Exosome cryoprotectant and application thereof in exosome storage

Abstract

The invention relates to the technical field of exosomes, in particular to an exosome cryoprotectant and application thereof in exosome storage. The invention provides an exosome cryoprotectant. The exosome cryoprotectant is prepared from the following components in concentration: 25 mM of a Hanx balanced salt solution, 10-40 mM of trehalose, 4 mM of glutathione, 1 mM of vitamin E, 1-20 g / L of human serum albumin, 1 mM of proline, 1 mM of glycine, 100-300 g / L of glycerol and 10-40 g / L of hyaluronic acid. The exosome cryoprotectant provided by the invention can effectively maintain the activity of the exosome under a low-temperature storage condition, and the composition of the exosome and the cryoprotectant can be stably stored at 4 DEG C,-20 DEG C and-80 DEG C; in addition, the exosome cryoprotectant provided by the invention effectively delays the degradation of the exosome at the temperature of 4 DEG C, so that the long-term storage of the exosome at the temperature of 4 DEG C is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and in particular to an exosome cryoprotectant and application thereof in exosome storage. Background Art

[0002] Mesenchymal stem cells (MSCs) have attracted extensive attention in recent years due to their multidirectional differentiation potential and immunomodulatory effects in regenerative medicine and tissue engineering. Exosomes secreted by MSCs play a vital role in intercellular communication, tissue repair and disease treatment. Exosomes have significant biological effects in wound repair, immunomodulation and the treatment of various diseases through the bioactive molecules (such as proteins, lipids, RNA, etc.) they carry. Therefore, a reasonable storage method is crucial to ensure its effectiveness in clinical and research.

[0003] Currently, cryopreservation of MSCs exosomes is usually carried out by low-temperature freezing (-80°C) or liquid nitrogen (-196°C) storage, which helps to significantly slow down the degradation of exosomes and reduce oxidative damage. However, ice crystals may be generated during the freezing process, destroying the exosome membrane structure, and repeated freezing and thawing operations will also lead to loss of their function. Therefore, it is crucial to add cryoprotectants to avoid damage.

[0004] In short-term storage, a 4°C environment is usually suitable for preserving exosomes within a few days to a week, especially during production and transportation. 4°C conditions can effectively slow down the degradation of exosomes, maintain their structure and function, and avoid damage caused by frequent freezing and thawing. Compared with ultra-low temperature storage, 4°C storage is more convenient, economical, and does not require expensive equipment. It is suitable for short-term transportation and temporary storage. However, under current technical conditions, the storage time at 4°C is relatively limited and is mainly suitable for short-term needs; if long-term storage or long-distance transportation is required, a lower temperature storage method is required, such as -80°C or liquid nitrogen storage.

[0005] Although storage at 4°C can slow down the degradation rate, the degradation rate is still higher than that at ultra-low temperature conditions such as -80°C. In particular, the proteins and RNA in the exosomes may suffer structural damage and loss of biological activity over time. In addition, at 4°C, exosomes are still exposed to the risk of oxidative stress and enzymatic degradation. Oxygen may cause lipid membrane oxidation, and degradative enzymes such as extracellular proteases may remain active at this temperature, further damaging the exosome membrane structure. Summary of the invention

[0006] In order to solve the above problems, the present invention provides an exosome cryoprotectant and its application in exosome storage. The exosome cryoprotectant provided by the present invention can effectively maintain the activity of exosomes under low-temperature storage conditions, and the composition of exosomes and cryoprotectants can be stably stored at 4°C, -20°C and -80°C.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides an exosome cryoprotectant, comprising components with the following concentrations: 25 mM Hanks' balanced salt solution, 10-40 mM trehalose, 4 mM glutathione, 1 mM vitamin E, 1-20 g / L human serum albumin, 1 mM proline, 1 mM glycine, 100-300 g / L glycerol and 10-40 g / L hyaluronic acid.

