Storage liquid for maintaining activity of apoptotic body and preparation and storage method of storage liquid

Abstract

The invention belongs to the technical field of biology, and particularly relates to storage liquid for maintaining the activity of apoptotic bodies and a preparation and storage method of the storage liquid. The storage solution is prepared from human serum albumin, trehalose, glycerol, dimethyl sulfoxide, ectoine, vitamin C, histidine, glucose, arginine hydrochloride, proline and sodium chloride. By adding the storage liquid for maintaining the activity of the apoptotic bodies into the apoptotic bodies, the activity of the stored apoptotic bodies can be prolonged to seven days or even longer. The storage solution can be used as a cryopreservation solution as well as a freeze-drying protective agent, and is wide in applicable temperature range including-80 DEG C to 4 DEG C. The storage solution can reduce the storage and transportation cost of apoptotic bodies and expand the application range of the apoptotic bodies. By using the storage liquid, the stability of the apoptotic bodies in the storage process can be improved, the integrity effect of the apoptotic bodies can be kept, and industrial application of the apoptotic bodies is greatly facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a storage solution for maintaining the activity of apoptotic bodies and a configuration and storage method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have great therapeutic potential in regulating immune responses and reducing inflammation because they can differentiate into different cell types. However, the direct use of MSCs has the risks of difficulty in proliferation at the site of injury, immune rejection, and potential tumorigenicity, which limits their clinical translational application. The cell-free therapy based on stem cell technology has provided a new therapeutic strategy in the field of regenerative medicine. Among them, apoptosis is a form of programmed cell death that is essential for growth and tissue homeostasis. Apoptotic bodies (ABs) are extracellular vesicles released by dying cells in the final stage of apoptosis. The latest progress of apoptotic bodies has elucidated the potential of immune regulation, virus clearance, vascular protection, tissue regeneration, and disease diagnosis of apoptotic bodies. In addition, apoptotic bodies can be used as drug carriers to enhance drug stability, cellular uptake, and targeted therapeutic efficacy. Apoptotic bodies have broad potential in the diagnosis, prognosis, and treatment of a variety of diseases, including cancer, systemic inflammatory diseases, cardiovascular diseases, and tissue regeneration.

[0003] The extraction process of apoptotic bodies of mesenchymal stem cells is complicated and not easy to preserve. The conventional use and storage method is to place the apoptotic bodies in PBS phosphate buffer at 2~8℃ and freeze at -80℃. Although PBS phosphate buffer can provide it with osmotic pressure and pH value similar to human body fluids, it still has certain limitations in maintaining the stability of apoptotic bodies. The activity of apoptotic bodies can only be maintained for three days or even shorter. First of all, whether it is refrigerated, frozen or freeze-dried PBS, it cannot provide more efficacy to prevent the aggregation of apoptotic bodies, changes in biological characteristics, markers, and reduction in content and activity. This makes the apoptotic bodies have a short storage time, high storage and transportation costs, and a narrow range of applications. Secondly, the current progress of apoptotic bodies is mainly in the early research stage. Even if a few products have entered the early clinical stage, they are mainly used for disease diagnosis, prognosis monitoring, etc. If a safe, effective, stable, and suitable formula for apoptotic bodies can be developed, it can be used for the early development of apoptotic bodies and can also be used in preparations for clinical use. Therefore, there is an urgent need for a preparation that can prolong the activity of apoptotic bodies. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of ectoine and the like in the preservation of apoptotic bodies.

[0005] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a storage solution for maintaining the activity of apoptotic bodies comprises human serum albumin, trehalose, glycerol, dimethyl sulfoxide, ectoine, vitamin C, histidine, glucose, arginine hydrochloride, proline, and sodium chloride.

[0006] Preferably, the storage solution is composed of the following components, and the concentration of each component is 1~10 g / L human serum albumin, 10~50 g / L trehalose, 30%~50% (v / v) glycerol, 5%~10% (v / v) dimethyl sulfoxide, 0.1~100 g / L tetrahydropyrimidine, 0.1~1.0 g / L vitamin C, 0.1~1.0 g / L histidine, 1~5 g / L glucose, 5~20 g / L arginine hydrochloride, 5~25 g / L proline, and 0.1~1.5 g / L sodium chloride.

[0007] Preferably, the storage solution is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0008] The second aspect of the present invention is a method for preparing a storage solution for maintaining the activity of apoptotic bodies, characterized in that it includes the following steps: dissolving the components of the storage solution in physiological saline by shaking according to concentration requirements, filtering the components, and mixing the filtered components to obtain a storage solution for maintaining the activity of apoptotic bodies.

