Method for improving exosome stability

Abstract

The invention relates to the technical field of exosome preservation, in particular to a method for improving exosome stability, which comprises the following preparation steps: a first preservation solution: adding 15% of ultrafiltration purified human plasma extract, 2% of novel saccharide derivative, 0.8% of functional amino acid combination and 0.05% of specific exosome active substance protective agent into a basic buffer solution; the second preservation solution is prepared by taking a special cell culture medium as a solvent and adding 25% of sheep serum, 4% of polysaccharide substances, 1.5% of trace element mixed solution and 0.1% of antioxidant polypeptide; the third preservation solution is prepared by utilizing a buffer system with specific osmotic pressure and pH value, and adding 30% of pig placenta extract, 6% of cyclodextrin derivative, 2% of protein stabilizer and 0.2% of enzyme activity protective agent aiming at enzymes possibly existing in the exosome; the stability of the exosome under different preservation conditions is effectively improved, and the requirements of the exosome in the fields of biomedical research, clinical diagnosis and treatment and the like are better met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of exosome preservation, in particular to a method for improving the stability of exosomes. BACKGROUND

[0002] Exosomes are a kind of extracellular vesicles secreted by cells with a diameter of 30-150nm, having a double-membrane structure, and containing active ingredients such as proteins, liposomes and nucleic acids. Exosomes contain abundant cholesterol and sphingomyelin, and the nucleic acids carried can target the mRNA level in cells, and have therapeutic potential for various diseases. However, exosomes face many problems during preservation, and the existing methods for improving the stability of exosomes and exosome preservation solutions have certain deficiencies. Some patent solutions are not fine enough in the selection of components and the ratio of concentrations, and lack targeted protection for exosomes of different sources and different application scenarios; the storage conditions are not studied in depth, and it is difficult to guarantee the stability of exosomes in complex or extreme environments; when evaluating the stability of exosomes, the evaluation of their overall function and biological activity is not comprehensive enough.

[0003] According to the search, Chinese Patent Publication No. CN116897922A discloses a method for improving the stability of exosomes and an exosome preservation solution. The exosome preservation solution is obtained by mixing pectin and / or sodium alginate with a phosphate buffer. When used, the exosome preservation solution is first heated to form a colloidal solution, and then fresh exosomes are added. After sealing, the exosomes can be stored at room temperature for a long time. The protective solution can increase the charge of the exosome system, avoid changes in the particle size, concentration and activity of the exosomes, protect the integrity and stability of the exosomes, and enable the exosomes to maintain their biological activity and the stability of their contents for a long time. However, the exosome preservation solution in this solution does not fully consider the exosomes of different sources and different application scenarios in terms of component selection and concentration ratio. For exosomes from specific cells, the existing formula cannot provide targeted protection. The study on the long-term stability of exosomes under different storage conditions is not in-depth, and only conventional cryopreservation and ordinary solution conditions are mentioned, which cannot meet the demand for guaranteeing the stability of exosomes in complex or extreme environments such as large temperature fluctuations and long storage time. SUMMARY

