Preserving fluid for improving cold storage stability of exosome and preparation method of preserving fluid

Abstract

The invention discloses a preserving fluid for improving the cold storage stability of exosomes and a preparation method thereof, the preserving fluid comprises glucose, glycerol, albumin and a buffer solution in a specific proportion, and stable preservation of the exosomes is realized through multiple protection mechanisms. The preparation method comprises the following steps: mixing the components in sequence at room temperature, filtering and sterilizing. The invention also provides a preservation method of the exosome, which comprises the following steps: mixing the exosome and the preservation solution in proportion, and preserving in an environment of-20 DEG C. Experiments prove that the exosome can be stably preserved for more than 6 months at the temperature of-20 DEG C by the preservation solution, and the structure and the function of the exosome are kept stable in the repeated freezing and thawing process. The preservation solution has the advantages of being good in protection effect, easy and convenient to prepare, low in cost and the like, and a practical new scheme is provided for storage and transportation of the exosome.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and particularly to a preservation solution for long-term preservation of exosomes under -20°C conditions and a preparation method thereof. Background Art

[0002] Exosomes are nano-scale membrane vesicles with important biological functions secreted by cells. Due to their unique biological properties, they show broad application prospects in biomarker research, drug delivery system development, and regenerative medicine. These nano-scale vesicles carry a variety of bioactive substances such as proteins and nucleic acids, and are of great value in disease diagnosis, treatment, and basic research. However, due to the complexity of the exosome structure and its sensitivity to environmental factors, its storage stability has always been one of the key issues restricting its clinical transformation, and there is an urgent need to develop more effective preservation solutions.

[0003] At present, the storage methods of exosomes mainly include -80℃ ultra-low temperature storage, 4-8℃ short-term storage and freeze-dried storage. -80℃ ultra-low temperature storage is the most commonly used method in the laboratory, which can better maintain the activity of exosomes. However, this method not only requires special ultra-low temperature equipment and high storage costs, but also it is difficult to ensure a continuous low temperature environment during the transportation of large quantities of samples.

[0004] Although short-term storage at 4-8°C is simple and convenient, exosomes are less stable at this temperature and are prone to degradation. Therefore, they are only suitable for short-term experimental research and cannot meet the long-term storage requirements of clinical applications. Although freeze-dried storage can achieve long-term storage at room temperature, the freeze-drying process is complex and the cost is high, which also limits its wide application.

[0005] Given that -20°C is the most common storage condition in laboratories and medical institutions, if exosomes can be stored stably for a long time at this temperature, it will greatly reduce storage costs and improve the feasibility of clinical applications. However, there is currently a lack of reliable -20°C exosome storage solutions, especially protective measures under repeated freezing and thawing conditions. Conventional cell preservation fluids (such as culture media containing 10% DMSO) are not ideal for protecting exosomes, mainly because the membrane structure and content characteristics of exosomes are significantly different from those of cells. In addition, simple carbohydrate protective agents have weak anti-freeze and thaw resistance, and the effect of long-term preservation of exosomes is limited.

[0006] Therefore, developing a special preservation solution for exosomes that is suitable for -20°C conditions, can withstand multiple freeze-thaw cycles, and can be stored for a long time has important scientific significance and application value. Summary of the invention

[0007] The purpose of the present invention is to provide a preservation solution and a preparation method thereof that can preserve exosomes for a long time at -20°C, so as to solve the technical problems in the prior art that exosomes have poor stability and rapid activity loss under conventional refrigeration conditions.

[0008] In view of the deficiencies of the prior art and actual needs, the present invention provides an exosome preservation solution and a preparation method and application thereof, and designs a new exosome preservation solution formula with a simple formula that can stably preserve exosomes for a long time.

[0009] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an exosome preservation solution, comprising glucose, albumin, glycerol and a buffer.

