Exosome protection solution and preparation method thereof

Abstract

The application discloses an exosome protection solution and a preparation method thereof, and relates to the technical field of biology. The exosome protection solution comprises the following components in terms of concentration: 0.01-0.1 g / mL of HEPEs, 0.01-0.1 g / mL of trehalose, 0.01-0.1 g / mL of mannitol, 8-10 g / L of recombinant human blood albumin, 0.01-0.1 g / mL of glycine and 0.01-0.1 g / mL of poloxamer 188. The preparation method of the exosome protection solution comprises the following steps: S1, dissolving HEPEs in pure water, and stirring to prepare an HEPEs buffer solution; S2, adding trehalose, mannitol and glycine into the HEPEs buffer solution, and forming a mixed solution after complete dissolution; S3, adding recombinant human blood albumin and poloxamer 188 into the mixed solution and stirring uniformly; and S4, mixing the protection solution with exosomes at a ratio of 1:1. The exosome protection solution provided by the application can store exosomes in a temperature range of-80 DEG C to 4 DEG C, and meets the requirement of long-term storage of exosomes under different temperature environments. The protection solution can effectively improve the stability and integrity of exosomes in the long-term storage process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biology, in particular to an exosome protection solution and a preparation method thereof. BACKGROUND

[0002] Exosomes are 40-200nm extracellular vesicles, which can be used as a new mode of intercellular information exchange, and can regulate various biological processes under physiological and pathological conditions by carrying active factors such as miRNA, mRNA and protein. miRNA is one of the main factors of exosomes, which can regulate gene expression by binding to target genes, resulting in mRNA translation inhibition or transcript degradation. Exosomal miRNA not only has important significance in the prevention and control of diseases, but also more and more studies attempt to alleviate or treat diseases through exosomal miRNA; for example, studies have shown that miR-26a-5p, miR-126-3p and miR-222-3p have the effects of reducing inflammation, promoting cell proliferation and effectively treating skin damage.

[0003] The International Society for Extracellular Vesicles recommends suspending exosomes in PBS and storing them in a -80℃ refrigerator, but under this condition, the membrane of exosomes is easily affected by physical and chemical factors (such as pH, temperature and osmotic pressure) and changes, resulting in phenomena such as exosome membrane fusion, rupture, shrinkage and membrane inversion, and even destruction of lipids, proteins and nucleic acids, and miRNA degradation; and with the passage of time, the particle number of exosomes gradually decreases, and the protein amount and miRNA expression decrease. In addition, the clinical transformation of exosomes in the treatment of diseases has a market prospect, and ultra-low temperature transportation will cause huge additional economic burden.

[0004] The prior art has the following main disadvantages:

[0005] 1. The storage temperature range of the exosome protection solution is narrow, which is difficult to meet the long-term storage requirements under different temperature environments;

[0006] 2. Exosomes are prone to aggregation, fusion and rupture during long-term storage, resulting in a decrease in the stability and integrity of exosomes;

[0007] 3. The composition of the existing exosome protection solution is relatively complex, and the preparation process is complicated and inconvenient to operate;

[0008] 4. There is a problem that the exosome protection solution has limited protection effect on exosomes, and the biological activity of exosomes cannot be effectively maintained;

[0009] 5. Some exosome protection solutions contain animal-derived components, which have potential safety hazards and poor batch consistency.

[0010] Patent CN111226902A discloses an exosome protection solution and an exosome preservation method. The exosome protection solution includes bovine serum albumin, trehalose, glycerol, dimethyl sulfoxide and phosphate buffer. The exosome protection solution and the preservation method can effectively maintain the integrity, biological activity and content stability of the exosome. However, dimethyl sulfoxide has cytotoxicity, and its toxicity to tissues and cells depends on the exposure time, temperature and concentration; in addition, bovine serum albumin as a heterologous excipient is subject to certain restrictions in drug production and declaration. Therefore, the above exosome protection solution and exosome preservation method are mainly suitable for scientific research and cannot meet the needs of clinical treatment.

[0011] Patent CN118435936A discloses an exosome cryopreservation protection solution and a preparation method. The preparation raw materials of the protection solution include PBS, GAGs sugar chain, polyethylene glycol 6000, human serum albumin and propylene glycol. The exosome protection solution can maintain the protein content of the exosome and maintain the activity of the exosome. However, the exosome protection solution is only suitable for storing exosomes in a low-temperature environment of-20℃ and cannot meet the storage requirements of exosomes under-80℃ and 4℃ conditions.