[0004] The present application aims to provide a method for improving the stability of exosomes to solve the problems of poor stability of exosomes in the existing technology during preservation, including insufficient component optimization, limited storage conditions and incomplete stability evaluation.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions: A method for improving the stability of exosomes, comprising the following preparation steps: S1. Exosomes are collected from cell culture supernatant via ultracentrifugation, and from biological samples such as plasma and urine using a specific kit, with the source and batch labeled. S2, First preservation solution: 15% ultrafiltration purified human plasma extract, 2% novel carbohydrate derivatives, 0.8% functional amino acid combination, and 0.05% specific exosome active substance protectant added to the basic buffer solution; The second preservation solution uses a specially prepared cell culture medium as a solvent, and adds 25% sheep serum, 4% polysaccharides, 1.5% trace element mixture, and 0.1% antioxidant peptides. The third type of preservation solution: using a buffer system with specific osmotic pressure and pH value, adding 30% porcine placental extract, 6% cyclodextrin derivative, 2% protein stabilizer, and 0.2% enzyme activity protectant for enzymes that may be present in exosomes; S3. The collected exosomes are added to the first preservation solution, the second preservation solution and the third preservation solution under sterile conditions and mixed gently. S4. Dispense the preservation solution containing exosomes into strictly sterilized cryovials or vials, ensuring that the number of exosomes and the volume of preservation solution are consistent in each container, and remove as much air as possible before sealing. S5. Short-term: Exosome samples containing the first type of preservation solution are placed in a room temperature, low light and moderate humidity environment; exosome samples containing the second type of preservation solution are placed in a constant temperature refrigerator; exosome samples containing the third type of preservation solution are placed in a refrigerator. Interim: The three types of samples were placed in frozen bags with temperature monitoring tags and stored in a -20°C freezer. The placement time and temperature curve were recorded. During storage, the samples were briefly thawed weekly using a gradient thawing method. The structural integrity and functional activity indicators, such as membrane protein expression and internal nucleic acid integrity, were tested before being refrozen. Long-term: Using a controlled-rate cooling programmable cooling box or a gas phase liquid nitrogen tank, a small amount of samples are taken from the ultra-low temperature freezer or the liquid nitrogen tank every month. The exosomes are revived by thawing at -30°C for 5 minutes and then rewarming with stirring in a 25°C water bath for 3 minutes. The biological function is comprehensively evaluated through cell uptake experiments and detection of the effect on target cell function. S6. At different storage time points, observe the morphological and structural integrity of exosomes and whether there is aggregation or breakage using transmission or scanning electron microscopy; measure particle size distribution using dynamic light scattering to determine stability; measure the expression level of marker proteins on the surface of exosomes using Western blotting, and measure the internal nucleic acid content and integrity using quantitative PCR; co-culture exosomes with target cells and evaluate the impact on the biological behavior of target cells through cell viability, apoptosis detection and gene expression profiling analysis to reflect the biological activity and functional stability of exosomes.

[0006] As a preferred embodiment of the present invention, the novel sugar derivative of the first preservation solution in step S2 is maleic acid modified maltose.

[0007] As a preferred scheme of the present application, the polysaccharide in the second storage solution in step S2 is astragalus polysaccharide.

[0008] As a preferred scheme of the present application, the trace element mixture in the second storage solution in step S2 comprises iron, zinc and manganese.

[0009] As a preferred scheme of the present application, the protein stabilizer in the third storage solution in step S2 is polylysine.

[0010] As a preferred scheme of the present application, for short-term storage, the exosome sample in the first storage solution in step S2 is stored in an environment with normal temperature of 20-25℃, relatively dark light and stable humidity of 40%-60%.

[0011] As a preferred scheme of the present application, for short-term storage, the exosome sample in the second storage solution in step S2 is placed in a constant-temperature refrigerator at 4℃ with relative humidity of about 60%.

[0012] As a preferred scheme of the present application, for short-term storage, the exosome sample in the third storage solution in step S2 is placed in a 4℃ refrigerator equipped with a gas monitoring device.

[0013] As a preferred scheme of the present application, for medium-term storage, the exosome sample is thawed by gradient rewarming, i.e., transferred from -20℃ to -5℃ first and kept for 10 minutes, and then transferred to 4℃ and kept for 10 minutes.

[0014] As a preferred scheme of the present application, for long-term storage, the cooling rate of the exosome sample from room temperature to -80℃ is 1℃ / minute.

[0015] Compared with the prior art, the present application has the following beneficial effects: In the present application, the storage solution formula is optimized, the component and concentration ratio are adjusted according to the source and application scenario of the exosome, the protection effect on the exosome is improved, the storage conditions are finely controlled, suitable environment and treatment methods are provided in different storage stages, and the stability of the exosome under various environments is ensured, a comprehensive stability evaluation system can accurately reflect the quality and activity changes of the exosome, thereby effectively improving the stability of the exosome under different storage conditions, and better meeting the needs of the exosome in the fields of biomedical research, clinical diagnosis and treatment, etc. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0017] The raw materials used in the examples and comparative examples are, if not particularly limited, publicly known in the art, for example, can be directly purchased or prepared according to the publicly known preparation method.