[0009] Preferably, the exosome cryoprotectant comprises components in the following concentrations: Hanks' balanced salt solution 25 mM, trehalose 15-30 mM, glutathione 4 mM, vitamin E 1 mM, human serum albumin 2-10 g / L, proline 1 mM, glycine 1 mM, glycerol 100-200 g / L and hyaluronic acid 15-30 g / L.

[0010] Preferably, the molecular weight of the hyaluronic acid is less than 10 kDa.

[0011] Preferably, the solvent of the exosome cryoprotectant is PBS buffer.

[0012] The present invention provides the use of the exosome cryoprotectant described in the above technical solution in the storage and / or transportation of exosomes.

[0013] Preferably, the storage and / or transportation temperature is -80°C to 4°C.

[0014] Preferably, the exosomes are mesenchymal stem cell-derived exosomes.

[0015] Preferably, the application further comprises: mixing the exosomes and the exosome cryoprotectant, placing the mixture in a container, and sealing the container after filling with an inert gas.

[0016] Preferably, the container is a light-proof container.

[0017] Preferably, the container is a container having a chlorinated organopolysiloxane (Sigmacote) coating on the inner wall.

[0018] Beneficial effects:

[0019] The present invention provides an exosome cryoprotectant, comprising the following components in concentration: 25mM Hanks' balanced salt solution, 10-40mM trehalose, 4mM glutathione, 1mM vitamin E, 1-20g / L human albumin, 1mM proline, 1mM glycine, 100-300g / L glycerol and 10-40g / L hyaluronic acid. The exosome cryoprotectant obtained by compounding appropriate components of the present invention can effectively maintain the activity of exosomes under low-temperature storage conditions, and the composition of exosomes and cryoprotectants can be stably stored under 4°C, -20°C and -80°C; in addition, the exosome cryoprotectant provided by the present invention effectively delays the degradation of exosomes at 4°C, thereby realizing long-term storage of exosomes at 4°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The particle count results of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 1 week;

[0022] Figure 2 The particle size count results of exosomes stored in different protective solutions at +4℃, -20℃, and -80℃ for 1 week;

[0023] Figure 3 The particle count results of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 6 weeks;

[0024] Figure 4 The particle size count results of exosomes stored in different protective solutions at +4℃, -20℃, and -80℃ for 6 weeks;

[0025] Figure 5 The particle count results of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 12 weeks;

[0026] Figure 6 The particle size count results of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 12 weeks;

[0027] Figure 7 The small RNA content of exosomes stored in different protective solutions at +4℃, -20℃, and -80℃ for 12 weeks;

[0028] Figure 8 miRNA content of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 12 weeks;

[0029] Fig. 9 The full-length DNA content of exosomes stored in different protective solutions at +4°C, -20°C, and -80°C for 12 weeks;

[0030] Fig.10 The results of specific marker expression of exosomes stored in ExoCS protective solution + 4°C for 12 weeks;

[0031] Fig.11 To examine the structural integrity of exosomes under the conditions of +4°C, -20°C, and -80°C in ExoCS protective solution by transmission electron microscopy;

[0032] Fig.12 The biological activity of exosomes after storage in ExoCS protective solution at +4℃, -20℃, and -80℃ for different lengths of time and then re-thawed;

[0033] Fig.13 The results of protein quantitative analysis of exosomes in each bottle using BCA in Example 9;

[0034] Among them, * means P≤0.05, ** means P≤0.01, ** means P≤0.001, and ns means P>0.05. DETAILED DESCRIPTION

[0035] The present invention provides an exosome cryoprotectant, comprising components with the following concentrations: 25 mM Hanks' balanced salt solution, 10-40 mM trehalose, 4 mM glutathione, 1 mM vitamin E, 1-20 g / L human serum albumin, 1 mM proline, 1 mM glycine, 100-300 g / L glycerol and 10-40 g / L hyaluronic acid.

[0036] Unless otherwise specified, the present invention has no special requirements on the sources of the components in the exosome cryoprotectant, and commercially available products known to those skilled in the art may be used.