[0009] Preferably, the filtration uses a filter with a pore size of 0.2-0.3 μm.

[0010] Preferably, the pH value of the storage solution is 6.5-7.5.

[0011] Preferably, the storage solution is used at a temperature of -80°C to 4°C.

[0012] In a third aspect of the present invention, a method for storing apoptotic bodies comprises the following steps: extracting apoptotic bodies, adding the apoptotic bodies to the storage solution, mixing, and obtaining a preserved sample; adding the preserved sample to a cryopreservation tube and placing it in a programmed cooling box, and storing it in a -80°C refrigerator for later use.

[0013] Preferably, the volume ratio of the apoptotic bodies to the storage solution is 1:30-100.

[0014] The apoptotic bodies described above are apoptotic bodies of deciduous dental pulp stem cells (stem cells from human exfoliated deciduous teeth, SHEDs).

[0015] In the present invention, the storage solution includes a cryoprotectant, an excipient, an antioxidant, a pH regulator, a stabilizer and an osmotic pressure regulator according to their functions.

[0016] The reason why cryoprotectants can maintain the stability of apoptotic bodies is that, in aqueous solution, water molecules interact with certain polar groups on the surface of apoptotic bodies, causing the apoptotic bodies to be preferentially hydrated. In the initial stage of the freezing process, cryoprotectants are selectively excluded from the vicinity of apoptotic bodies. This exclusion of solute molecules increases the free energy of unfolding, which is conducive to maintaining a stable natural state. Second, the presence of cryoprotectants leads to an increase in the viscosity of the frozen concentrate, which reduces the mobility of the molecules and slows down all dynamic processes. If external conditions are not conducive to the stability of apoptotic bodies, the cryoprotectants bound to the protein surface are more likely to be removed first, which is conducive to the maintenance of the natural protein conformation.

[0017] Human serum albumin, glycerol, dimethyl sulfoxide, and ectoine are used as cryoprotectants in the present invention. In body fluids, human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules, and can maintain normal osmotic pressure of blood. Clinically, human serum albumin can be used to treat shock and burns, to supplement blood loss caused by surgery, accidents, or massive bleeding, and can also be used as a plasma volume expander. In the present invention, human serum albumin increases the protein concentration in the apoptotic body preservation solution system to prevent proteases from hydrolyzing apoptotic body membrane proteins. Glycerol makes the preservation solution antifreeze and will not freeze at -20°C, preventing structural damage to apoptotic bodies caused by freezing and thawing during storage and use. Dimethyl sulfoxide can be used as an antifreeze when preserving cells at deep low temperatures (-196°C). It can quickly penetrate the membrane of apoptotic bodies and enter the apoptotic bodies, lowering the freezing point and further preventing the preservation solution from freezing. At the same time, it increases the ion concentration in the membrane of apoptotic bodies and reduces the formation of ice crystals in cells, thereby protecting the stability of the apoptotic body structure and its contents. It is currently a widely used cell cryoprotectant. Tetrahydropyrimidine can help cells resist freezing conditions and improve the protective effect of cryopreservation of cells, tissues, and organs. When used in combination with dimethyl sulfoxide, its dosage can be reduced, its toxicity to cells can be reduced, and the cryopreservation effect can be improved.

[0018] Trehalose is used as a carbohydrate excipient in the present invention, which has a magical protective effect on organisms, because trehalose can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration, effectively protecting protein molecules from denaturation and inactivation, thereby maintaining the life process and biological characteristics of living organisms.

[0019] Vitamin C has the effects of anti-oxidation and anti-free radicals. The present invention adopts vitamin C as an antioxidant.

[0020] During formulation of frozen or lyophilized products, pH changes due to buffer crystallization must be considered. Histidine is alkaline in aqueous solution and can be used as a pH adjuster. In addition, histidine can bind to compounds such as Cu 2+ and Fe 2+ The ions such as ions that easily catalyze the oxidative modification of antibody proteins, so histidine can also act as an antioxidant. In the present invention, histidine is selected as a pH regulator.

[0021] Glucose is used to replenish the energy requirements of apoptotic bodies.