[0010] In the present invention, a new formula of exosome preservation solution is designed. Glucose prevents the imbalance of cell hydration of exosomes under low temperature conditions, and reduces the damage to the membrane structure of exosomes during repeated freezing and thawing. Glycerol avoids the damage caused by ice crystal formation during the freezing process and reduces the damage to exosomes caused by freeze-thaw cycles. Albumin regulates the osmotic pressure of the exosome preservation solution and stabilizes the surface structure of the exosomes. The buffer provides a stable pH environment. The four components work together to preserve exosomes for a long time at -20°C without affecting their activity.

[0011] The physical and chemical indicators of the exosome preservation solution include: pH range: 7.2-7.4; isotonicity: 280-320mOsm / kg; appearance: clear and transparent, free of visible impurities; quality requirements: in compliance with the sterility inspection standards specified in the Chinese Pharmacopoeia.

[0012] The glucose concentration is 0.5% to 5% (w / v), preferably 2% (w / v).

[0013] The concentration of glycerol in the exosome preservation solution is 5% to 10% (v / v), preferably 7% (v / v).

[0014] The albumin includes human serum albumin and / or bovine serum albumin.

[0015] The purity of albumin in the exosome preservation solution is not less than 98%, and the concentration is 0.1% to 1% (v / v), preferably 0.5% (v / v).

[0016] The buffer comprises PBS buffer and / or HEPES buffer, wherein the concentration of the HEPES buffer is 10-50 mM, preferably 25 mM.

[0017] In a second aspect, the present invention provides a method for preparing the exosome preservation solution according to the first aspect, the preparation method comprising: (1) Prepare buffer to the required concentration; (2) Add glucose to the buffer and fully dissolve it; (3) Add glycerol to fully dissolve; (4) Slowly add albumin at low temperature and stir at low speed; (5) Sterilize by filtering using a 0.22 μm filter membrane, and aseptically aliquot and seal.

[0018] In the preparation method: (1) During the glucose and glycerol dissolution stage, the stirring speed is maintained at 200-300 rpm and the temperature is controlled at 20-25°C; (2) During the albumin addition phase, the stirring speed should not exceed 100 rpm and the temperature should be controlled at 4-8°C; (3) The sterilization and filtration operation is carried out in a clean environment with a cleanliness level of 100; (4) The packaging operation is carried out under laminar flow conditions.

[0019] Preferably, the prepared preservation solution is stored at 4-8°C.

[0020] In a third aspect, the present invention provides a method for preserving exosomes, the method comprising: setting the mixing volume ratio of exosomes to preservation solution to 1:1 to 1:5 (v / v), preferably 1:2.

[0021] Gentle manipulation should be used when mixing exosomes with the preservation solution.

[0022] The exosomes are stored by freezing at -20 ± 2°C.

[0023] The exosomes can be stored for 1 day to 6 months.

[0024] The storage method further comprises a thawing step: placing the stored exosome sample in a constant temperature water bath at 28 ± 0.5°C for rapid thawing.

[0025] The stored exosomes can withstand 1 to 6 freeze-thaw cycles.

[0026] In a fourth aspect, the present invention provides use of the exosome preservation solution described in the first aspect in the preservation of exosomes.

[0027] The exosomes include, but are not limited to, exosomes isolated from biological fluids; exosomes from tissues; exosomes collected from culture media; and exosome preparations from multiple species.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an exosome preservation solution, which synergistically protects exosomes through multiple mechanisms and achieves long-term stable preservation at -20°C. It has obvious improvements in preservation conditions, protection effects and ease of use, and provides a practical new solution for the preservation of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graph shows the changes in exosome particle size at different storage times.

[0030] Figure 2 The graph shows the changes in exosome particle concentration at different storage times.

[0031] Figure 3 Transmission electron microscopy results of exosomes at different storage times.

[0032] Figure 4 The graph shows the change in exosome particle size after repeated freezing and thawing.

[0033] Figure 5 The graph shows the changes in exosome particle concentration after repeated freezing and thawing.

[0034] Figure 6 Transmission electron microscopy results of exosomes after repeated freezing and thawing.