[0012] Therefore, it is of great significance to develop an exosome protection solution and a preparation method thereof capable of stably storing exosomes for the future clinical transformation of exosomes. SUMMARY

[0013] Therefore, the present application aims to overcome the shortcomings of the prior art, and the main purpose is to provide an exosome protection solution and a preparation method thereof. The exosome protection solution provided by the present application can store exosomes in a temperature range of-80℃ to 4℃, meeting the needs of long-term storage of exosomes under different temperature conditions. The exosome protection solution provided by the present application can effectively improve the stability and integrity of exosomes during long-term storage, can maintain exosome particles for more than 12 months, and effectively avoids the occurrence of phenomena such as exosome aggregation, fusion and rupture. The exosome protection solution provided by the present application adopts a simple mixing and dissolving preparation process, is easy to operate and is easy to mass-produce.

[0014] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0015] An exosome protection solution includes the following components in terms of concentration: 0.01-0.1g / mL of HEPEs, 0.01-0.1g / mL of trehalose, 0.01-0.1g / mL of mannitol, 8-10g / L of recombinant human blood albumin, 0.01-0.1g / mL of glycine and 0.01-0.1g / mL of poloxamer 188.

[0016] As a preferred solution: the protective solution comprises the following components in terms of concentration: 0.01 g / mL of HEPEs, 0.01 g / mL of trehalose, 0.01 g / mL of mannitol, 8 g / L of recombinant human blood albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

[0017] The preparation method of the exosome protective solution comprises the following steps:

[0018] S1, dissolving HEPEs in pure water, stirring to prepare HEPEs buffer solution;

[0019] S2, adding trehalose, mannitol and glycine to the HEPEs buffer solution, forming a mixed solution after complete dissolution, cooling to room temperature, and storing at 2-6℃;

[0020] S3, adding recombinant human blood albumin and poloxamer 188 to the mixed solution and stirring uniformly, filtering with a filter to form a protective solution, and storing the protective solution at 2-6℃;

[0021] S4, mixing the protective solution with exosomes at a ratio of 1:1 and storing at-80 to 4℃.

[0022] As a preferred solution: in step S1, the HEPEs buffer solution is prepared by stirring at 37℃ and 50 rpm / min.

[0023] As a preferred solution: in step S2, trehalose, mannitol and glycine are added to the HEPEs buffer solution at 50℃ and 80 rpm / min.

[0024] As a preferred solution: in step S3, the filter membrane of the filter has a pore diameter of 0.22μm.

[0025] As a preferred solution: in step S2, the mixed solution is stored in a 4℃ refrigerator overnight; and in step S3, the protective solution is stored in a 4℃ refrigerator.

[0026] The present application has obvious advantages and beneficial effects compared with the prior art, specifically, from the above technical solution, the exosome protective solution and the preparation method thereof provided by the present application have the following advantages:

[0027] 1. The exosome protective solution provided by the present application can store exosomes at a temperature range of-80℃ to 4℃, meeting the demand for long-term storage of exosomes under different temperature environments;

[0028] 2、The exosome protective solution can effectively improve the stability and integrity of the exosome in the long-term storage process, can maintain the exosome particles for more than 12 months, and effectively avoids the occurrence of exosome aggregation, fusion and rupture and the like;

[0029] 3、The exosome protective solution has clear components and is safe and non-toxic, the adjuvants meet the pharmaceutical standards, the recombinant human serum albumin produced by the genetic engineering technology does not contain animal-derived components, the batch consistency is more stable, and the safety is higher;

[0030] 4、The exosome protective solution can effectively maintain the biological activity of the exosome, and ensure the integrity of the structure and function of the exosome in the long-term storage process;

[0031] 5、The exosome protective solution adopts a simple mixing and dissolving preparation process, is easy to operate, and is easy to mass produce;

[0032] 6、The exosome protective solution can store the exosome under the condition of-80 DEG C to 4 DEG C, improves the stability of the exosome storage, effectively alleviates the degradation of the exosome protein and particles, maintains the normal vesicle size, and effectively protects the low-temperature activity of the exosome.