[0018] The present application provides a technical solution: A method for improving the stability of exosomes, comprising the following preparation steps: S1, collecting exosomes from cell culture supernatant by ultracentrifugation, plasma and urine biological samples with a specific kit, marking the source and batch; S2, the first preservative solution: adding 15% ultrafiltration purified human plasma extract, 2% new sugar derivatives, 0.8% functional amino acid combination, 0.05% specific exosome active material protective agent to the basic buffer solution; The second preservative solution: adding 25% sheep serum, 4% polysaccharide material, 1.5% trace element mixed solution, 0.1% antioxidant polypeptide to the special cell culture medium as solvent; The third preservative solution: adding 30% pig placenta extract, 6% cyclodextrin derivative, 2% protein stabilizer, 0.2% enzyme activity protective agent for possible enzymes in exosomes to the buffer system with specific osmotic pressure and pH value; S3, adding the collected exosomes to the first preservative solution, the second preservative solution and the third preservative solution respectively in a sterile environment and mixing gently; S4, the preservative solution containing exosomes is divided into strictly sterilized cryogenic tubes or schott bottles, the exosome quantity and preservative solution volume of each container are consistent, and air is discharged as much as possible before sealing; S5, short term: the exosome sample containing the first preservative solution is monitored every 2 hours for appearance, particle size distribution and surface potential in a normal temperature, relatively dark light and moderate humidity environment; the exosome sample containing the second preservative solution is detected for active ingredient change every 4 hours in a constant temperature refrigerator at 4℃ and relative humidity of about 60%; the exosome sample containing the third preservative solution is placed in a refrigerated cabinet equipped with gas monitoring device at 4℃, and the state and related performance indicators are recorded every 6 hours; Medium-term: three samples are put into a cold storage bag with a temperature monitoring label in a -20℃ low-temperature refrigerator, and the time of putting in and the temperature curve are recorded. Every week, the samples are taken out for short-term thawing by the gradient rewarming method, and after the structural integrity and functional activity indexes such as membrane protein expression and internal nucleic acid integrity are detected, the samples are re-frozen. Long-term: the samples are stored in a -80℃ ultra-low temperature refrigerator after being cooled from room temperature to -80℃ at a controllable cooling rate; or the samples are directly stored in a gas phase liquid nitrogen tank. Every month, a small amount of samples are taken out from the ultra-low temperature refrigerator or the liquid nitrogen tank, thawed at -30℃ for 5 minutes, and then rewarmed in a 25℃ water bath for 3 minutes to reactivate the exosomes. The biological functions of the exosomes are comprehensively evaluated by cell uptake experiments, detection of the effects on target cell functions, and the like. S6, at different storage time nodes, the exosome morphological structure integrity and aggregation rupture are observed by transmission electron microscopy or scanning electron microscopy; the stability is judged by measuring the particle size distribution by dynamic light scattering instrument; the exosome surface marker protein expression amount is measured by Western blotting, and the internal nucleic acid content and integrity are measured by quantitative PCR; the exosomes are co-cultured with target cells, and the effects on the biological behavior of target cells are evaluated by cell viability, apoptosis detection and gene expression profile analysis to reflect the biological activity and functional stability of the exosomes.

[0019] In this embodiment, the novel saccharide derivative of the first preservative solution in step S2 is maleic acid modified maltose.

[0020] In this embodiment, the polysaccharide substance in the second preservative solution in step S2 is astragalus polysaccharide.

[0021] In this embodiment, the trace element mixture in the second preservative solution in step S2 contains iron, zinc and manganese.

[0022] In this embodiment, the protein stabilizer in the third preservative solution in step S2 is polylysine.

[0023] In this embodiment, during short-term storage, the exosome samples in the first preservative solution of step S2 are stored in an environment with normal temperature of 20-25℃, relatively dark light and stable humidity of 40%-60%.

[0024] In this embodiment, during short-term storage, the exosome samples in the second preservative solution of step S2 are placed in a constant-temperature refrigerator at 4℃ and with relative humidity of about 60%.

[0025] In this embodiment, during short-term storage, the exosome samples in the third preservative solution of step S2 are placed in a 4℃ refrigerator equipped with a gas monitoring device.

