Exosome protection liquid and preparation method thereof

Abstract

The invention discloses an exosome protection solution and a preparation method thereof, and relates to the technical field of biology, and the exosome protection solution comprises the following components by concentration: 0.01 to 0.1 g / mL of HEPEs, 0.01 to 0.1 g / mL of trehalose, 0.01 to 0.1 g / mL of mannitol, 8 to 10 g / L of recombinant human serum albumin, 0.01 to 0.1 g / mL of glycine and 0.01 to 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, trehalose, mannitol and glycine are added into the HEPEs buffer solution, and a mixed solution is formed after the trehalose, the mannitol and the glycine are completely dissolved; s3, adding recombinant human serum albumin and poloxamer 188 into the mixed solution, and uniformly stirring; s4, mixing the protective liquid and the exosome according to a ratio of 1: 1; the exosome protection liquid provided by the invention can be used for preserving the exosome in a temperature range of-80 DEG C to 4 DEG C, so that the requirement of preserving the exosome for a long time in different temperature environments is met; the protection liquid can effectively improve the stability and integrity of the exosome in the long-term storage process.

Description

Technical Field

[0001] The present invention relates to biological field technology, and in particular to an exosome protective solution and a preparation method thereof. Background Art

[0002] Exosomes are a type of extracellular vesicles with a size of 40-200 nm. Exosomes can be used as a new mode of information exchange between cells. They can regulate various biological processes under physiological and pathological conditions by carrying active factors such as miRNA, mRNA and proteins. MiRNA is one of the main factors for the function of exosomes. They regulate gene expression by binding to target genes, resulting in mRNA translation inhibition or transcript degradation. Exosomal miRNA is not only of great significance in the prevention and control of diseases, but more and more studies are trying to alleviate or treat diseases through exosomal miRNA; for example, studies have shown that miRNAs such as miR-26a-5p, miR-126-3p and miR-222-3p can reduce inflammation levels, promote cell proliferation, and effectively treat skin injuries.

[0003] The International Extracellular Vesicle Association recommends suspending exosomes with PBS and storing them in a -80℃ refrigerator. However, under this condition, the membrane of exosomes is easily affected by the physical and chemical factors of exosomes (such as pH, temperature and osmotic pressure), resulting in changes, such as exosome membrane fusion, rupture, shrinkage and membrane flipping, and even lipid, protein and nucleic acid damage, and miRNA degradation. Moreover, as time goes by, the number of exosome particles gradually decreases, and the amount of protein and miRNA expression decreases. In addition, the clinical transformation of the use of exosomes to treat diseases has market prospects, and ultra-low temperature transportation will cause a huge additional economic burden.

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

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

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

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

[0008] 4. There is a problem that the exosome protection solution has limited protective effect on exosomes and fails to effectively maintain the biological activity of exosomes;

[0009] 5. Some exosome protective fluids contain animal-derived ingredients, which pose potential safety risks and have poor consistency between batches.

[0010] Patent CN111226902A discloses an exosome protective solution and a method for preserving exosomes. The exosome protective solution includes bovine serum albumin, trehalose, glycerol, dimethyl sulfoxide and phosphate buffer. The exosome protective solution and the preservation method can effectively maintain the integrity, biological activity and stability of the contents of the exosomes. However, dimethyl sulfoxide is cytotoxic, 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-mentioned exosome protective 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 raw materials for preparing 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 exosomes and keep the exosome activity. However, the exosome protection solution is only suitable for storing exosomes at a low temperature of -20°C, and cannot meet the storage conditions of exosomes at -80°C and 4°C.

[0012] Therefore, the development of an exosome protective solution that can stably store exosomes and its preparation method is of great significance for the future clinical transformation of exosomes. Summary of the invention

[0013] In view of this, the present invention aims at the deficiencies in the prior art, and its main purpose is to provide an exosome protective solution and a preparation method thereof. The exosome protective solution provided by the present invention can preserve exosomes within a temperature range of -80°C to 4°C, meeting the demand for long-term preservation of exosomes under different temperature environments; the exosome protective solution provided by the present invention can effectively improve the stability and integrity of exosomes during long-term preservation, and can maintain the preservation of exosome particles for more than 12 months, effectively avoiding the occurrence of exosome aggregation, fusion and rupture; the exosome protective solution provided by the present invention adopts a simple mixed dissolution preparation process, which is easy to operate and easy to mass produce.