[0022] In the present invention, the storage solution also includes amino acids, which can stabilize apoptotic bodies by interacting with proteins on apoptotic bodies or by hydration. The specific situation needs to be determined according to the type of amino acids and the characteristics of the apoptotic bodies themselves. Amino acids are usually used together with sucrose or trehalose, and are rarely used alone in freeze-dried formulations. Existing literature reports that the stabilizing effect of amino acids is lower than that of traditional sugar cryoprotectants, and a large number of literature reports that the addition of amino acids can enhance the stabilizing effect of sugars, thereby making them suitable secondary stabilizers in the formulation; therefore, the present invention uses proline and arginine hydrochloride as secondary stabilizers.

[0023] The apoptotic bodies maintain a certain osmotic pressure during the storage process, which is not only necessary for the survival of the apoptotic bodies, but also related to maintaining the water balance inside and outside the apoptotic bodies. During the formulation design of freeze-dried products, the change of water inside the preparation product caused by the change of external temperature, which causes the sudden increase of the osmotic pressure inside the apoptotic bodies and affects its activity, must be considered. Sodium chloride is one of the commonly used osmotic pressure regulators. Studies have shown that glycerol can regulate the osmotic pressure inside and outside cells. Therefore, the present invention uses sodium chloride and glycerol together as osmotic pressure regulators.

[0024] The beneficial effects of the present invention are as follows: by adding a storage solution for maintaining the activity of apoptotic bodies in the present invention to apoptotic bodies, the activity of apoptotic bodies can be extended to seven days or even longer after preservation. The storage solution can be used as both a freezing solution and a freeze-drying protective agent, and has a wide applicable temperature range, including -80°C to 4°C. The storage solution can reduce the storage and transportation costs of apoptotic bodies and expand the scope of application of apoptotic bodies. The use of the above storage solution can improve the stability of apoptotic bodies during storage and maintain the integrity of apoptotic bodies, which greatly facilitates the industrial application of apoptotic bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0026] Figure 1 The bar graphs of cell survival rates on the 3rd and 7th days obtained by CCK-8 detection in the present invention (in the figure, (a) is apoptotic bodies stored at 4°C, and (b) is apoptotic bodies stored at -80°C). DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1 This example is a storage solution for maintaining the activity of apoptotic bodies of the present invention, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0029] Comparative Example 1 This comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L tetrahydropyrimidine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0030] Comparative Example 2 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0031] Comparative Example 3 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0032] Comparative Example 4 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0033] Comparative Example 5 This comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0034] Comparative Example 6 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0035] Comparative Example 7 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0036] Comparative Example 8 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

[0037] Comparative Example 9 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L proline, and 0.5 g / L sodium chloride.

[0038] Comparative Example 10 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, and 0.5 g / L sodium chloride.

[0039] Comparative Example 11 The present comparative example provides a storage solution for maintaining the activity of apoptotic bodies, which is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, and 5 g / L proline.

[0040] Experimental Example 1 Method for extracting apoptotic bodies from stem cells from human exfoliated deciduous teeth (SHEDs): 1) Culture cells: The ex vivo deciduous teeth were stored in PBS containing 300 U / mL penicillin and 300 U / mL streptomycin (3% double antibody). The ex vivo teeth were rinsed with PBS containing 3% double antibody, the surface soft tissue was scraped, the pulp was removed with a pulp needle, and the apical 1 / 3 of the pulp and the congested pulp were cut off. After the pulp tissue was rinsed with PBS containing 3% double antibody for 5 times, it was transferred to an EP tube containing collagenase (collagenase I and collagenase II were mixed at a ratio of 1:1, 1 mg / mL) and cut into pieces with sterile scissors. After mixing, it was digested in a 37°C constant temperature incubator for about 10 minutes, and then the digestion was terminated with an equal volume of culture medium containing 10% FBS in α-MEM. The centrifuge program was set to 1500 rpm and centrifuged for 5 minutes. After centrifugation, discard the supernatant, resuspend the precipitate with a small amount of culture medium containing 20% ​​FBS in α-MEM, transfer it to a culture dish, and after the tissue blocks adhere to the wall, add an appropriate amount of culture medium containing 20% ​​FBS in α-MEM. Place it in an incubator with 5% CO2, 37°C, and saturated humidity. After 3-7 days, the cells will crawl out. When the cell density reached 90%, discard the old culture medium, rinse the cells twice with PBS, add an appropriate amount of 0.25% trypsin (containing EDTA) to digest for about 2 min, observe the cells shrink and round under a microscope, add 2 times the volume of trypsin containing 10% FBS culture medium to terminate digestion, gently blow the attached cells that have not yet fallen off to make them fall off, transfer the cell suspension to a centrifuge tube, set the centrifuge program to 1000 rpm, centrifuge for 3 min, discard the supernatant, retain the cell pellet, add α-MEM culture medium containing 10% FBS, 100 U / mL penicillin and 100 U / mL streptomycin to resuspend the cell pellet and blow and mix, and aspirate the cell suspension into a culture dish and pass it at a ratio of 1:3. Select 3rd to 5th generation SHEDs with good growth status for the experiment.