[0037] As an embodiment, the concentration of trehalose in the exosome cryoprotectant is 15-30 mM; as another embodiment, the concentration of trehalose in the exosome cryoprotectant is 20-25 mM; as another embodiment, the concentration of trehalose in the exosome cryoprotectant is 25 mM. The present invention can effectively maintain the integrity of exosome vesicles by compounding trehalose at an appropriate concentration.

[0038] As an embodiment, the concentration of human albumin in the exosome cryoprotectant is 2-10 g / L; as another embodiment, the concentration of human albumin in the exosome cryoprotectant is 2-5 g / L; as another embodiment, the concentration of human albumin in the exosome cryoprotectant is 2 g / L. The present invention can maintain the degree of exosome dispersion and reduce fusion by compounding human albumin at an appropriate concentration.

[0039] As an embodiment, the concentration of glycerol in the exosome cryoprotectant is 100-200 g / L; as another embodiment, the concentration of glycerol in the exosome cryoprotectant is 100-150 g / L; as another embodiment, the concentration of glycerol in the exosome cryoprotectant is 100 g / L. The present invention can reduce the damage of low temperature to exosomes by compounding glycerol with an appropriate concentration.

[0040] As an embodiment, the concentration of hyaluronic acid in the exosome cryoprotectant is 15-30 g / L; as another embodiment, the concentration of hyaluronic acid in the exosome cryoprotectant is 20-25 g / L; as another embodiment, the concentration of hyaluronic acid in the exosome cryoprotectant is 20 g / L. As an embodiment, the molecular weight of the hyaluronic acid is less than 10 kDa. The present invention can provide strong moisture and continuous hydration by compounding hyaluronic acid of an appropriate concentration, maintain the hydration force of the protective liquid, make the protective agent more stable, and facilitate long-term storage.

[0041] The present invention can achieve the effects of anti-oxidation and maintaining the components of RNA in exosome vesicles by compounding glutathione, vitamin E, proline and glycine at appropriate concentrations.

[0042] As an embodiment, the solvent of the exosome cryoprotectant is PBS buffer.

[0043] The exosome cryoprotectant obtained by compounding appropriate components of the present invention can effectively maintain the activity of exosomes under low-temperature storage conditions, and the composition of exosomes and cryoprotectants can be stably stored at 4°C, -20°C and -80°C. In addition, the exosome cryoprotectant provided by the present invention effectively delays the degradation of exosomes at 4°C, thereby realizing long-term storage of exosomes at 4°C.

[0044] Based on the above advantages, the present invention provides the use of the exosome cryoprotectant described in the above technical solution in the storage and / or transportation of exosomes.

[0045] As an embodiment, the storage and / or transportation temperature is -80°C to 4°C. As another embodiment, the storage and / or transportation temperature is -80°C, -20°C or 4°C.

[0046] As an embodiment, the exosomes are mesenchymal stem cell-derived exosomes.

[0047] As an embodiment, when the exosomes are stored for a long time or transported over long distances, after the exosomes and the exosome cryoprotectant are mixed, the stability of the exosomes can be further improved by using nitrogen protection and strictly controlling the transportation temperature, thereby achieving long-term storage or long-distance transportation of the exosomes at 4°C.

[0048] As an embodiment, the application further includes: after mixing the exosomes and the exosome cryoprotectant, placing them in a container, and sealing them after filling with an inert gas. As another embodiment, the inert gas can be nitrogen. As an embodiment, the container is a light-proof container. As another embodiment, the container can be a light-proof glass bottle. As another embodiment, the container is a container with a Sigmacote (MERCK, SL-2) coating on the inner wall. As another embodiment, the container is a light-proof container with a Sigmacote coating on the inner wall.

