Cell preserving fluid and application thereof in exosome storage
By using a specific formulation of cell preservation solution, the limitations and high costs of exosome preservation equipment have been overcome, enabling efficient preservation of exosomes at low temperatures, reducing equipment requirements and transportation costs, while maintaining the integrity of the vesicle structure.
Patent Information
- Application Number
- CN202511370169.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2026-01-02
AI Technical Summary
Existing methods for preserving exosomes require ultra-low temperature freezing, which involves limited specialized equipment and high power consumption. Traditional methods pose risks of contamination and are costly, while freeze-drying is expensive and damages the vesicle structure after reconstitution.
The cell preservation solution used was a mixture of serum-free mesenchymal stem cell culture medium, polyethylene glycol 400 and dextran in a specific ratio, with the addition of PBS buffer and dimethyl sulfoxide for the preservation of exosomes. The volume ratio was 1:5~10, and the mixture was kept at pH 6.5-7.0.
This technology enables the preservation of exosomes at normal low temperatures, reducing equipment requirements and transportation costs, minimizing the risk of contamination during freeze-thaw cycles, and maintaining the integrity of the vesicle structure.
Smart Images
Figure CN121242018A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological storage, and particularly relates to a cell preservation solution and application thereof in various exosome storage. BACKGROUND
[0002] Human stem cells are cells from embryos, fetuses or adults that have the ability of unlimited self-renewal and proliferation under certain conditions, can produce daughter cells with the same phenotype and genotype, and can also produce specialized cells constituting tissues and organs of the body, and can also differentiate into progenitor cells.
[0003] Mesenchymal stem cells are a kind of multipotent stem cells, which have all the common properties of stem cells, i.e. self-renewal and multiple differentiation ability.
[0004] Exosomes are membrane outer bilayer lipid vesicles secreted by cells, with a diameter of about 30-200 nm, containing proteins, liposomes, polysaccharides and RNA, etc. They can transport nucleic acids, lipids and proteins, and participate in intercellular information exchange.
[0005] In recent years, with the increasing in-depth research of various new technologies on Chinese herbal medicines, exosomes of Chinese herbal medicines have gradually become a new direction and a new hotspot of research, including exosome research on honeysuckle, ganoderma, etc.
[0006] In the prior art, various exosomes need to be stored at ultra-low temperature (below -80℃) to ensure that their biological activity is not reduced and their vesicle structure is relatively complete. However, special medical-grade ultra-low temperature freezers have limited volume and high power consumption, and are not easy to be widely promoted. The traditional method is to use centrifugation to remove the refrigerated liquid and then use the frozen liquid again. This method increases the risk of contamination of exosomes, and the whole process cost is high. There are also reports of exosome solution freeze-drying production process, but the corresponding cost of production and packaging is high, and the reconstitution of freeze-dried powder has a destructive effect on the vesicles. SUMMARY
[0007] The purpose of the present application is to overcome the above-mentioned problems of inconvenient exosome preservation and high cost, and to provide a cell preservation solution which can significantly maintain the cell activity during cold storage or freezing stage.
[0008] The second aspect of the present application is to provide a preparation method of the above-mentioned cell preservation solution.
[0009] The third aspect of the present application is to provide the application of the above-mentioned cell preservation solution in exosome storage.
[0010] The purpose of the present application is achieved by the following way: A cell preservation solution, comprising a mesenchymal stem cell serum-free culture medium, polyethylene glycol 400 and dextran, three The mass ratio of the three is 3-4:1-2:1-2.
[0011] Preferably, the mass ratio of the three is 3:1:1.
[0012] Preferably, the dextran is 400,000 units.
[0013] Preferably, the medium is mesenchymal stem cell serum-free medium NC0103.
[0014] The preparation method of the above-mentioned cell preservation solution comprises the following steps: The mesenchymal stem cell serum-free medium, polyethylene glycol 400 and dextran are mixed according to a mass ratio of 3-4:1-2:1-2, then PBS buffer solution is added to a concentration of 20-50 mmol / L, the pH is maintained at 6.5-7.0, and 10% dimethyl sulfoxide of the total volume of the mixed solution is added, and the mixture is fully vortexed and mixed to obtain the cell preservation solution.
[0015] The above-mentioned cell preservation solution can be applied in exosome storage.
[0016] When applied, the cell preservation solution exosome solution is mixed at a volume ratio of 1:5-10.
[0017] Preferably, the particle size of the exosome solution is 30-300 nm, which accounts for more than 85%, and the particle concentration is not less than 2*10 10 / mL.
[0018] Preferably, the cell preservation solution and the above-mentioned exosome solution are mixed at a volume ratio of 1:5.
[0019] Preferably, the exosome is a stem cell exosome or a plant exosome.
[0020] The beneficial effects of the present application compared with the prior art are: The various components of the cell preservation solution provided by the present application jointly act on the cell activity in the cold storage or freezing stage, and have a significant maintenance effect. The preservation solution of the present application can be simply converted from an ultralow-temperature storage state to a general low-temperature storage state, which reduces the risk of quality decline of the exosome caused by repeated freezing and thawing, and reduces the cost of production, storage and transportation related logistics, compared with the traditional method of removing the cold storage solution by centrifugation and then using the freezing solution again. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 The electron microscope image of the human stem cell exosome to which the cell preservation solution of Example 1 is added and which is stored at-4℃ for 90 days.