[0014] To achieve the above purpose, the present invention adopts the following technical solutions:

[0015] An 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 albumin, 0.01-0.1 g / mL of glycine and 0.01-0.1 g / mL of poloxamer 188.

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

[0017] The method for preparing an exosome protective solution comprises the following steps:

[0018] S1. Dissolve HEPEs in pure water and stir to prepare HEPEs buffer solution;

[0019] S2. Add trehalose, mannitol and glycine to the HEPEs buffer, dissolve them completely to form a mixed solution, cool to room temperature, and store at 2-6°C;

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

[0021] S4. Mix the protective solution and exosomes in a 1:1 ratio and store at -80 to 4°C.

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

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

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

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

[0026] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that the present application provides an exosome protection solution and a preparation method thereof:

[0027] 1. The exosome protection solution provided by the present invention can preserve exosomes in a temperature range of -80°C to 4°C, meeting the demand for long-term preservation of exosomes in different temperature environments;

[0028] 2. The exosome protection solution provided by the present invention can effectively improve the stability and integrity of exosomes during long-term storage, can maintain the storage of exosome particles for more than 12 months, and effectively avoid the occurrence of exosome aggregation, fusion and rupture;

[0029] 3. The exosome protection solution provided by the present invention has clear ingredients and is safe and non-toxic. The auxiliary materials all meet the pharmaceutical standards. The recombinant human serum albumin produced by genetic engineering technology does not contain animal-derived components, and the consistency between batches is more stable and the safety is higher.

[0030] 4. The exosome protection solution provided by the present invention can effectively maintain the biological activity of exosomes and ensure the structural and functional integrity of exosomes during long-term storage;

[0031] 5. The exosome protection solution provided by the present invention adopts a simple mixed dissolution preparation process, which is easy to operate and easy to mass produce;

[0032] 6. Exosome protection solution can preserve exosomes at -80℃ to 4℃, improve the storage stability of exosomes, effectively alleviate the degradation of exosome proteins and particles, maintain their normal vesicle size, and effectively protect the low-temperature activity of exosomes.

[0033] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the electron microscopic result of storage of exosomes at -80°C for 0, 1, 3, 6, 9 and 12 months in Comparative Example 1 of the present invention;

[0035] Figure 2 The electron microscopy results of storing exosomes at -80°C to 4°C for 1 month in Examples 1-3 of the present invention and Comparative Example 1;

[0036] Figure 3 The electron microscopy results of storing exosomes at -80°C to 4°C for 3 months in Examples 1-3 of the present invention and Comparative Example 1 are shown;

[0037] Figure 4 The electron microscopy results of storing exosomes at -80°C to 4°C for 12 months in Examples 1-3 of the present invention and Comparative Example 1;

[0038] Figure 5 This is a Western Blot detection diagram of surface markers of exosomes stored at -80°C to 4°C for 3 months in Examples 1-3 of the present invention and Comparative Example 1;

[0039] Figure 6This is a Western Blot detection diagram of surface markers of exosomes stored at -80°C to 4°C for 12 months in Examples 1-3 of the present invention and Comparative Example 1;

[0040] Figure 7 The results of the fluorescence quantitative PCR detection of miR-26a-5p, miR-126-3p and miR-222-3p in the samples of freshly extracted exosomes suspended in Examples 1-3 of the present invention and Comparative Example 1 are shown;

[0041] Figure 8 The results of fluorescent quantitative PCR detection of miR-26a-5p, miR-126-3p and miR-222-3p contents in Examples 1-3 of the present invention and Comparative Example 1 at -80°C to 4°C for 12 months. DETAILED DESCRIPTION

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

[0043] The protective solution includes 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 albumin, 0.01 g / mL of glycine and 0.01 g / mL of poloxamer 188.