[0049] Exosomes are wrapped by lipid bilayer membranes, which contain functional molecules such as proteins and RNA, and these components are easily damaged by external environmental factors. First, lipids and proteins in exosomes are highly sensitive to oxygen, and are easily oxidized when exposed to oxygen, resulting in membrane structure destruction and functional molecule degradation, thereby affecting the stability and biological function of exosomes. The present invention can effectively exclude oxygen, inhibit the occurrence of oxidative stress reaction, and delay the degradation process of exosomes by sealing with nitrogen. In addition, light, especially ultraviolet rays, can trigger photooxidation reactions, destroy nucleic acids, proteins and lipids in exosomes, and cause loss of activity. The present invention further uses opaque glass bottles to effectively block light, especially ultraviolet rays, to prevent the effect of light damage on the activity of exosomes. The present invention further uses Sigmacote coating to provide a hydrophobic silanized protective layer for the inner wall of the glass bottle, significantly reducing the adsorption between the exosomes and the bottle wall. Due to the small particle size of the exosomes and the certain charge on the surface, it is easy to physically adsorb on the inner wall of ordinary glass containers, resulting in a reduced recovery rate and partial loss of exosomes. Sigmacote coating reduces the interaction between exosomes and glass walls and nonspecific adsorption by changing the physicochemical properties of the glass surface, which helps improve the recovery efficiency of exosomes during preservation and ensures the integrity and quantitative accuracy of the samples.

[0050] In order to further illustrate the present invention, an exosome cryoprotectant provided by the present invention and its application in exosome storage are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] A cryoprotectant for exosome storage, the specific composition of which is shown in Table 1.

[0053] Table 1 Formulation of cryoprotectants for exosome storage

[0054]

[0055]

[0056] Example 2

[0057] The cell supernatant of umbilical cord mesenchymal stem cells hUC-MSC-0214 cultured for 14 days under the conditions of 3DFiberCell bioreactor Rooster EVPro was harvested; the umbilical cord mesenchymal stem cells hUC-MSC-0214 are disclosed in Chinese patent CN115074320A; the isolation and amplification methods of the umbilical cord mesenchymal stem cells hUC-MSC-0214 can be found in Chinese patent CN115678842A; the 3DFiberCell bioreactor and its use method can be found in Chinese patent CN115074320A.

[0058] The cell supernatant was passed through a low speed centrifugation step (700×g for 5 minutes) to remove cells and debris, and then centrifuged at 2000×g for 20 minutes to remove larger particles and debris to obtain a pre-cleared cell culture supernatant. The pre-cleared cell culture supernatant was then passed through a 0.22 μm bottle top vacuum filter to remove any larger particles to obtain a low particle cell supernatant.

[0059] The low particle cell supernatant was subjected to TFF filtration (300 kDa membrane packaged tangential flow filtration or hollow fiber tangential flow filter) and then EV Sartorius strong anion EV purification monolithic column combined with elution chromatography purifies EVs to obtain exosomes. For specific methods, please refer to Chinese patent CN115960821A.

[0060] Example 3

[0061] use EV, the EV sample (i.e., exosomes) purified in Example 2 was collected by 280 nm UV absorbance chromatography and concentrated to a final volume of 500 μL (Millipore).

[0062] The concentrated EV samples were resuspended in different preservation solutions (PBS buffer, ExoCS and ExoCap), and the EVs resuspended in each preservation solution were stored at +4°C, -20°C and -80°C, respectively; the ExoCS was the cryoprotectant prepared in Example 1; the ExoCap was a commercial exosome storage solution purchased from MBL International, with the product number MEX-USB.

[0063] After the exosomes were stored in different preservation solutions for 1 week, the nanoparticle size and concentration were detected using a nanoparticle tracking analyzer (NTA). Figure 1 and Figure 2 .