[0026] In this embodiment, during medium-term storage, the exosome samples are thawed by the gradient rewarming method, i.e., first transferred from -20℃ to -5℃ and kept for 10 minutes, and then transferred to 4℃ and kept for 10 minutes.

[0027] In this embodiment, the cooling rate of the exosome sample from room temperature to -80°C is 1°C / min during long-term storage. Example 1

[0028] Sample collection preparation: From a certain cell culture supernatant, exosome samples were obtained by ultracentrifugation. The obtained exosomes were labeled as a specific batch, and detailed source information was recorded for subsequent research and quality control.

[0029] Improved preparation of preservation solution: Prepare the base buffer and add the ultrafiltration purified human plasma extract to it to make the mass fraction of the preservation solution reach 15%. Then add 2% maleic acid modified maltose, 0.8% glutathione and arginine mixture, and 0.05% protective agent for specific active substances in the exosome sample, mix thoroughly to obtain the preservation solution of Example 1.

[0030] Mixing of exosomes and preservation solution: In a sterile laboratory environment, the collected exosomes are slowly added to the preservation solution of Example 1, and gentle stirring is used to ensure that the exosomes are fully mixed with the preservation solution without damaging the structure of the exosomes.

[0031] Packaging and initial treatment: The exosome-containing preservation solution is carefully divided into pre-sterilized cryogenic tubes, and the number of exosomes and the volume of preservation solution in each cryogenic tube are strictly consistent. Before sealing the cryogenic tube, use professional equipment to try to expel the air in the tube to the maximum extent to minimize the potential impact of oxygen on the exosomes.

[0032] Optimized storage conditions: Short-term storage (48 hours, 4°C): Place the cryogenic tubes containing exosomes and the preservation solution of Example 1 in a thermostat with temperature and humidity adjustment functions, set the environmental temperature to 4°C, and the humidity to about 60%, and the light is relatively dark. Every 4 hours, use dynamic light scattering to detect the particle size distribution of the exosomes, observe the morphology and structure of the exosomes by electron microscope, and record the data in detail.

[0033] Medium-term storage (1 month, -20°C): After the sample is placed in a frozen bag with a temperature monitoring label, it is placed in a -20°C freezer. Every week, take out a sample and thaw it according to the gradient thawing method, i.e. first transfer from -20°C to -5°C for 10 minutes, then to 4°C for 10 minutes. After thawing, detect the expression of exosome surface marker proteins by Western blotting, and detect the content and integrity of internal nucleic acids (miRNA, mRNA, etc.) by quantitative PCR. After the detection is completed, the sample is re-frozen to -20°C freezer.

[0034] Long-term storage (3 months, -80°C): The samples were cooled from room temperature to -80°C at a rate of 1°C / min using a programmable cooling box, and then transferred to a -80°C ultra-low temperature freezer for long-term storage. Every month, a small amount of sample was taken out of the ultra-low temperature freezer, and the exosomes were revived according to the rewarming procedure of first thawing at -30°C for 5 minutes, and then quickly transferring to a 25°C water bath for stirring and rewarming for 3 minutes. The revived exosomes were co-cultured with specific target cells, and the cell viability was detected by MTT experiment, the cell apoptosis was detected by Annexin V-FITC double staining method, and the gene expression profile was analyzed by RNA-sequencing, to comprehensively evaluate the biological function of the exosomes, and record the experimental data.

[0035] Exosome stability evaluation: At specific time nodes, i.e. at the end of 48 hours of short-term storage, at the end of 1 month of medium-term storage, and at the end of 3 months of long-term storage, the exosome samples of Example 1 were evaluated for stability. The morphological structure integrity of the exosomes was observed in detail using a microscope (transmission electron microscope and scanning electron microscope), the particle size distribution range of the exosomes was accurately measured by dynamic light scattering instrument, and the particle size change rate data was analyzed. The retention rate of the surface marker protein of the exosomes was accurately detected by Western blotting, and the internal nucleic acid (RNA integrity value RIN) and other indicators were obtained by quantitative PCR technology. The influence of the exosomes on the biological behavior of target cells was comprehensively evaluated by cell viability detection (MTT experiment), cell apoptosis detection (Annexin V-FITC double staining method), and gene expression profile analysis (RNA-sequencing), to reflect the biological function activity of the exosomes and the accuracy rate data of the influence on the gene expression of target cells. The data are recorded as follows: Short-term storage (48 hours, 4°C): The exosome particle size change rate was ±5%, the marker protein retention rate was 90%, and the nucleic acid integrity (RNA integrity value RIN) was 8.0.