[0035] Figure 7 Schematic diagram of the comprehensive score of exosomes after repeated freezing and thawing. DETAILED DESCRIPTION

[0036] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0037] Example 1: Preparation of exosome preservation solution This example provides a typical exosome preservation solution preparation method. The specific steps are as follows: 1. Preparation of buffer: HEPES buffer (25 mM) was selected as the basic buffer system. HEPES was dissolved in 80% volume of water for injection and the pH was adjusted to 7.3 ± 0.1 with sodium hydroxide solution. This concentration of HEPES can maintain a stable pH environment during freezing.

[0038] 2. Addition of glucose: Add glucose to the above buffer to a final concentration of 2% (w / v). Stir at 250 rpm for 15 minutes at room temperature (23 ± 2°C) to ensure that the glucose is completely dissolved. This concentration of glucose can provide sufficient cell protection without causing abnormal osmotic pressure.

[0039] 3. Addition of glycerol: Keeping the same temperature, slowly add glycerol to the solution to a final concentration of 7% (v / v). Continue stirring at 250 rpm for 10 minutes until the solution is uniform and transparent. The addition of glycerol significantly improves the antifreeze performance of the solution.

[0040] 4. Addition of albumin: After cooling the solution at 4°C for 30 minutes, human serum albumin was slowly added to a final concentration of 0.5% (v / v). Gently stir with a low-speed stirrer (80 rpm) for 12 minutes. This step requires special attention to the control of temperature and stirring speed to prevent protein denaturation.

[0041] 5. Sterilization, filtration and packaging: Sterilize the prepared preservation solution with a 0.22μm filter membrane in a clean workbench. Then dispense into sterile cryopreservation tubes, 0.5mL per tube, seal and store at 4℃ for later use. The finished product should be used within 3 months.

[0042] 6. The preservation solution shall be sent for testing of physical and chemical parameters and quality indicators.

[0043] Table 1 Physical and chemical indicators and quality inspection of exosome preservation fluid

[0044] The test results showed that the prepared preservation solution was clear and transparent, with no visible particles. The pH value was 7.31, the osmotic pressure was 298 mOsm / kg, the sterility test was qualified, the endotoxin content was 0.21 EU / mL, and all indicators met the quality requirements. After being stored at 4°C for 3 months, there was no significant change in the properties of the solution.

[0045] Example 2: Exosome preservation experiment This example verifies the protective effect of the preservation solution on exosomes.

[0046] 1. Exosome sample preparation: Exosomes were isolated from the culture supernatant of human umbilical cord mesenchymal stem cells. The specific steps were as follows: serum-free culture medium was collected after 72 hours of cell culture, and cell debris was removed by centrifugation at 300g for 10 minutes and 2000g for 20 minutes, and the supernatant was ultracentrifuged at 120,000g for 70 minutes to obtain exosome precipitate, which was then resuspended in PBS to obtain the exosome sample.

[0047] 2. Save processing: The purified exosomes were divided into three groups and mixed with various preservation solutions at a volume ratio of 1:2: Experimental group: preservation solution of the present invention Control group 1: sucrose-based preservation solution (6.85% sucrose, 2% trehalose, PBS, pH 7.4) Control group 2: Commercially available cell freezing solution (70% culture medium, 20% FBS, 10% DMSO) Control group 3: saline Each group of samples was packaged and stored at -20°C.

[0048] 3. Sampling time point: Samples were taken for analysis at 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months of storage, with 3 parallel samples taken at each time point.

[0049] 4. Morphological analysis: Transmission electron microscopy was used to observe the morphological structure of exosomes.

[0050] 5. Particle size distribution analysis: The particle size distribution of exosomes was determined using a nanoparticle tracking analyzer (NTA).

[0051] 6. Protein marker detection: The expression of exosome-specific markers CD81, TSG101, CD63 and CD9 was detected by Western blot.

[0052] 7. Overall rating: The comprehensive score under each group of conditions was the sum of the change in the number and concentration of exosome particles, the exosome electron microscopy morphology score, and the exosome protein marker detection score.