[0033] In order to more clearly illustrate the structural features and effects of the present application, the following will be combined with the drawings and specific examples to be described in detail. DRAWINGS

[0034] Figure 1 The electron microscope result diagram of the exosome stored at-80 DEG C for 0, 1, 3, 6, 9 and 12 months of the present application comparative example 1;

[0035] Figure 2 The electron microscope result diagram of the exosome stored at-80 DEG C to 4 DEG C for 1 month of the present application examples 1-3 and comparative example 1;

[0036] Figure 3 The electron microscope result diagram of the exosome stored at-80 DEG C to 4 DEG C for 3 months of the present application examples 1-3 and comparative example 1;

[0037] Figure 4 The electron microscope result diagram of the exosome stored at-80 DEG C to 4 DEG C for 12 months of the present application examples 1-3 and comparative example 1;

[0038] Figure 5 The surface marker Western Blot detection diagram of the exosome stored at-80 DEG C to 4 DEG C for 3 months of the present application examples 1-3 and comparative example 1;

[0039] Figure 6Figure of surface marker Western Blot detection of exosomes stored at-80℃ to 4℃ for 12 months for Examples 1-3 and Comparative Example 1 of the present application;

[0040] Figure 7 Figure of results of detection of miR-26a-5p, miR-126-3p and miR-222-3p content in samples of fresh extracted exosomes by fluorescent quantitative PCR for Examples 1-3 and Comparative Example 1 of the present application;

[0041] Figure 8 Figure of results of detection of miR-26a-5p, miR-126-3p and miR-222-3p content by fluorescent quantitative PCR for Examples 1-3 and Comparative Example 1 of the present application for exosomes stored at-80℃ to 4℃ for 12 months. DETAILED DESCRIPTION

[0042] As shown in the present application, Figures 1 to 8 An exosome protection solution includes the following components in concentration: 0.01-0.1 g / mL of HEPEs, 0.01-0.1 g / mL of trehalose, 0.01-0.1 g / mL of mannitol, 8-10 g / L of recombinant human blood albumin, 0.01-0.1 g / mL of glycine and 0.01-0.1 g / mL of poloxamer 188.

[0043] The protection solution includes the following components in concentration: 0.01 g / mL of HEPEs, 0.01 g / mL of trehalose, 0.01 g / mL of mannitol, 8 g / L of recombinant human blood albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

[0044] The preparation method of the exosome protection solution includes the following steps:

[0045] S1, dissolving HEPEs in pure water to prepare HEPEs buffer by stirring;

[0046] S2, adding trehalose, mannitol and glycine to the HEPEs buffer, forming a mixed solution after complete dissolution, cooling to room temperature and storing at 2-6℃;

[0047] S3, adding recombinant human blood albumin and poloxamer 188 to the mixed solution and stirring uniformly, filtering with a filter to form the protection solution, and storing the protection solution at 2-6℃;

[0048] S4, mixing the protection solution with exosomes at a ratio of 1:1 and storing at-80 to 4℃.

[0049] The HEPEs buffer solution was prepared by stirring at 37℃ and 50rpm / min in step S1.

[0050] In step S2, trehalose, mannitol and glycine were added to the HEPEs buffer solution at 50℃ and 80rpm / min.

[0051] In step S3, the filter membrane of the filter had a pore size of 0.22μm.

[0052] In step S2, the mixed solution was stored in a refrigerator at 4℃ overnight; in step S3, the protective solution was stored in a refrigerator at 4℃.

[0053] In the embodiments of the present application, HEPEs and glycine were purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd., trehalose and poloxamer 188 were purchased from BASF (China) Co., Ltd., mannitol and recombinant human serum albumin were purchased from Shanghai Macklin Biochemical Technology Co., Ltd.

[0054] The protective solution excipient meets the pharmaceutical standard and is suitable for a wide temperature range of-80℃ to 4℃, which can significantly improve the stability and integrity of the exosomes, ensure that the particles are well preserved for 12 months, and avoid phenomena such as aggregation, fusion and rupture.

[0055] To achieve the above-mentioned objectives, the present application adopts a scientific technical solution and provides an exosome protective solution, which comprises HEPEs, trehalose, mannitol, recombinant human serum albumin, glycine and poloxamer 188; the protective solution can provide comprehensive protection for exosomes during storage.

[0056] HEPEs is an amphoteric ion buffer, which is one of the important components of the protective solution, and its main function is to stabilize the pH value of the water phase inside and outside the exosomes. Since HEPEs is a buffer pair that is not easy to penetrate the biological membrane, it can effectively prevent the pH fluctuation of the internal and external environment of the exosomes, thereby maintaining the stability of the exosome structure.

[0057] Trehalose is a non-reducing disaccharide, which plays a key role as an excipient in the protective solution. It can protect the structure of biological macromolecules from being destroyed, and at the same time, it can make high-concentration proteins exist in a stable colloidal form by wrapping them in nanoclusters formed by it. In addition, trehalose can inhibit the formation of large ice crystals during freezing, making the ice crystals smaller and more delicate, thereby reducing the damage to the surrounding tissues. This property not only improves the preservation effect of the product in a low-temperature state, but also alleviates the speed during thawing and reduces the dehydration phenomenon after freeze-thawing.