[0036] Medium-term storage (1 month, -20°C): The exosome membrane structure integrity rate was 85%, the functional protein activity retention rate was 82%, and the internal nucleic acid active ingredient retention rate was 75%.

[0037] Long-term storage (3 months, -80°C): The biological function activity of the exosomes (measured by cell uptake rate) was 78%, and the accuracy rate of the influence on the gene expression of target cells (compared with the initial state) was 80%. Example 2

[0038] Sample collection preparation: From the fresh plasma sample, exosomes were extracted using a special kit, and the source and batch information of the exosomes were also carefully labeled to ensure the traceability of the entire experimental process.

[0039] Improved preservation solution preparation: A special cell culture medium was used as the solvent, 25% sheep serum was added, followed by the addition of 4% astragalus polysaccharides, 1.5% trace element mixed solution containing iron, zinc and manganese, and 0.1% antioxidant polypeptide. The mixture was stirred thoroughly to ensure uniform dispersion of all components, and the preservation solution of Example 2 was prepared.

[0040] Exosome and preservation solution mixing: Under strict sterile operating conditions, the collected exosomes were mixed with the preservation solution of Example 2 in a predetermined ratio. Gentle stirring was used to ensure that the exosomes and the preservation solution were fully mixed while minimizing damage to the exosomes.

[0041] Packaging and initial processing: As in Example 1, the exosome-containing preservation solution was accurately dispensed into sterile cryogenic tubes. The number of exosomes and the volume of preservation solution in each tube were controlled to be consistent, and the air in the tubes was removed to reduce oxidation.

[0042] Optimized storage conditions: Short-term storage (48 hours, 4°C): The sample was placed in a constant temperature refrigerator at 4°C. A humidity adjusting device was installed in the refrigerator to ensure that the relative humidity was stable at about 60%. Every 4 hours, a specific detection instrument and method were used to detect the exosomes, and detailed data such as changes in the appearance of the exosomes and changes in active ingredients were recorded.

[0043] Medium-term storage (1 month, -20°C): As in Example 1, the sample was placed in a cryogenic bag and stored in a -20°C refrigerator. Every week, the sample was thawed according to the gradient thawing method, and the structural integrity and functional activity of the exosomes were detected. After completion, the sample was refrozen.

[0044] Long-term storage (3 months, -80°C): According to the long-term storage steps of Example 1, the sample was first programmed to -80°C and stored in an ultra-low temperature refrigerator. Every month, the exosomes were revived according to the established thawing program and biological function evaluation and other related experiments were performed. The results were recorded in detail.

[0045] Exosome stability evaluation: The evaluation was performed at the corresponding time nodes of short-term, medium-term and long-term storage, and the relevant data were obtained and recorded as follows: Short-term storage (48 hours, 4°C): The exosome particle size change rate was ±4%, the marker protein retention rate was 92%, and the nucleic acid integrity (RNA integrity value RIN) was 8.5.

[0046] Medium-term storage (1 month, -20°C): The exosome membrane structure integrity rate was 88%, the functional protein activity retention rate was 85%, and the internal nucleic acid active ingredient retention rate was 80%.

[0047] Long term storage (3 months, -80°C): exosome biological functional activity (measured as cell uptake rate) was 82% and accuracy of influence on target cell gene expression (compared to initial state) was 84%. Example 3

[0048] Sample collection preparation: exosomes were extracted from urine samples by specific kits.