[0044] The method for preparing an exosome protective solution comprises the following steps:

[0045] S1. Dissolve HEPEs in pure water and stir to prepare HEPEs buffer solution;

[0046] S2. Add trehalose, mannitol and glycine to the HEPEs buffer, dissolve them completely to form a mixed solution, cool to room temperature, and store at 2-6°C;

[0047] S3, adding recombinant human albumin and poloxamer 188 to the mixed solution, stirring evenly, filtering with a filter to form a protective solution, and storing the protective solution at 2-6°C;

[0048] S4. Mix the protective solution and exosomes in a 1:1 ratio and store at -80 to 4°C.

[0049] In step S1, HEPEs buffer was prepared by stirring at 37° C. and 50 rpm / min.

[0050] In step S2, trehalose, mannitol and glycine are added to the HEPEs buffer at 50° C. and 80 rpm / min.

[0051] The filter membrane pore diameter of the filter used in step S3 is 0.22 μm.

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

[0053] In the examples 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., and mannitol and recombinant human albumin were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0054] The protective liquid excipient meets pharmaceutical standards and is suitable for a wide temperature range of -80°C to 4°C. It can significantly improve the stability and integrity of exosomes, ensuring that their particles are well preserved within 12 months and avoiding aggregation, fusion and rupture.

[0055] In order to achieve the above objectives, the present invention adopts a scientific technical solution and provides an exosome protection solution, whose components include HEPEs, trehalose, mannitol, recombinant human serum albumin, glycine and poloxamer 188; the protection solution can provide comprehensive protection for exosomes during the storage process.

[0056] HEPEs is a zwitterionic buffer. As one of the important components of the protective solution, its main function is to stabilize the pH value of the aqueous phase inside and outside the exosomes. Since HEPEs is a buffer pair that does not easily pass through biological membranes, it can effectively prevent pH fluctuations in the internal and external environments of exosomes, thereby maintaining the stability of the exosome structure.

[0057] Trehalose is a non-reducing disaccharide that plays a key role as an excipient in the protective solution. It can protect the structure of biological macromolecules from damage, and at the same time, by encapsulating high concentrations of proteins in the nanoclusters they form, it allows them to exist in a stable colloidal form. In addition, trehalose can inhibit the formation of large ice crystals during the freezing process, making the ice crystals smaller and more delicate, thereby reducing damage to surrounding tissues. This property can not only improve the preservation effect of the product at low temperatures, but also slow down the thawing speed and reduce dehydration after freeze-thaw.

[0058] Mannitol also plays an important role in exosome cryopreservation. It prevents protein aggregation by forming an amorphous structure, thereby 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 and ensure their stability during cryopreservation.

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

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

[0061] Poloxamer 188 is a nonionic linear copolymer with surfactant properties, which is usually used to improve the stability and solubility of drugs. In the exosome protection solution, poloxamer 188 reduces or eliminates the adsorption of proteins on different contact interfaces, reduces the aggregation and damage of exosomes during the freezing process, and thus maintains the biological activity and structural integrity of exosomes.

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

[0063] Example 1

[0064] The present embodiment provides an exosome protective solution, wherein the raw materials for preparing the exosome protective solution include, in terms of mass 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 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 protective solution, wherein the raw materials for preparing the exosome protective solution 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 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, wherein the raw materials for preparing the exosome protection solution 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 albumin, 0.1 g / mL of glycine, and 0.1 g / mL of poloxamer 188.

[0069] Comparative Example 1

[0070] In this comparative example, PBS solution recommended by the International Extracellular Vesicle Association was used to preserve exosomes.

[0071] Comparative Example 2

[0072] The present embodiment provides an exosome protection solution, wherein the raw materials for preparing the exosome protection solution 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 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, wherein the raw materials for preparing the exosome protection solution 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 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 protective solution, wherein the raw materials for preparing the exosome protective solution 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 albumin, 0.05 g / mL of glycine, and 0.05 g / mL of poloxamer 188.

[0077] The preparation methods of the exosome protective solutions in Examples 1 to 3 and Comparative Examples 2 to 4 are all:

[0078] S1. Dissolve HEPEs in pure water and stir to prepare HEPEs buffer solution;

[0079] S2. Add trehalose, mannitol and glycine to the HEPEs buffer, dissolve them completely to form a mixed solution, cool to room temperature, and store at 2-6°C;

[0080] S3, adding recombinant human albumin and poloxamer 188 to the mixed solution, stirring evenly, filtering with a filter to form a protective solution, and storing the protective solution at 2-6°C;

[0081] S4. Mix the protective solution and exosomes in a 1:1 ratio and store at -80 to 4°C.