[0058] Mannitol also plays an important role in exosome cryopreservation. It prevents protein aggregation by forming amorphous structures, effectively reducing the formation and growth of ice crystals during the freezing process. This mechanism can protect the structure and function of exosomes in a low-temperature environment, ensuring their stability during cryopreservation.

[0059] Recombinant human serum albumin is another important component of the protective solution. As the most abundant protein in plasma, it can prevent protein denaturation and aggregation during freeze-thawing by forming non-covalent bonds with other protein molecules, thereby protecting the structural and functional integrity of exosomes. However, due to the risk of inconsistent composition between batches of blood-derived albumin and the potential contamination of blood-derived pathogens such as HIV and hepatitis B virus, the present invention chooses to use recombinant human serum albumin produced by genetic engineering technology. This recombinant protein does not contain animal-derived components, has higher batch consistency and safety, and can effectively avoid the potential risk of blood-derived contamination.

[0060] Glycine, as an auxiliary component in the protective solution, can lower the freezing point of the solution, thereby slowing down the formation rate of ice crystals. In addition, glycine has good hydration ability and can form stable hydration with protein molecules to prevent protein denaturation due to water loss during freeze-thawing. This property makes glycine play a significant role in improving the freeze-thawing effect.

[0061] Poloxamer 188 is a non-ionic linear copolymer with surfactant properties, commonly used to improve the stability and solubility of drugs. In the exosome protective solution, poloxamer 188 reduces or eliminates protein adsorption on different contact interfaces, reducing exosome aggregation and damage during freezing, thereby maintaining the biological activity and structural integrity of exosomes.

[0062] In summary, the present invention develops an exosome protective solution through a scientific and reasonable technical solution. The components of the protective solution are carefully selected to effectively solve the stability and integrity problems encountered by exosomes during storage. This protective solution is suitable for a wide temperature range and can significantly extend the shelf life of exosomes, providing reliable technical support for long-term storage and application of exosomes.

[0063] Example 1

[0064] The present embodiment provides an exosome protective solution, the preparation raw materials of the exosome protective solution include: 0.01 g / mL of HEPEs, 0.01 g / mL of trehalose, 0.01 g / mL of mannitol, 8 g / L of recombinant human serum albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

[0065] Example 2

[0066] The present embodiment provides an exosome protection solution, the preparation raw materials of which include, in terms of mass concentration, 0.05 g / mL of HEPEs, 0.05 g / mL of trehalose, 0.1 g / mL of mannitol, 10 g / L of recombinant human blood albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

[0067] Example 3

[0068] The present embodiment provides an exosome protection solution, the preparation raw materials of which include, in terms of mass concentration, 0.1 g / mL of HEPEs, 0.1 g / mL of trehalose, 0.1 g / mL of mannitol, 10 g / L of recombinant human blood albumin, 0.1 g / mL of glycine and 0.1 g / mL of poloxamer 188.

[0069] Comparative Example 1

[0070] The present comparative example uses the solution PBS recommended by the International Society for Extracellular Vesicles to store exosomes.

[0071] Comparative Example 2

[0072] The present embodiment provides an exosome protection solution, the preparation raw materials of which include, in terms of mass concentration, 0.01 g / mL of HEPEs, 0.01 g / mL of trehalose, 0.05 g / mL of mannitol, 9 g / L of recombinant human blood albumin, 0.1 g / mL of glycine and 0.1 g / mL of poloxamer 188.

[0073] Comparative Example 3

[0074] The present embodiment provides an exosome protection solution, the preparation raw materials of which include, in terms of mass concentration, 0.05 g / mL of HEPEs, 0.05 g / mL of trehalose, 0.05 g / mL of mannitol, 9 g / L of recombinant human blood albumin, 0.05 g / mL of glycine and 0.05 g / mL of poloxamer 188.

[0075] Comparative Example 4

[0076] The present embodiment provides an exosome protection solution, the preparation raw materials of which include, in terms of mass concentration, 0.1 g / mL of HEPEs, 0.1 g / mL of trehalose, 0.01 g / mL of mannitol, 8 g / L of recombinant human blood albumin, 0.05 g / mL of glycine and 0.05 g / mL of poloxamer 188.