[0064] Depend on Figure 1 It can be seen that in PBS preservation solution, the particle concentration ratio of storage value to fresh value decreased significantly with decreasing temperature: 92%, 70% and 71% at 4℃, -20℃ and -80℃, respectively, indicating that PBS cannot effectively maintain the stability of exosome particles, especially at lower temperatures. In ExoCS preservation solution, the particle concentration of storage value / fresh value was 112%, 115% and 112% at 4℃, -20℃ and -80℃, respectively, which not only remained stable but even increased slightly, indicating that ExoCS can well protect the particle structure of exosomes at various temperatures. Similarly, the particle concentration ratio of ExoCap preservation solution under different temperature conditions also showed extremely high stability, with the storage value / fresh value ratio at 4℃, -20℃ and -80℃ being 114%, 109% and 108%, respectively. These data show that compared with PBS, ExoCS and ExoCap have better preservation effects and can significantly maintain the integrity and concentration of exosome particles under low-temperature storage conditions.

[0065] Depend on Figure 2It can be seen that different preservation solutions show certain differences under different temperature conditions. For PBS preservation solution, the average particle size of exosomes was 95.6 (±3.0) nm, 100.0 (±4.6) nm and 99.3 (±4.2 nm) at 4 ° C, -20 ° C and -80 ° C, respectively, indicating that under low temperature conditions, PBS preservation solution failed to maintain the size consistency of exosomes, especially at -20 ° C, the particle size increased slightly. In ExoCS preservation solution, the average particle size of exosomes was 95.0 (±3.0) nm, 95.3 (±2.1) nm and 94.7 (±3.1) nm at 4 ° C, -20 ° C and -80 ° C, respectively, showing high stability and small changes in particle size, indicating that ExoCS has a good protective effect on exosomes at various temperatures. Similarly, in ExoCap preservation solution, the average particle size of exosomes at 4°C, -20°C and -80°C was 94.2 (±1.7) nm, 97.2 (±4.6) nm and 96.5 (±3.5) nm, respectively, with a small change range, indicating that ExoCap has good temperature stability. In general, ExoCS and ExoCap are better at maintaining the particle size stability of exosomes under different temperature conditions than PBS.

[0066] Example 4

[0067] The method is similar to that of Example 3, except that the same source samples were stored at 4°C, -20°C or -80°C for 6 weeks and the same measurements were performed. Figure 3 and Figure 4 .

[0068] Depend on Figure 3 It can be seen that under different storage solutions and storage temperatures, the particle concentration ratio of exosome storage value to fresh value showed significant differences with changes in temperature and storage solution. In PBS storage solution, the concentration of exosomes was 48%, 24% and 44% at 4°C, -20°C and -80°C, respectively. The exosome concentration decreased significantly at -20°C, indicating that PBS had a poor effect on maintaining the exosome concentration at lower temperatures. In ExoCS storage solution, the exosome concentration was 87%, 72% and 85% at 4°C, -20°C and -80°C, respectively, showing good stability, especially at 4°C and -80°C, where a higher concentration was maintained, indicating that the protective effect of ExoCS on exosomes was relatively stable. In the ExoCap preservation solution, the exosome concentrations were 85%, 68% and 75% at 4°C, -20°C and -80°C, respectively, with little change. A relatively high concentration was also maintained at 4°C and -80°C, further indicating that the stability of ExoCap under low-temperature storage is similar to that of ExoCS, and can effectively maintain the concentration of exosomes.

[0069] Depend on Figure 4It can be seen that after 6 weeks of storage in different preservation solutions (PBS, ExoCS, ExoCap) and temperatures (4°C, -20°C, -80°C), the average particle size of exosomes showed different trends. In PBS preservation solution, the exosome particle size was 108.7 (±4.9) nm, 141.7 (±4.0) nm and 132.6 (±3.1) nm at 4°C, -20°C and -80°C, respectively, indicating that as the storage temperature decreases, the particle size increases. The particle size increase range at -20°C is higher than that at -80°C, which may be due to the fusion of vesicles, indicating that PBS may have poor structural stability for exosomes under low temperature conditions. In ExoCS preservation solution, the particle sizes of exosomes were 127.7 (±5.7) nm, 117.0 (±4.6) nm and 123.3 (±2.5) nm, respectively, showing better temperature stability, and the particle size changes were small at different storage temperatures, indicating that ExoCS can better protect the structural integrity of exosomes. For ExoCap preservation solution, the exosome particle sizes were 123.1 (±11.3) nm, 126.3 (±17.6) nm and 124.7 (±4.7) nm at 4°C, -20°C and -80°C, respectively, and the particle size changes were small, indicating that ExoCap has a good protective effect on the stability of exosome particle size.