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

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

[0084] The filter membrane pore diameter of the filter used in step S3 is 0.22 μm.

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

[0086] Example of exosome extraction and detection:

[0087] 1. Exosome Extraction

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

[0089] 2. Exosome Detection Methods

[0090] 1. Determination of exosome protein concentration

[0091] The protein concentration of exosome samples was detected using the Micro BCA protein quantification kit (Thermo Fisher Scientific, USA, Cat. No. 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 multiples of 10, 20, and 50 times;

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

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

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

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

[0098] 2. Determination of exosome particle size and particle concentration

[0099] The particle size and concentration of exosomes were detected using a nanoparticle tracking analyzer (Malvern Panalytical, UK, model Nanosight300)

[0100] 1) Preparation for the machine

[0101] Install the pipeline of the nanoparticle size tracking analyzer, clean the pipeline with PBS, and calibrate the instrument.

[0102] 2) Sample preparation

[0103] The test samples were diluted with PBS 500 times, 1000 times and 2000 times, respectively, that is, the ratio of exosome stock solution to PBS was 1:499, 1:999 and 1:1999.

[0104] 3) Sample testing

[0105] Use a syringe to draw the sample, exhaust all the bubbles in the syringe, and inject the sample to be tested into the sample pool for on-machine testing.

[0106] 3. Exosome morphology detection

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

[0108] 2) Gently place the copper mesh on the sample droplet to be tested and adsorb for 10 minutes;

[0109] 3) Move it onto a drop of 4% paraformaldehyde, fix it for 10 minutes, and then wash the copper grid twice with DPBS;

[0110] 4) Move it onto a drop of 2.5% pentanediol, fix it for 10 minutes, and then wash the copper grid with DPBS 5 times;

[0111] 5) Move it onto a drop of 3% uranyl acetate, negatively stain for 30 minutes, move it onto filter paper, absorb excess liquid, and let it dry for 30 minutes.

[0112] 6) Use a 120kV cryo-electron microscope equipped with a digital camera and image analysis software for observation and detection.

[0113] 4. Detection of exosome surface marker proteins

[0114] 1) Protein sample preparation

[0115] The samples were fully lysed on ice with RIPA lysis buffer containing PMSF, and after adding Loading Buffer, they were placed in a 100°C metal bath and boiled for 10 min.

[0116] 2) Protein electrophoresis and membrane transfer

[0117] The samples to be tested were added to the wells of SDS-PAGE precast gel (Shanghai Bio-Tech, Cat. No. P0465S), and after electrophoresis and transfer, the PVDF membrane was blocked with 5% bovine serum albumin at room temperature for 2 h.

[0118] 3) Antibody incubation

[0119] The blocked strips were incubated with GM130 (Abcam, UK, Catalog No. ab52649), ALIX (Abcam, UK, Catalog No. ab186429), Calnexin (Abcam, UK, Catalog No. ab92573), HSP70 (Abcam, UK, Catalog No. ab181606), TSG101 (Abcam, UK, Catalog No. ab125011), CD63 (Abcam, UK, Catalog No. ab134045), GAPDH (CST, USA, Catalog No. 2118S) at 4°C overnight; after primary antibody incubation, the strips were washed 3 times with PBST, 5 min each time; the strips were incubated with HRP-labeled goat anti-rabbit IgG (H+L) (Shanghai Biyuntian, Catalog No. A0208) secondary antibody at 37°C for 2 h;

[0120] 4) Band development

[0121] The strips incubated with the secondary antibody were washed three times with PBST, each time for 5 min; the strips were exposed and developed using an ECL luminescence kit (Shanghai Biotech, catalog number P0018AS).

[0122] 5. Detection of miR-26a-5p, miR-26-3p and miR-222-3p expression by fluorescence quantitative PCR

[0123] Total RNA was extracted from the exosome solution using miRNeasy Micro Kit (QIAGEN, USA, Cat. No. 217084), reverse transcribed using cDNA synthesis kit (Shanghai Bioengineering), and qRT-PCR was performed using miRNA fluorescence quantitative kit on a fluorescence quantitative PCR system (Roche, Switzerland). U6 was used as an internal reference gene. The relative expression level was then calculated using the 2-ΔΔCT method.