[0077] The preparation methods of the exosome protection solutions in the above-mentioned Example 1 to Example 3 and Comparative Examples 2-4 are all as follows:

[0078] S1, dissolve HEPEs in pure water, stir to prepare HEPEs buffer solution;

[0079] S2, add trehalose, mannitol and glycine to the HEPEs buffer solution, form a mixed solution after complete dissolution, cool to room temperature, and store at 2-6°C;

[0080] S3, add recombinant human serum albumin and poloxamer 188 to the mixed solution and stir uniformly, filter to form a protective solution using a filter, and store the protective solution at 2-6°C;

[0081] S4, mix the protective solution with the exosomes at a ratio of 1:1, and store at -80 to 4°C.

[0082] In this step S1, the HEPEs buffer solution is prepared by stirring at 37°C and 50 rpm / min.

[0083] In this step S2, trehalose, mannitol and glycine are added to the HEPEs buffer solution at 50°C and 80 rpm / min.

[0084] In this step S3, the filter membrane of the filter used has a pore diameter of 0.22 μm.

[0085] In this step S2, the mixed solution is stored overnight in a 4°C refrigerator; in this step S3, the protective solution is stored in a 4°C refrigerator.

[0086] Exosome extraction and detection example:

[0087] I. Exosome extraction

[0088] The method for extracting exosomes derived from umbilical cord mesenchymal stem cells is as follows: P3-P5 generation of umbilical cord mesenchymal stem cells is selected, and the cell supernatant is collected after 3 days of culture using serum-free medium. The collected 2L cell supernatant is centrifuged at 500g for 10min, filtered with a 70μm cell screen to remove cell debris and dead cells, and the centrifuged medium is concentrated by tangential flow filtration and washed with PBS; the concentrated medium is centrifuged at 1000g, 2000g and 10000g for 10min, 20min and 30min respectively, and after each centrifugation, a 70μm cell screen is used for filtration to remove apoptotic vesicles and large vesicles; the medium is centrifuged twice at 120000g and 100min. The exosome precipitate is completely dissolved in 1.6mL PBS to obtain an exosome sample.

[0089] II. Exosome detection method

[0090] 1. Exosome protein concentration determination

[0091] Protein concentration detection of exosome sample by Micro BCA Protein Quantification Kit (Thermo Fisher Scientific, USA, item number 23235)

[0092] 1) Dilute the BSA standard in the kit to obtain 8 standards with protein concentrations of 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.0025 mg / mL, 0.00125 mg / mL, 0.00625 mg / mL, 0.003125 mg / mL and 0 mg / mL;

[0093] 2) Dilute the sample to be tested with PBS solution at dilution ratios of 10, 20 and 50;

[0094] 3) Mix working solution A, working solution B and working solution at a ratio of 25:24:1 to prepare the mixed working solution;

[0095] 4) Mix the standard and the sample to be tested with the working solution at 50 μL and 200 μL, respectively, and incubate at 37°C for 30 min;

[0096] 5) After the reaction is completed, use the microplate reader to measure the OD values of the standard and the sample to be tested at a wavelength of 570 nm.

[0097] 6) Calculate the protein concentration of the sample to be tested according to the standard curve.

[0098] 2. Exosome particle size and particle concentration determination

[0099] Use Nanoparticle Tracking Analysis (Malvern Panalytical, UK, Model Nanosight300) to detect the particle size and particle concentration of exosomes

[0100] 1) Machine preparation

[0101] Install the pipeline of the Nanoparticle Tracking Analysis and use PBS to clean the pipeline and calibrate the instrument.

[0102] 2) Sample preparation

[0103] Dilute the sample to be tested with PBS at 500 times, 1000 times and 2000 times, i.e. the ratio of exosome stock solution to PBS is 1:499, 1:999 and 1:1999.

[0104] 3) Sample detection

[0105] Use a syringe to suck the sample, expel the bubbles in the syringe, and inject the sample to be tested into the sample pool for machine detection.

[0106] 3. Exosome morphology detection

[0107] 1) Dilute the sample to be tested 5 times with PBS to prepare 20 μL of sample dilution solution;

[0108] 2) Gently place the copper mesh onto the sample droplet to be tested and allow it to absorb for 10 minutes;

[0109] 3) Transfer it onto a droplet of 4% paraformaldehyde solution, fix for 10 min, and then wash the copper mesh twice with DPBS;

[0110] 4) Transfer it onto a drop of 2.5% pentanediol solution, fix for 10 min, and then wash the copper mesh 5 times with DPBS;

[0111] 5) Transfer it onto a drop of 3% uranium acetate solution, negatively stain for 30 minutes, then transfer it to filter paper, absorb the excess liquid, and air dry for 30 minutes.