[0049]

[0050]

[0051] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary and that certain changes and modifications can be made thereto without departing from the principles and spirit of the application. The scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for improving exosome stability, characterized in that, The preparation steps include the following: S1. Exosomes are collected from cell culture supernatant via ultracentrifugation, and from biological samples such as plasma and urine using a specific kit, with the source and batch labeled. S2, First preservation solution: 15% ultrafiltration purified human plasma extract, 2% novel carbohydrate derivatives, 0.8% functional amino acid combination, and 0.05% specific exosome active substance protectant added to the basic buffer solution; The second preservation solution uses a specially prepared cell culture medium as a solvent, and adds 25% sheep serum, 4% polysaccharides, 1.5% trace element mixture, and 0.1% antioxidant peptides. The third type of preservation solution: using a buffer system with specific osmotic pressure and pH value, adding 30% porcine placental extract, 6% cyclodextrin derivative, 2% protein stabilizer, and 0.2% enzyme activity protectant for enzymes that may be present in exosomes; S3. The collected exosomes are added to the first preservation solution, the second preservation solution and the third preservation solution under sterile conditions and mixed gently. S4. Dispense the preservation solution containing exosomes into strictly sterilized cryovials or vials, ensuring that the number of exosomes and the volume of preservation solution are consistent in each container, and remove as much air as possible before sealing. S5. Short-term: Exosome samples containing the first type of preservation solution are placed in an environment with room temperature, low light and moderate humidity; exosome samples containing the second type of preservation solution are placed in a constant temperature refrigerator; exosome samples containing the third type of preservation solution are placed in a refrigerator. Interim: The three types of samples were placed in frozen bags with temperature monitoring tags and stored in a -20°C freezer. The placement time and temperature curve were recorded. During storage, the samples were briefly thawed weekly using a gradient thawing method. The structural integrity and functional activity indicators, such as membrane protein expression and internal nucleic acid integrity, were tested before being refrozen. Long-term: Using a controlled-rate cooling programmable cooling box or a gas phase liquid nitrogen tank, a small amount of samples are taken from the ultra-low temperature freezer or the liquid nitrogen tank every month. The exosomes are revived by thawing at -30°C for 5 minutes and then rewarming with stirring in a 25°C water bath for 3 minutes. The biological function is comprehensively evaluated through cell uptake experiments and detection of the effect on target cell function. S6. At different storage time points, observe the morphological and structural integrity of exosomes and whether there is aggregation or breakage using transmission or scanning electron microscopy; measure particle size distribution using dynamic light scattering to determine stability; measure the expression level of marker proteins on the surface of exosomes using Western blotting, and measure the internal nucleic acid content and integrity using quantitative PCR; co-culture exosomes with target cells and evaluate the impact on the biological behavior of target cells through cell viability, apoptosis detection and gene expression profiling analysis to reflect the biological activity and functional stability of exosomes.

2. The method for improving exosome stability according to claim 1, characterized in that: In step S2, the novel sugar derivative of the first preservation solution is maleic acid-modified maltose.

3. The method for improving exosome stability according to claim 1, characterized in that: In step S2, the polysaccharide in the second preservation solution is Astragalus polysaccharide.

4. The method for improving exosome stability according to claim 1, characterized in that: The trace element mixture in the second preservation solution in step S2 contains iron, zinc, and manganese.

5. The method for improving exosome stability according to claim 1, characterized in that: The protein stabilizer in the third preservation solution described in step S2 is polylysine.

6. The method for improving exosome stability according to claim 1, characterized in that: For short-term storage, the first type of preservation solution exosome sample in step S2 is stored in an environment with a normal temperature of 20-25°C, low light, and stable humidity of 40%-60%.

7. The method for improving exosome stability according to claim 1, characterized in that: For short-term storage, the exosome sample in the second type of preservation solution in step S2 is placed in a constant temperature refrigerator at 4°C and a relative humidity of about 60%.

8. The method for improving exosome stability according to claim 1, characterized in that: For short-term storage, the third type of preservation solution exosome sample from step S2 is placed in a 4°C refrigerator equipped with a gas monitoring device.

9. The method for improving exosome stability according to claim 1, characterized in that: During interim storage, the exosome samples were thawed using a gradient refreezing method, that is, first transferred from -20°C to -5°C and kept for 10 minutes, and then transferred to 4°C and kept for 10 minutes.

10. A method for improving exosome stability according to claim 1, characterized in that: During long-term storage, the exosome sample was cooled from room temperature to -80°C at a rate of 1°C / minute.

Images