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

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

[0126] Table 1 records the detection of protein concentration, particle size and particle concentration of the three batches of exosomes stored in Comparative Example 1 at 0, 1, 3, 6, 9 and 12 months in a -80°C refrigerator. The results show that the changes in protein concentration, particle concentration and particle size of the 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 downward.

[0127] Figure 1 The morphological tests were performed on the three batches of exosomes stored in the -80°C refrigerator at 0, 1, 3, 6, 9 and 12 months in Comparative Example 1. The results showed that over time, the exosomes of each batch underwent aggregation, rupture and fusion to varying degrees at each time point.

[0128] Based on the above research results, when stored at low temperature for a long time, the changes in protein concentration, particle concentration and particle size of exosomes in comparative example 1 using PBS solution as a protective agent were unstable, and there was a tendency for vesicle degradation, and vesicles would aggregate, rupture and fuse. Therefore, it is very important to develop a preservative that can preserve exosomes over a wide temperature range and for a long time for future product transportation, storage and transformation.

[0129] Table 1: Test results of protein concentration, particle size and particle concentration of three batches of exosomes stored in Comparative Example 1 at 0, 1, 3, 6, 9 and 12 months

[0130]

[0131] Effect Example 2: Initial screening of exosome protective solution

[0132] The exosome dilution was mixed with Examples 1 to 3 and Comparative Examples 1 to 4 in a 1:1 ratio, and placed in refrigerators at -80°C, -20°C, and 4°C, respectively. The protein concentration, particle size, and particle concentration were detected after 7, 14, and 28 days.

[0133] 1. 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 endosomal samples in Examples 1 to 3 and Comparative Examples 1 to 4 after 7, 14 and 28 days of storage in -80°C, -20°C and 4°C refrigerators, respectively.

[0135] Table 2: Protein concentration, particle concentration and particle size test results

[0136]

[0137]

[0138]

[0139] The above results show that compared with Comparative Examples 1 to 4, at -80°C, -20°C and 4°C, the exosome samples preserved in Examples 1 to 3 have good stability in protein concentration, particle concentration and particle size after 7, 14 and 28 days of preservation, low degree of degradation, and particle size maintained within the size range of exosome vesicles. However, the exosomes in Comparative Example 1 degraded rapidly in protein and particles at different temperatures, and the concentration of exosomes in Comparative Examples 2 to 4 changed unstably. In summary, the protective solutions of Examples 1 to 3 can more stably preserve exosome samples for 28 days at -80°C to 4°C.

[0140] Effect Example 3: Re-verification of exosome protective solution

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

[0142] 1. Exosome protein concentration, particle concentration and particle size results

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

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

[0145]

[0146]

[0147]

[0148] The results show that compared with the comparative example 1 preserved with traditional PBS, the exosome samples preserved in Examples 1 to 3 have good stability in protein concentration, particle concentration and particle size after 1 month, 3 months and 12 months under the conditions of -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 it still has a good effect in preventing particle degradation. In summary, the protective solutions of Examples 1 and 2 are more suitable for stably preserving exosome samples for 12 months under the conditions of -80°C to 4°C, especially when preserved in Example 1, the exosome protein and particle concentrations are higher.

[0149] 2. Morphology Detection

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

[0151] Figure 2 , 3The results of and 4 show that compared with the comparative example 1 preserved in PBS, the exosomes in the protective solution of Examples 1 to 3 can maintain the saucer-shaped vesicle structure of the phospholipid bilayer after being stored at -80°C to 4°C for 1 month, 3 months and 12 months, with clear membrane vesicle edges and intact structure. Under the conditions of -80°C to 4°C, the exosomes preserved in Examples 1 to 3 are morphologically stable within 1-3 months, especially the exosomes preserved in Example 1 have a more complete structure.

[0152] 3. Surface marker detection

[0153] Figure 5 and Figure 6 The figures respectively show the Western Blot detection results of surface markers of exosome samples after being stored for 3 months and 12 months at -80°C, -20°C and 4°C using the protective solutions of Examples 1 to 3 and Comparative Example 1.