[0070] Example 5

[0071] The method is similar to that of Example 3, except that the same source samples were stored at 4°C, -20°C or -80°C for 12 weeks and the same measurements were performed. Figure 5 and Figure 6 .

[0072] Depend on Figure 5 It can be seen that in the PBS preservation solution, the particle concentration ratio of the exosome storage value to the fresh value decreased significantly with the decrease in temperature. Specifically, the concentration was 66% at 4°C, decreased to 33% at -20°C, and further decreased to 27% at -80°C, indicating that PBS has a poor effect on the preservation of exosome concentration under low temperature conditions. In the ExoCS preservation solution, the exosome concentration ratio was relatively stable, with concentrations of 81%, 62% and 67% at 4°C, -20°C and -80°C, respectively, showing a better low-temperature protection effect. Especially at -80°C, ExoCS can effectively maintain the concentration level of exosomes. In the ExoCap preservation solution, the exosome concentration was 75%, 46% and 69% at 4°C, -20°C and -80°C, respectively. Although there was a certain decrease at -20°C, the concentration ratio could still be maintained at 69% at -80°C, indicating that ExoCap also has good stability in ultra-low temperature environments.

[0073] Depend on Figure 6It can be seen that after the exosomes were stored for 12 weeks in different preservation solutions (PBS, ExoCS, ExoCap) and temperatures (4°C, -20°C, -80°C), the average particle size of the exosomes showed different trends. In the PBS preservation solution, the exosome particle size was 106.3 (±2.1) nm when stored at 4°C, increased to 145.3 (±3.8) nm at -20°C, and slightly decreased to 133.0 (±4.4) nm at -80°C. This shows that in the PBS storage solution, the particle size stability of the exosomes in the low temperature environment is poor, especially at -20°C, the particle size increases significantly. In contrast, the ExoCS preservation solution shows better particle size stability. At 4℃, -20℃ and -80℃, the exosome particle sizes were 131.7 (±4.7) nm, 129.3 (±4.1) nm and 127.3 (±1.5) nm, respectively, showing a relatively consistent particle size distribution, especially at -80℃, the particle size fluctuation was minimal, indicating that ExoCS can maintain the particle size stability of exosomes at different storage temperatures. For ExoCap preservation solution, the exosome particle sizes were 120.7 (±14.5) nm, 134.5 (±12.3) nm and 127.8 (±1.6) nm at 4℃, -20℃ and -80℃, respectively. Although the particle size changes greatly at 4℃ and -20℃, the particle size fluctuation is small at -80℃, showing good stability.

[0074] In general, the particle number and particle size of ExoCS under different freezing temperature conditions fluctuate the least, especially when frozen at 4°C for 12 weeks, the concentration and particle size of exosomes can still be maintained, indicating that it has a good stabilizing effect on exosomes. ExoCap can also effectively maintain particle size stability and particle concentration at -80°C, while PBS preservation solution has poor exosome stability under low temperature conditions, especially at -20°C, the particle size increases significantly, and is not suitable as a cryopreservation solution for exosomes.

[0075] In summary, compared with PBS, ExoCS and ExoCap have better effects on maintaining the concentration of exosomes at different temperatures, especially showing stronger stability at -80°C. This shows that ExoCS and ExoCap are more suitable for long-term cryopreservation of exosomes, which helps to maintain their concentration and biological activity.