[0112] 6) Observation and detection were performed using a 120kV cryo-electron microscope equipped with a digital camera and image analysis software.

[0113] 4. Detection of exosome surface marker proteins

[0114] 1) Protein sample preparation

[0115] The test sample was fully lysed on ice using RIPA lysis buffer with added PMSF, and after adding Loading Buffer, it was boiled in a 100°C metal bath for 10 min.

[0116] 2) Protein electrophoresis and transfer

[0117] The sample to be tested was added into the well of the SDS-PAGE precast gel (Shanghai Beyotime, item number P0465S), and after electrophoresis and membrane transfer, the PVDF membrane was blocked with 5% bovine serum albumin and left at room temperature for 2 hours.

[0118] 3) Antibody incubation

[0119] The blocked bands were incubated overnight at 4°C with GM130 (Abcam UK, catalog number ab52649), ALIX (Abcam UK, catalog number ab186429), Calnexin (Abcam UK, catalog number ab92573), HSP70 (Abcam UK, catalog number ab181606), TSG101 (Abcam UK, catalog number ab125011), CD63 (Abcam UK, catalog number ab134045), and GAPDH (CST USA, catalog number 2118S). After incubation with primary antibody, the bands were washed three times with PBST for 5 min each time. The bands were then incubated with HRP-labeled goat anti-rabbit IgG (H+L) (Shanghai Beyotime, catalog number A0208) secondary antibody for 2 h at 37°C.

[0120] 4) Strip development

[0121] The strip after the secondary antibody incubation was washed with PBST for 3 times, 5 min each time; and the strip was exposed and developed with an ECL luminescent kit (Shanghai Biyun Tian, item number P0018AS).

[0122] 5. Fluorescent quantitative PCR detection of miR-26a-5p, miR-26-3p and miR-222-3p expression

[0123] Total RNA was extracted from the exosome solution using a miRNeasy Micro Kit (QIAGEN, USA, item number 217084), reverse transcribed by a cDNA synthesis kit (Shanghai Sangon), and subjected to qRT-PCR using a miRNA fluorescent quantitative kit on a fluorescent quantitative PCR system (Roche, Switzerland). U6 was used as an internal reference gene. Then the relative expression level was calculated by the 2-ΔΔCT method.

[0124] Effect Example 1: Long-term stability monitoring of exosomes

[0125] Three batches of umbilical cord mesenchymal stem cell-derived exosomes were used, named I, II and III, and mixed with Comparative Example 1 at a ratio of 1:1, and placed in a -80°C refrigerator. Protein concentration, particle size and particle concentration and morphology were detected at 0, 1, 3, 6, 9 and 12 months.

[0126] Table 1 records the detection of protein concentration, particle size and particle concentration of three batches of exosomes stored in Comparative Example 1 in a -80°C refrigerator at 0, 1, 3, 6, 9 and 12 months. The results show that the changes of protein concentration, particle concentration and particle size of three batches of exosomes stored in Comparative Example 1 at different time points are different, sometimes increasing and sometimes decreasing, but the overall trend is still declining.

[0127] Figure 1 is the morphology detection of three batches of exosomes stored in Comparative Example 1 in a -80°C refrigerator at 0, 1, 3, 6, 9 and 12 months. The results show that with the passage of time, the exosomes of each batch have different degrees of aggregation, rupture and fusion at each time point.

[0128] Based on the above research results, when long-term cryopreservation is used, the changes of protein concentration, particle concentration and particle size of exosomes in Comparative Example 1 using PBS solution as a protective agent are unstable, there is a trend of vesicle degradation, and the vesicles will have aggregation, rupture and fusion. Therefore, it is very important to develop a preservative for storing exosomes with a wide temperature range and a long time for future product transportation, storage and transformation.

[0129] Table 1: Results of protein concentration, particle size and particle concentration detection of 3 batches of preserved exosomes of Comparative Example 1 at 0, 1, 3, 6, 9 and 12 months

[0130]

[0131] Effect Example 2: Primary screening of exosome protection solution

[0132] The exosome diluent was mixed with Examples 1 to 3 and Comparative Examples 1 to 4 at 1:1, and placed in -80°C, -20°C and 4°C refrigerators, respectively. Protein concentration, particle size and particle concentration detection was performed after 7, 14 and 28 days.

[0133] I. Exosome protein concentration, particle concentration and particle size results

[0134] Table 2 records the results of protein concentration, particle concentration and particle size detection of exosome samples in Examples 1 to 3 and Comparative Examples 1 to 4 stored at -80°C, -20°C and 4°C, respectively, after 7, 14 and 28 days.