[0154] Figure 5 and Figure 6 The results show that under the conditions of -80°C, -20°C and 4°C, the exosomes in the protective solutions of Examples 1 to 3 and Comparative Example 1 still detected the expression of positive surface markers ALIX, HSP70 and CD63 after storage for 3 months and 12 months, and the expression of negative surface markers GM130 and Calnexin was not detected. This shows that the protective solutions of Examples 1 to 3 can stably preserve exosomes for up to 12 months, especially the positive marker expression of the exosomes stored in Example 1 at -80°C to 4°C is higher.

[0155] 4. Detection of miR-26a-5p, miR-126-3p and miR-222-3p expression by quantitative PCR

[0156] Figure 7 It means that after the freshly extracted exosomes are suspended with the protective solution of Examples 1 to 3 and Comparative Example 1, the content of miR-26a-5p, miR-126-3p and miR-222-3p in the sample is detected by fluorescent quantitative PCR. The results show that there is no obvious expression difference in the content of miR-26a-5p, miR-126-3p and miR-222-3p in the exosomes suspended with Examples 1 to 3 and Comparative Example 1.

[0157] Figure 8It is shown that after the exosome samples were stored at -80°C, -20°C and 4°C for 12 months using the protective solution of Examples 1 to 3 and Comparative Example 1, the expression of miR-26a-5p, miR-126-3p and miR-222-3p was detected by fluorescent quantitative PCR. The results showed that the contents of these three miRNAs in the exosomes stored in Comparative Example 1 decreased significantly, while the contents of miR-26a-5p, miR-126-3p and miR-222-3p in the samples stored in Examples 1 to 3 were all higher than those in Comparative Example 1.

[0158] In summary, the cryoprotectant liquid freezing examples provided in Examples 1 to 3 of the present application are provided to illustrate the present invention in order to help understand the present invention, and do not limit the present invention.

[0159] The design emphasis of the present invention is:

[0160] 1. The exosome protection solution provided by the present invention can preserve exosomes in a temperature range of -80°C to 4°C, meeting the demand for long-term preservation of exosomes in different temperature environments;

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

[0162] 3. The exosome protection solution provided by the present invention has clear ingredients and is safe and non-toxic. The excipients all meet the pharmaceutical standards. The recombinant human serum albumin produced by genetic engineering technology does not contain animal-derived ingredients, and the consistency between batches is more stable and the safety is higher.

[0163] 4. The exosome protection solution provided by the present invention can effectively maintain the biological activity of exosomes and ensure the structural and functional integrity of exosomes during long-term storage;

[0164] 5. The exosome protection solution provided by the present invention adopts a simple mixed dissolution preparation process, which is easy to operate and easy to mass produce;

[0165] 6. Exosome protection solution can preserve exosomes at -80℃ to 4℃, improve the storage stability of exosomes, effectively alleviate the degradation of exosome proteins and particles, maintain their normal vesicle size, and effectively protect the low-temperature activity of exosomes.

[0166] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An exosome protection solution, characterized in that: The composition includes 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 albumin, 0.01-0.1 g / mL of glycine and 0.01-0.1 g / mL of poloxamer 188.

2. The exosome protection solution according to claim 1, characterized in that: The composition includes the following components in terms of concentration: 0.01 g / mL HEPEs, 0.01 g / mL trehalose, 0.01 g / mL mannitol, 8 g / L recombinant human albumin, 0.01 g / mL glycine and 0.01 g / mL poloxamer 188.

3. A method for preparing an exosome protection solution according to any one of claims 1 to 2, characterized in that: The steps include: S1. Dissolve HEPEs in pure water and stir to prepare HEPEs buffer solution; S2. Add trehalose, mannitol and glycine to the HEPEs buffer, dissolve them completely to form a mixed solution, cool to room temperature, and store at 2-6°C; S3, adding recombinant human albumin and poloxamer 188 to the mixed solution, stirring evenly, filtering with a filter to form a protective solution, and storing the protective solution at 2-6°C; S4. Mix the protective solution and exosomes in a 1:1 ratio and store at -80 to 4°C.

4. The method for preparing an exosome protective solution according to claim 3, characterized in that: In the step S1, HEPEs buffer is prepared by stirring at 37° C. 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 at 50° C. and 80 rpm / min.

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

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

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