[0053] Table 2 Comprehensive scores of exosomes in experimental groups at different storage times

[0054] Table 3 Comprehensive scores of exosomes in control group 1 at different storage times

[0055] Table 4 Control group 2-comprehensive score of exosomes at different storage times

[0056] Table 5 Comprehensive scores of exosomes in control group 3 at different storage times

[0057] Table 3-5 and Figure 1-3 The results showed that different preservation solutions had significant differences in the protective effects of exosomes. During the 6-month storage at -20°C: The experimental group (preservation solution of the present invention) showed better protective effect. Figure 1-2 The results showed that during the entire 6-month storage period, the particle size of the exosomes in the experimental group remained stable within the range of 110±5 nm, and the particle number concentration changed slightly. Figure 3) showed that when stored for 1 week, the exosomes were typical round or oval vesicles with double-layer membrane structure, with a diameter of about 30-150nm, complete membrane structure and uniform morphology. As the storage time was extended to 6 months, the exosomes still maintained good morphological characteristics, without obvious damage, aggregation or deformation. As shown in Table 3, from 1 week to 6 months, the various indicators of exosomes remained stable, and the comprehensive score of this group remained at a high level, showing a good long-term preservation effect.

[0058] The exosome particle size and concentration of control group 1 (sucrose-based preservation solution), control group 2 (commercially available cell freezing solution) and control group 3 (normal saline) changed greatly during the 6-month storage period, the membrane structure was damaged, and the exosomes were degraded. The exosome index scores showed a gradual downward trend: the initial protective effect was good, but gradually decreased over time. At 6 months, each index dropped to 4-7 points, and the protective effect was significantly reduced.

[0059] In summary, the preservation solution of the present invention has significant advantages over traditional preservation solutions. It can maintain the morphological integrity, quantitative stability and functional characteristics of exosomes for a long time (at least 6 months) at -20°C, providing a better solution for the storage and transportation of exosomes.

[0060] Example 3: Evaluation of the effect of repeated freezing and thawing This example verifies the protective effect of the preservation solution on exosomes under repeated freezing and thawing conditions.

[0061] 1. Preparation of exosomes, same as Example 2 2. Save processing: The purified exosomes were divided into three groups and mixed with various preservation solutions at a volume ratio of 1:2: Experimental group: preservation solution of the present invention Control group 1: sucrose-based preservation solution (6.85% sucrose, 2% trehalose, PBS, pH 7.4) Control group 2: Commercially available cell freezing solution (70% culture medium, 20% FBS, 10% DMSO) Control group 3: saline 3. Freeze-thaw experiment design: The stored exosome samples were subjected to multiple freeze-thaw cycles. Each cycle included: a) Take the sample out from -20℃ and thaw it quickly in a 28℃ water bath; b) After standing at room temperature for 10 minutes, return to -20°C and freeze overnight; c) A total of 6 freeze-thaw cycles were performed, and samples were taken for analysis after each cycle.

[0062] 4. Morphological analysis: Transmission electron microscopy was used to observe the morphological structure of exosomes.

[0063] 5. Particle size distribution analysis: The particle size distribution of exosomes was determined using a nanoparticle tracking analyzer (NTA).

[0064] 6. Protein marker detection: The expression of exosome-specific markers CD81, TSG101, CD63 and CD9 was detected by Western blot.

[0065] 7. Overall rating: The comprehensive score under each group of conditions was the sum of the change in the number and concentration of exosome particles, the exosome electron microscopy morphology score, and the exosome protein marker detection score.

[0066] Table 6 Experimental group - comprehensive score of repeated freezing and thawing exosomes

[0067] Table 7 Control group 1-comprehensive score of repeated freezing and thawing exosomes

[0068] Table 8 Control group 2-comprehensive score of repeated freezing and thawing exosomes

[0069] Table 9 Control group 3-repeated freezing and thawing exosome comprehensive score

[0070] Table 6-9 and Figure 4-6 The results showed that different preservation solutions had different protective effects on exosomes under repeated freezing and thawing conditions.

