chem B\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 5695\u0026ndash;5703 (2019).\u003c/li\u003e\n\u003cli\u003eFry, L. J. \u003cem\u003eet al.\u003c/em\u003e Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts. \u003cem\u003eTransfusion\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1834\u0026ndash;1842 (2013).\u003c/li\u003e\n\u003cli\u003eZaloga, J. \u003cem\u003eet al.\u003c/em\u003e Different storage conditions influence biocompatibility and physicochemical properties of iron oxide nanoparticles. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 9368\u0026ndash;9384 (2015).\u003c/li\u003e\n\u003cli\u003eWelsh, J. A. \u003cem\u003eet al.\u003c/em\u003e Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. \u003cem\u003eJ of Extracellular Vesicle\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e12404 (2024).\u003c/li\u003e\n\u003cli\u003eTanaka, S., Naruse, Y., Terasaka, K. \u0026amp; Fujioka, S. Concentration and Dilution of Ultrafine Bubbles in Water. \u003cem\u003eColloids and Interfaces\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 50 (2020).\u003c/li\u003e\n\u003cli\u003eTominaga, N. \u003cem\u003eet al.\u003c/em\u003e Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood\u0026ndash;brain barrier. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 6716 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Extracellular vesicles, Preservation, Cryopreservation, Freeze-Thaw Cycles, Storage Temperature","lastPublishedDoi":"10.21203/rs.3.rs-7586495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7586495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Extracellular vesicles (EVs) are crucial mediators of intercellular communication and have significant diagnostic and therapeutic potential. However, their preservation poses considerable challenges. This study examined the stability of EVs derived from PC3 cells when stored in PBS, HBSS, or HBSS with 5% glucose under long-term storage conditions (4 °C, -30 °C, and -80 °C) and subjected to up to six freeze-thaw cycles. The physicochemical properties of EVs, including their concentration, size, morphology, and protein content, were assessed over 90-day period. The findings indicate that storage at -30 °C significantly compromised EV integrity across all buffers, resulting in particle loss and morphological degradation. Although storage at -80 °C better preserved particle concentrations, morphological instability persisted at this temperature. In contrast, storage at 4 °C in PBS was most effective for maintaining morphology, albeit with a reduction in protein content over time. Repeated freeze-thaw cycles, particularly at -30 °C, caused substantial damage. These results show that storage temperature, duration, and suspension buffer affect EV integrity, providing useful information for developing improved preservation methods.","manuscriptTitle":"Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 12:28:53","doi":"10.21203/rs.3.rs-7586495/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3c5320bd-29cf-48bb-9d7c-0c605f2ca0a7","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55427374,"name":"Biological sciences/Biological techniques"},{"id":55427375,"name":"Biological sciences/Biophysics"},{"id":55427376,"name":"Biological sciences/Biotechnology"},{"id":55427377,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2025-11-14T15:53:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 12:28:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7586495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7586495","identity":"rs-7586495","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}