[0135] Table 2: Results of protein concentration, particle concentration and particle size detection

[0136]

[0137]

[0138]

[0139] The above results show that, compared with Comparative Examples 1 to 4, the exosome samples stored in Examples 1 to 3 have better protein concentration, particle concentration and particle size stability, lower degradation degree and particle size maintained within the exosome vesicle size range after 7, 14 and 28 days of storage at -80°C, -20°C and 4°C. The proteins and particles of the exosomes of Comparative Example 1 degrade rapidly at different temperatures, and the exosome concentrations of Comparative Examples 2 to 4 are unstable. In summary, the protection solution of Examples 1 to 3 can more stably store exosome samples for 28 days at -80°C to 4°C.

[0140] Effect Example 3: Secondary verification of exosome protection solution

[0141] The exosome diluent was mixed with Examples 1 to 3 and Comparative Example 1 at 1:1, and placed in -80°C, -20°C and 4°C refrigerators, respectively. After 1 month, 3 months and 12 months, the protein concentration, particle size and particle concentration and morphology were detected, the expression of the inner surface marker was detected at 3 months and 12 months, and the contents of miR-26a-5p, miR-126-3p and miR-222-3p were detected at 12 months.

[0142] I. Exosome protein concentration, particle concentration and particle size results

[0143] Table 3 records the results of detecting the protein concentration, particle concentration and particle size of the exosome samples of Examples 1 to 3 and Comparative Example 1 stored at -80°C, -20°C and 4°C refrigerators after 1 month, 3 months and 12 months, respectively.

[0144] Table 3: Detection results of protein concentration, particle concentration and particle size

[0145]

[0146]

[0147]

[0148] The results show that, compared with the traditional PBS preserved Comparative Example 1, the exosome samples preserved in Examples 1 to 3 have better stability of protein concentration, particle concentration and particle size after 1 month, 3 months and 12 months at -80°C, -20°C and 4°C. Among them, the exosome protein concentration and particle concentration of Examples 1 and 2 degrade less, and the particle size remains within the vesicle size range, and the preservation effect is the best. Although the protein concentration of Example 3 degrades faster, the particle concentration changes stably, and still has a good effect of preventing particle degradation. In summary, the protective solution of Examples 1 and 2 is more suitable for stable storage of exosome samples for 12 months at -80°C to 4°C, especially when preserved with Example 1, the exosome protein and particle concentration is higher.

[0149] II. Morphology detection

[0150] Figure 2 、 3 and 4 respectively represent the electron microscope results of the exosome samples preserved for 1 month, 3 months and 12 months at -80°C, -20°C and 4°C using the protective solution of Examples 1 to 3 and Comparative Example 1.

[0151] Figure 2 、 3The results of 1 and 4 show that the exosomes in the protective solution of Example 1 to Example 3 can maintain the phospholipid bilayer teabag-like vesicle structure after being stored at -80°C to 4°C for 1 month, 3 months and 12 months, and the membrane bubble edge is clear and the structure is complete, compared with the comparative example 1 stored in PBS. The exosomes stored in the protective solution of Example 1 to Example 3 are morphologically stable within 1-3 months at -80°C to 4°C, and the exosomes stored in the protective solution of Example 1 are more complete in structure.

[0152] III. Surface marker detection

[0153] Figure 5 and Figure 6 The results of 1 and 4 show that the exosomes in the protective solution of Example 1 to Example 3 can maintain the phospholipid bilayer teabag-like vesicle structure after being stored at -80°C to 4°C for 1 month, 3 months and 12 months, and the membrane bubble edge is clear and the structure is complete, compared with the comparative example 1 stored in PBS. The exosomes stored in the protective solution of Example 1 to Example 3 are morphologically stable within 1-3 months at -80°C to 4°C, and the exosomes stored in the protective solution of Example 1 are more complete in structure.

[0154] Figure 5 and Figure 6 The results of 1 and 4 show that the exosomes in the protective solution of Example 1 to Example 3 can maintain the phospholipid bilayer teabag-like vesicle structure after being stored at -80°C to 4°C for 1 month, 3 months and 12 months, and the membrane bubble edge is clear and the structure is complete, compared with the comparative example 1 stored in PBS. The exosomes stored in the protective solution of Example 1 to Example 3 are morphologically stable within 1-3 months at -80°C to 4°C, and the exosomes stored in the protective solution of Example 1 are more complete in structure.