[0071] After six cycles of freezing and thawing in the experimental group (preservation solution of the present invention), the exosomes maintained excellent stability. Transmission electron microscopy showed that the exosomes maintained a typical round or cup-shaped structure, with good membrane integrity and no obvious damage. The particle size distribution was stable within the range of 110±10 nm, and the particle number concentration changed little. Within 6 repeated freeze-thaw cycles, all indicators of the exosomes remained stable, and the comprehensive score of the group remained at a high level, showing a good anti-freeze-thaw protection effect.

[0072] After freeze-thaw cycles, the particle size distribution of control group 1 (sucrose-based preservation solution), control group 2 (commercially available cell freezing solution) and control group 3 (normal saline) was unstable, and the exosomes were damaged to a certain extent, with broken particles. The exosome index score showed a gradual downward trend.

[0073] In summary, the preservation solution of the present invention can more effectively protect the structural integrity of exosomes during repeated freezing and thawing compared with traditional preservation solutions, and has obvious technical advantages.

[0074] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A preservation solution for improving the cold storage stability of exosomes, characterized in that: The exosome preservation solution comprises glucose, albumin, glycerol and buffer.

2. The preservation solution for improving the cold storage stability of exosomes according to claim 1, characterized in that: The physicochemical indicators of the preservation solution include: 2.1 pH range: 7.2-7.4; 2.2 Isotonicity: 280-320 mOsm / kg; 2.3 Appearance: Clear and transparent, without visible impurities; 2.4 Quality requirements: Conform to the sterility test standards specified in the Chinese Pharmacopoeia.

3. The preservation solution for improving the cold storage stability of exosomes according to claim 1, characterized in that the concentration of each component is: 3.1 Glucose concentration is 0.5% to 5% w / v; 3.2 The volume fraction of glycerol is 5% to 10% v / v; 3.3 The albumin volume fraction is 0.1% to 1% v / v; the albumin purity is not less than 98%; 3.4 Buffer: PBS buffer pH 7.4 and / or HEPES buffer, wherein the concentration of HEPES buffer is 10 to 50 mM.

4. A method for preparing a preservation solution for improving the cold storage stability of exosomes according to any one of claims 1 to 3, characterized in that: The following steps are involved: 4.1 Prepare the buffer solution to the required concentration according to the formula ratio and adjust the pH to 7.2 to 7.4; 4.2 At 20-25°C, slowly add glucose to the buffer solution and fully dissolve it while stirring at 200-300 rpm. 4.3 Continue to add glycerol under the same conditions and stir until it is completely dissolved; 4.4 Cool the mixture to 4-8°C and slowly add albumin while stirring at a low speed not exceeding 100 rpm; 4.5 In a clean environment with cleanliness reaching Class 100, use a 0.22 μm filter membrane for sterilization filtration; 4.6 Perform sterile packaging and seal in a laminar flow biosafety cabinet.

5. The method for preparing a preservation solution for improving the cold storage stability of exosomes according to claim 4, characterized in that: The following quality control requirements are also included: 5.1 Raw materials should meet medical grade requirements; 5.2 Monitor the ambient temperature and humidity during the entire preparation process; 5.3 Finished products are subject to sterility testing and endotoxin testing; 5.4 The prepared preservation solution can be stably stored at 4 to 8°C for 3 months.

6. A method for using the preservation solution for improving the cold storage stability of exosomes according to any one of claims 1 to 3, characterized in that: 6.1 The optimal mixing volume ratio of exosomes to preservation solution is 1:2 v / v. Mixing should be done gently. 6.2 The mixed samples were stored at -20 ± 2 ℃; 6.3 The storage period is 1 day to 6 months; 6.4 Thawing should be done by rapid thawing in a constant temperature water bath at 28 ± 0.5 ℃; 6.5 It can withstand 6 freeze-thaw cycles under specified storage conditions.

7. Use of the preservation solution for improving the cold storage stability of exosomes according to any one of claims 1 to 3 in the preservation of exosomes, characterized in that: The exosomes include but are not limited to: 7.1 Exosomes isolated from biological fluids of human or animal origin; 7.2 Exosomes from tissue culture; 7.3 Exosomes isolated from cell culture supernatant; 7.4 Exosomes from recombinant engineered bacteria.

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