[0041] The pulp can also be removed under sterile conditions, and about 2 mm of the pulp tissue at the apex of the tooth is removed and cut into pieces. Type I collagenase (3 mg / ml) is used to digest the pulp at 37°C for 10 min, and complete culture medium is added to terminate the digestion. The suspension is centrifuged and the supernatant is discarded. A small amount of complete culture medium is added to prepare a suspension and inoculated into a 6-well plate. A cover glass is pressed on the pulp tissue, and an appropriate amount of culture medium is added. The suspension is placed in a constant temperature incubator at 37°C and 5% CO2 for culture. The medium is changed once after 24 hours, and then every 3 days until the cells in the culture flask are fused into a monolayer (70%~80% confluence rate), and then 0.25% trypsin is added to digest at room temperature for 3~5 min, which is the primary culture of SHEDs. Add α-MEM culture medium containing 10% FBS or 20% FBS high-glucose DMEM cell culture medium to completely eliminate the effect of 0.25% trypsin, centrifuge, and discard the supernatant; continue to add α-MEM culture medium containing 10% FBS or 20% FBS high-glucose DMEM cell culture medium to the precipitated cells at a ratio of 1:2 or 1:3 to perform subculture of SHEDs, and the 3rd to 5th generation cells can be used for subsequent experiments.

[0042] 2) Inducing cell apoptosis: SHEDs were treated with α-MEM basal medium containing 0.1 μM staurosporine (STS) for 12 h to induce apoptosis.

[0043] 3) Collect the cell culture supernatant and centrifuge the supernatant: After collecting the supernatant, use differential centrifugation to obtain SHED-ABs. Centrifuge at 800 G for 10 min, then at 2000 G for 10 min at 4°C to remove cells and cell debris; collect the supernatant, centrifuge at 16000 G for 30 min at 4°C, and the resulting precipitate is the extracellular vesicles of ABs size. Gently rinse with PBS and repeat the above centrifugation steps to obtain the precipitate, and resuspend the precipitate in 100 µL PBS.

[0044] Example 2 This embodiment provides a method for cryopreservation of apoptotic bodies of deciduous tooth dental pulp stem cells, comprising the following steps: The method described in Experimental Example 1 was used to obtain apoptotic bodies of deciduous tooth pulp stem cells, and the apoptotic bodies of deciduous tooth pulp stem cells were added to a storage solution for maintaining the activity of apoptotic bodies described in Example 1, mixed well, and a preserved sample was obtained; the preserved sample was added to a cryopreservation tube and placed in a programmed cooling box, and stored in a -80°C refrigerator for later use.

[0045] Experimental Example 2 Investigation of the types and dosage of components in the storage solution The method described in Experimental Example 1 was used to obtain apoptotic bodies of deciduous dental pulp stem cells. Freshly extracted apoptotic bodies of deciduous dental pulp stem cells were used as Group A. The apoptotic bodies of deciduous dental pulp stem cells were added to a storage solution for maintaining the activity of apoptotic bodies described in Example 1 and Comparative Examples 1 to 11, respectively. After freezing and reviving by the method in Example 2, the revived apoptotic bodies were obtained as Groups B to M. Cell culture solutions were added to Groups A to M, respectively, and cells were cultured. The cells were cultured to about 60%, and UVA was used for irradiation for 5 minutes, and then the cells were cultured. Cells were counted after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days of culture. The results are shown in Table 1. It was found that the components of the reserve solution provided in the embodiment of the present invention were all effective ingredients. The apoptotic bodies were cryopreserved using the storage solution and storage method provided in the embodiment of the present invention. After preservation, the structure and activity of the apoptotic bodies can reach seven days or even longer, and the preservation rate is above 95%, which provides convenience for the industrial application of apoptotic bodies.