[0076] Example 6

[0077] The exosome samples were stored in different storage solutions and at different temperatures for 12 weeks using the method of Example 3, and the stability of RNA (miRNA and small RNA) in the exosome samples after storage was detected, and full-length cDNA was amplified from miRNA using TaKaRA cDNA synthesiskits. The details are as follows:

[0078] Total RNA was extracted using the Direct-zol RNA kit (Zymo Research) and quantified using the Quant-iTRiboGreen RNA assay kit (Thermo Fisher Scientific), and RNA species ranging from 5 to 200 bp were measured using the small RNA kit (DNF-470-0275) on an Agilent 2100 Bioanalyzer. RNA integrity was tested on an Agilent 2100 Bioanalyzer using the small RNA and High Sensitivity DNA kits (Agilent Technologies).

[0079] RNA and cDNA concentrations were calculated using Bioanalyzer software. For small RNA, all lengths (5 to 200 bp) were calculated, while for full-length cDNA, all fragments between 400 and 10,000 bp were calculated. To determine the stability of miRNA, the proportion of full-length cDNA was compared with the concentration of DNA fragments <400b. The results are shown in Figure 7 to Figure 9 .

[0080] Depend on Figure 7 and Figure 8 It can be seen that small RNA and miRNA degradation was significantly higher after storage in PBS at all temperatures compared to ExoCS and ExoCap, and small RNA and miRNA degradation in frozen samples (-20 / 80°C) was generally lower than that in exosomes stored at +4°C. When comparing samples stored at the respective temperatures, in all cases, exosomes stored in ExoCS buffer showed the least obvious signs of RNA degradation among the candidate buffers tested.

[0081] Depend on Fig. 9 It can be seen that the analysis of full-length cDNA amplified from the same set of samples generally showed that the absolute amount of RNA detected in frozen exosome samples in ExoCS and ExoCap protection solutions was higher than that in PBS storage solution, regardless of +4°C, -20°C or -80°C. At all temperatures, miRNA was the least stable after storage in PBS, and exosomes in ExoCS protection solution had the highest ratio of full-length cDNA under all storage temperature conditions.

[0082] In summary, ExoCS protective solution can maintain the concentration and particle size of exosomes within 12 weeks under storage conditions of +4℃, -20℃ or -80℃, while avoiding RNA degradation caused by long-term low-temperature storage and ensuring the biological activity of exosomes. It can achieve the same low-temperature storage protection effect of exosomes as the commercial product ExoCap, especially the biological activity of exosomes stored at 4℃ is more stable.

[0083] Example 7

[0084] The present invention used Protein Simple Wes to detect the expression levels of specific marker proteins of exosomes and fresh exosomes after ExoCS protective solution was stored at 4°C for 12 weeks. The results are shown in Fig.10 , where F (Fresh) refers to fresh exosomes, and S (Storage) refers to exosomes after low-temperature storage for 12 weeks.

[0085] Depend on Fig.10 It can be seen that under the conditions of ExoCS preservation solution stored at 4°C for 12 weeks, the exosome-specific positive markers CD9, CD63, CD81, TSG101, and Alix were all expressed, while the negative marker Calnexin was not expressed, proving that after the exosomes were stored at 4°C in ExoCS protective solution for 12 weeks, the exosomes still had complete membrane protein function.

[0086] Example 8

[0087] Transmission electron microscopy was used to detect the structure of exosomes in the exosome samples under the conditions of ExoCS protective solution and different storage temperatures, and at low temperature storage time points of 1 week, 6 weeks, and 12 weeks. Fig.11 .

[0088] Depend on Fig.11 It can be seen that ExoCS protective solution can ensure the structural integrity of exosomes within 12 weeks at +4℃, -20℃ or -80℃.

[0089] After the above samples were reconstituted at room temperature, the bioactivity was tested in the HSF cell migration experiment under the same dose conditions. The results are shown in Fig.12 , where Control is the PBS-treated group.

[0090] Depend on Fig.12 It can be seen that under different storage temperature conditions, exosomes stored at low temperature for 1 week, 6 weeks, and 12 weeks can promote the proliferation and migration of HSF, and exosomes have significant biological activity.