[0155] IV. Fluorescent quantitative PCR detection of the expression of miR-26a-5p, miR-126-3p and miR-222-3p

[0156] Figure 7 The results of 1 and 4 show that the exosomes in the protective solution of Example 1 to Example 3 can maintain the phospholipid bilayer teabag-like vesicle structure after being stored at -80°C to 4°C for 1 month, 3 months and 12 months, and the membrane bubble edge is clear and the structure is complete, compared with the comparative example 1 stored in PBS. The exosomes stored in the protective solution of Example 1 to Example 3 are morphologically stable within 1-3 months at -80°C to 4°C, and the exosomes stored in the protective solution of Example 1 are more complete in structure.

[0157] Figure 8The results show that the contents of the three miRNAs in the exosomes stored by Comparative Example 1 decrease significantly, while the contents of miR-26a-5p, miR-126-3p and miR-222-3p in the samples stored by Examples 1 to 3 are all higher than those in Comparative Example 1.

[0158] In summary, the cryoprotective solution provided by Examples 1 to 3 of the present application is described to help understand the present application, and does not limit the present application.

[0159] The design focus of the present application is:

[0160] 1. The exosome protective solution provided by the present application can store exosomes at a temperature range of -80 to 4 DEG C, meeting the needs of long-term storage of exosomes under different temperature environments;

[0161] 2. The exosome protective solution provided by the present application can effectively improve the stability and integrity of exosomes during long-term storage, can maintain exosome particles for more than 12 months, and effectively avoid the occurrence of exosome aggregation, fusion and rupture, etc.

[0162] 3. The exosome protective solution provided by the present application has clear and safe components, and the adjuvants meet the pharmaceutical standards. The recombinant human serum albumin produced by genetic engineering technology does not contain animal-derived components, has more stable batch consistency and higher safety.

[0163] 4. The exosome protective solution provided by the present application can effectively maintain the biological activity of exosomes, and ensure the integrity of the structure and function of exosomes during long-term storage.

[0164] 5. The exosome protective solution provided by the present application uses a simple mixing and dissolving preparation process, which is simple to operate and easy to mass-produce.

[0165] 6. The exosome protective solution can store exosomes at a temperature range of -80 to 4 DEG C, improve the stability of exosome storage, effectively alleviate the degradation of exosome proteins and particles, maintain the normal vesicle size, and effectively protect the low-temperature activity of exosomes.

[0166] The above is only a preferred embodiment of the present application, and does not limit the technical scope of the present application. Therefore, any slight modification, equivalent change and modification made according to the technical essence of the present application to the above embodiment all fall within the scope of the technical solution of the present application.

Claims

1. An exosome protection solution, characterized by: The composition includes the following components by concentration: 0.01-0.1 g / mL of HEPEs, 0.01-0.1 g / mL of trehalose, 0.01-0.1 g / mL of mannitol, 8-10 g / L of recombinant human blood albumin, 0.01-0.1 g / mL of glycine and 0.01-0.1 g / mL of poloxamer 188.

2. The exosome protection solution of claim 1, wherein: The composition includes the following components by concentration: 0.01 g / mL of HEPEs, 0.01 g / mL of trehalose, 0.01 g / mL of mannitol, 8 g / L of recombinant human blood albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

3. A method of preparing an exosome protection solution according to any one of claims 1-2, characterized by: The method comprises the following steps: S1, dissolving HEPEs in pure water, stirring to prepare HEPEs buffer solution; S2, adding trehalose, mannitol and glycine to the HEPEs buffer solution, forming a mixed solution after complete dissolution, cooling to room temperature, and storing at 2-6℃; S3, adding recombinant human blood albumin and poloxamer 188 to the mixed solution and stirring uniformly, filtering with a filter to form a protective solution, and storing the protective solution at 2-6℃; S4, mixing the protective solution with exosomes at a ratio of 1:1 and storing at -80 to 4℃.

4. The method for preparing an exosome protective solution according to claim 3, characterized in that: In the step S1, the HEPEs buffer solution is prepared by stirring at 37℃ and 50 rpm / min.

5. The method for preparing an exosome protective solution according to claim 3, characterized in that: In the step S2, trehalose, mannitol and glycine are added to the HEPEs buffer solution at 50℃ and 80 rpm / min.

6. The method for preparing an exosome protective solution according to claim 3, characterized in that: In the step S3, the filter membrane of the filter has a pore diameter of 0.22 μm.

7. The method for preparing an exosome protective solution according to claim 3, characterized in that: In the step S2, the mixed solution is stored in a 4℃ refrigerator overnight; and in the step S3, the protective solution is stored in a 4℃ refrigerator.

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