[0046] Table 1 Effects of different storage solutions for maintaining apoptotic body activity on the number of apoptotic bodies over time Experimental Example 3 Optimal temperature verification CCK-8 assay: PC12 cells were selected with a quantitative concentration of 2.0×10 3 The cells were inoculated into a 96-well cell culture plate, culture medium was added, and the cells were placed in a 37°C, 5% CO2 incubator for culture. After 12 h, the SHED-ABs stored at 4°C and -80°C in the storage solution described in Example 1 of the present invention were added and cultured for 12 h. 100 μL of the prepared CCK-8 solution (10% CCK-8 + culture medium) was added to each well, and the cells were placed in a cell culture incubator for 1 h. The absorbance value (OD value) at 450 nm was observed on a microplate reader. Figure 1 As shown, by comparison, it can be found that the activity preservation rate of apoptotic bodies (SHED-ABs) is better when frozen at -80°C.

[0047] Example 4 This example is another storage solution for maintaining the activity of apoptotic bodies of the present invention. A polymer composed of vinyl pyrrolidone and vinyl acetate in a composition ratio of 1:0.4 is added to any one of Comparative Examples 1-11 of the present invention. The concentration of the vinyl pyrrolidone and vinyl acetate is 10% (w / v), and the K value of the polymer is 20, and the weight average molecular weight is 5000. After the polymer described in this example is added to Comparative Examples 1-11 of the present invention, culture and cell counting are performed according to the experimental method of Experimental Example 2. The results are shown in Table 2. It is found that the preservation rate of apoptotic bodies in each group is increased, proving that the storage solution described in this example can stably preserve the activity of apoptotic bodies.

[0048] Table 2 Effects of storage solutions for maintaining apoptotic body activity on the number of apoptotic bodies over time after adding polymers The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A storage solution for maintaining the activity of apoptotic bodies, characterized in that: The storage solution comprises human serum albumin, trehalose, glycerol, dimethyl sulfoxide, ectoine, vitamin C, histidine, glucose, arginine hydrochloride, proline and sodium chloride.

2. A storage solution for maintaining the activity of apoptotic bodies according to claim 1, characterized in that: The storage solution is composed of the following components, and the concentration of each component is 1-10 g / L human serum albumin, 10-50 g / L trehalose, 30%-50% (v / v) glycerol, 5%-10% (v / v) dimethyl sulfoxide, 0.1-100 g / L tetrahydropyrimidine, 0.1-1.0 g / L vitamin C, 0.1-1.0 g / L histidine, 1-5 g / L glucose, 5-20 g / L arginine hydrochloride, 5-25 g / L proline, and 0.1-1.5 g / L sodium chloride.

3. A storage solution for maintaining the activity of apoptotic bodies according to claim 2, characterized in that: The storage solution is composed of the following components, and the concentration of each component is 5 g / L human serum albumin, 20 g / L trehalose, 30% (v / v) glycerol, 5% (v / v) dimethyl sulfoxide, 0.1 g / L ectoine, 0.5 g / L vitamin C, 0.5 g / L histidine, 1 g / L glucose, 5 g / L arginine hydrochloride, 5 g / L proline, and 0.5 g / L sodium chloride.

4. A method for preparing a storage solution for maintaining the activity of apoptotic bodies as claimed in any one of claims 1 to 3, characterized in that The following steps are involved: The components of the storage solution are dissolved in physiological saline by shaking according to the concentration requirements, and the components are filtered. The filtered components are mixed to prepare a storage solution for maintaining the activity of apoptotic bodies.

5. The method for preparing a storage solution for maintaining the activity of apoptotic bodies according to claim 4, characterized in that: The filtration is performed using a filter with a pore size of 0.2-0.3 μm.

6. A storage solution for maintaining the activity of apoptotic bodies according to claim 1, characterized in that: The pH value of the storage solution is 6.5-7.

5.

7. A storage solution for maintaining the activity of apoptotic bodies according to claim 1, characterized in that: The storage solution is used at a temperature of -80°C to 4°C.

8. A method for storing a storage solution for maintaining the activity of apoptotic bodies as claimed in any one of claims 1 to 3, characterized in that The following steps are involved: The apoptotic bodies are extracted by adding the apoptotic bodies to the storage solution and mixing to obtain a preserved sample; the preserved sample is added to a cryopreservation tube and placed in a programmed cooling box and stored in a -80°C refrigerator for later use.

9. The method for storing a storage solution for maintaining the activity of apoptotic bodies according to claim 8, characterized in that: The volume ratio of the apoptotic bodies to the storage solution is 1:30-100.

10. The method for storing a storage solution for maintaining the activity of apoptotic bodies according to claim 8, characterized in that: The apoptotic bodies are apoptotic bodies of deciduous tooth pulp stem cells.

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