[0091] In summary, ExoCS exosome cryopreservation solution can ensure the concentration, particle size, membrane protein function, structural integrity and biological activity of exosomes at +4℃, -20℃ or -80℃. It can achieve the same level of protection effect as the existing exosome cryopreservation solution ExoCap, especially the exosome protection effect under 4℃ storage condition is better than ExoCap. ExoCS preservation solution can also be used for long-term cryopreservation of exosomes at -120℃.

[0092] Example 9

[0093] The exosomes purified in Example 2 were concentrated to 1×10 10 The particles / bottle were stored in 4 mL ExoCS protective solution and divided into 4 groups, 3 bottles in each group, stored in different containers, namely:

[0094] (1) 5 mL glass vial, air-tight (referred to as Glass Tube);

[0095] (2) 5 mL light-proof glass vial, sealed with nitrogen (referred to as Glass Tube + N2);

[0096] (3) The inner wall of the 5 mL opaque glass bottle was coated with Sigmacote (MERCK, SL-2) and air-tight (denoted as GlassTube+Sigmacote);

[0097] (4) The inner wall of the 5 mL opaque glass bottle is coated with Sigmacote and sealed with nitrogen (referred to as Glass Tube+Sigmacote+N2).

[0098] The exosomes were stored at 4°C for 12 weeks under light conditions, and then the protein in each bottle was quantitatively analyzed using BCA. Fig.13 .

[0099] The results showed that compared with storing exosomes directly in glass containers, both nitrogen sealing and Sigmacote coating reduced protein loss, and combining the two methods could better slow down the degradation and adsorption of exosomes.

[0100] It can be seen that when selecting containers for preserving exosomes, the combined method of nitrogen sealing, light-proof glass bottles and Sigmacote coating can provide an ideal storage environment for exosomes with low oxygen, light avoidance and low adsorption, thereby maximizing the protection of their structural integrity and biological activity.

[0101] In summary, the combined application of nitrogen sealing, light-proof glass bottles and Sigmacote coating can effectively reduce oxidative damage, photodamage and adsorption loss, provide a stable, low-interference storage environment for exosomes, significantly extend the shelf life of exosomes, ensure their biological function and structural integrity, and meet the strict requirements of scientific research and clinical applications for high-quality exosomes.

[0102] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An exosome cryoprotectant, characterized in that: The invention comprises components with the following concentrations: 25 mM Hanks' balanced salt solution, 10-40 mM trehalose, 4 mM glutathione, 1 mM vitamin E, 1-20 g / L human serum albumin, 1 mM proline, 1 mM glycine, 100-300 g / L glycerol and 10-40 g / L hyaluronic acid.

2. The exosome cryoprotectant according to claim 1, characterized in that The exosome cryoprotectant includes components with the following concentrations: 25 mM Hanks' balanced salt solution, 15-30 mM trehalose, 4 mM glutathione, 1 mM vitamin E, 2-10 g / L human albumin, 1 mM proline, 1 mM glycine, 100-200 g / L glycerol and 15-30 g / L hyaluronic acid.

3. The exosome cryoprotectant according to claim 1 or 2, characterized in that The molecular weight of the hyaluronic acid is less than 10 kDa.

4. The exosome cryoprotectant according to claim 1 or 2, characterized in that The solvent of the exosome cryoprotectant is PBS buffer.

5. Use of the exosome cryoprotectant according to any one of claims 1 to 4 in the storage and / or transportation of exosomes.

6. The use according to claim 5, characterized in that: The storage and / or transportation temperature is -80°C to 4°C.

7. The use according to claim 5 or 6, characterized in that: The exosomes are exosomes derived from mesenchymal stem cells.

8. The use according to claim 5, characterized in that: The application also includes: mixing the exosomes and the exosome cryoprotectant, placing the mixture in a container, and sealing the container after filling it with an inert gas.

9. The use according to claim 8, characterized in that: The container is a light-proof container.

10. The use according to claim 8 or 9, characterized in that: The container is a container with a chlorinated organic polysiloxane coating on the inner wall.

Images