[0022] Figure 2The electron microscope image of ganoderma lucidum exosome with cell preservation solution of Example 1 stored at-4℃ for 90 days. DETAILED DESCRIPTION
[0023] The application is further explained by specific examples below.
[0024] In the following examples, mesenchymal stem cell serum-free medium NC0103 was purchased from Beijing Yookang Biological Technology Co., Ltd.; polyethylene glycol 400 (injection grade) was purchased from Nanjing Well Pharmaceutical Co., Ltd.; dextran (400,000 units) was purchased from Nanjing Taixin Biological Technology Co., Ltd. Example 1
[0025] The mesenchymal stem cell serum-free medium NC0103, polyethylene glycol 400 (injection grade), and dextran (400,000 units) were mixed in a mass ratio of 3:1:1, then PBS buffer was added to a concentration of 20 mmol / L, the pH was maintained at 6.5-7.0, and 10% dimethyl sulfoxide was added to the total volume of the mixed solution, and then vortexed and mixed evenly. Example 2
[0026] The mesenchymal stem cell serum-free medium NC0103, polyethylene glycol 400 (injection grade), and dextran (400,000 units) were mixed in a mass ratio of 4:2:2, then PBS buffer was added to a concentration of 50 mmol / L, the pH was maintained at 6.5-7.0, and 10% dimethyl sulfoxide was added to the total volume of the mixed solution, and then vortexed and mixed evenly. Test Example 1
[0027] 1. Effect on particle size and concentration The cell preservation solution of Example 1 was mixed with human mesenchymal stem cell exosomes (provided by Nanjing Zhongke Pharmaceutical Co., Ltd., with a particle size of 30-200 nm accounting for more than 85%, and a particle size concentration of more than 2×10 10 / mL) listed in Table 1 in a volume ratio of 1:5, and stored at-4℃ for 90 days. The particle size and concentration of the sample stored at-80℃ for 90 days were compared, and the results are shown in Table 1.
[0028]
[0029] 2. Effect on morphology From Figure 1 and Figure 2 The electron microscope images showed that the exosome vesicles were still complete and the edges were clear after being stored at-4℃ for 90 days with the cell preservation solution of Example 1.
[0030] Figure 2The preparation method of Ganoderma exosomes shown is as follows: 1. Clean, disinfect, and chop fresh Ganoderma to obtain pieces smaller than 1 cm. 3 Ganoderma lucidum blocks were soaked in PBS for 6 hours, then cut, ground, and juiced. The resulting filtrate was coarsely filtered through three layers of sterile gauze. The coarse filtrate was transferred to a sterile centrifuge tube for centrifugation at 1000 rpm for 20 minutes to remove impurities. The supernatant was then enzymatically hydrolyzed with a compound enzyme preparation containing 20% pectinase, 40% cellulase, 10% β-1,3-glucanase, and 30% ligninase at a ratio of 0.1-0.3% to obtain the enzymatic hydrolysate. The hydrolysate was then frozen, centrifuged, and filtered through a 100-mesh sieve to remove impurities. The filtrate was concentrated to obtain Ganoderma lucidum exosomes with a filtration diameter of 30-200 nm. The obtained Ganoderma lucidum exosomes were then sterilized by irradiation.
[0031] Impact on safety indicators Simultaneously, safety indicators of human mesenchymal stem cell exosomes T / CRHA001-2021 group standard - small extracellular vesicles derived from human mesenchymal stem cells were investigated after storage in the cell preservation solution at -4℃ for 90 days. The test results showed that endotoxin was <0.5 EU / ml, and the results for fungi, bacteria, and mycoplasma were negative, all meeting the requirements, indicating that the preservation solution was not contaminated at -4℃.
Claims
1. A cell preservation solution, characterized in that, The medium includes serum-free culture medium for mesenchymal stem cells, polyethylene glycol 400, and dextran, with a mass ratio of 3-4:1-2:1-2.
2. The cell preservation solution according to claim 1, characterized in that... The serum-free culture medium for mesenchymal stem cells, polyethylene glycol 400, and dextran were used in a mass ratio of 3:1:
1.
3. The cell preservation solution according to claim 1, characterized in that... The amount of dextran mentioned is 400,000 units.
4. The cell preservation solution according to claim 1, characterized in that... The culture medium is serum-free mesenchymal stem cell culture medium NC0103.
5. A method for preparing the cell preservation solution according to claim 1, characterized in that... Includes the following steps: Mix serum-free culture medium for mesenchymal stem cells, polyethylene glycol 400, and dextran at a mass ratio of 3-4:1-2:1-2. Then add PBS buffer to a concentration of 20-50 mmol / L, maintaining the pH at 6.5-7.
0. Finally, add 10% of the total volume of the above mixture with dimethyl sulfoxide and vortex thoroughly to mix.
6. The use of the cell preservation solution according to claim 1 in exosome storage.
7. The application according to claim 6, characterized in that... The exosomes mentioned are stem cell exosomes or plant exosomes.