Exosome preserving fluid and exosome preserving method

Abstract

The invention belongs to the technical field of biology, and relates to an exosome preserving fluid and an exosome preserving method. The preservation solution comprises glycerol with the concentration of 4-6% (V / V), trehalose with the concentration of 0.5-1.5% (W / V), albumin with the concentration of 0.05-0.2% (W / V), a sterile protective agent with the concentration of 0.01-0.03% (W / V), vitamin E with the concentration of 0.05-0.1% (W / V), ethylenediamine tetraacetic acid with the concentration of 0.05-0.2 mM, a protease inhibitor with the concentration of 0.05-0.1 mM and a basic solvent of 0.1 M phosphate buffer solution. The invention relates to an exosome preserving fluid and an exosome preserving method which are extremely low in toxic effect on a human body and can effectively preserve exosomes and maintain biological activity of the exosomes.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to an exosome preservation solution and a method for preserving exosomes. Background Art

[0002] Exosomes refer to small membrane vesicles (30 - 150 nm) containing complex RNA and proteins. Nowadays, they specifically refer to discoidal vesicles with a diameter of 40 - 100 nm. In 1983, exosomes were first discovered in sheep reticulocytes and were named "exosome" by Johnstone in 1987. A variety of cells can secrete exosomes under normal and pathological conditions. They mainly originate from multivesicular bodies formed by the invagination of lysosomal microparticles within cells and are released into the extracellular matrix after the fusion of the outer membrane of multivesicular bodies with the cell membrane. All cultured cell types can secrete exosomes (such as stem cells, immune cells, tumor cells, etc.), and exosomes naturally exist in body fluids, including blood, saliva, urine, cerebrospinal fluid, and milk. Exosomes are regarded as specifically secreted membrane vesicles that can participate in cell - to - cell communication. Their membranes are composed of a phospholipid bilayer and contain bioactive substances such as proteins, lipids, RNA (such as mRNA, miRNA), and DNA. The membrane is rich in cholesterol, sphingomyelin, and tetraspanins (such as CD63, CD81, CD9), and has high heterogeneity. Nowadays, the research on exosomes is deepening day by day. However, exosomes are not easy to preserve and are prone to decomposition after purification. How to preserve exosomes has become a major problem.

[0003] Therefore, to solve the above problems, the present invention provides an exosome preservation solution and a method for preserving exosomes. Summary of the Invention

[0004] Aiming at the above problems, the purpose of this application is to provide an exosome preservation solution and a method for preserving exosomes, specifically an exosome preservation solution and a method for preserving exosomes that have extremely low toxicity to the human body and can effectively preserve exosomes and maintain their biological activity.

[0005] To achieve the above - mentioned purpose, the present invention provides an exosome preservation solution. The preservation solution includes glycerol at a concentration of 4 - 6% (V / V), trehalose at 0.5 - 1.5% (W / V), albumin at 0.05 - 0.2% (W / V), a sterile protective agent at 0.01 - 0.03% (W / V), vitamin E at 0.05 - 0.1% (W / V), ethylenediaminetetraacetic acid at 0.05 - 0.2 mM, a protease inhibitor at 0.05 - 0.1 mM, and a basic solvent of 0.1 M phosphate buffer solution.

[0006] Furthermore, the albumin is at least one of bovine serum albumin or human serum albumin.

[0007] Further, the aseptic protectant is at least one of gentamicin, clavulanic acid, and sodium azide.

[0008] Further, the protease inhibitor is at least one of trypsin inhibitor, leucine aminopeptidase inhibitor, and phenylmethylsulfonyl fluoride.

[0009] The functions of the components of the above exosome preservation solution are as follows: Glycerol is used as an antifreeze agent. By forming hydrogen bonds between the hydroxyl groups of glycerol molecules and water molecules, the formation of ice crystals is reduced, and the mechanical damage caused by ice crystals is decreased. Secondly, glycerol embeds in the lipid bilayer, reducing membrane rigidity and maintaining the fluidity of the lipid membrane, preventing lipid solidification at low temperatures from causing membrane embrittlement and maintaining the fluidity of the lipid membrane. Trehalose is used as a vitrification inhibitor. By interfering with the arrangement of water molecules, it prevents the solution from entering the vitrified state, maintains the flexibility of the lipid membrane, prevents the lipid membrane from embrittling during cryopreservation, and keeps the discoidal structure of exosomes, having a synergistic antifreeze effect with glycerol. Bovine serum albumin has an anti-adhesion effect. It adsorbs on the inner wall of the cryopreservation tube through hydrophobic interaction, reducing the aggregation of exosomes caused by mechanical stress during cryopreservation. Secondly, it can occupy the potential adhesion sites on the membrane surface, reducing the adhesion between exosomes after cryopreservation, avoiding aggregation in the cryopreservation tube, and ensuring uniform dispersion after resuscitation. Sodium azide has an antibacterial effect. It destroys bacterial DNA through alkylation and inhibits bacterial proliferation. It has extremely low cytotoxicity to mammalian cells and meets the clinical-grade safety standard. Vitamin E is used as a fat-soluble antioxidant. It protects the unsaturated fatty acids in the exosome membrane from oxidative degradation by scavenging singlet oxygen and lipid peroxides. Ethylenediaminetetraacetic acid is used as a metal chelator. It binds to Fe 3+ / Cu 2+ to inhibit lipid peroxidation reactions, reduce the production of lipid peroxides, and protect unsaturated fatty acids. Phenylmethylsulfonyl fluoride is used as a protease inhibitor. It inactivates trypsin / elastase by covalently modifying serine residues, thereby inhibiting the degradation of exosome membrane proteins by endogenous proteases such as lysozyme; preventing the loss of exosome surface protein markers caused by protease activation during cryopreservation and protecting the integrity of membrane proteins. Phosphate buffer solution is used as a basic solvent to maintain the dynamic balance of pH. A stable pH environment prevents the folding or aggregation of proteins on the surface of the exosome membrane, thereby inhibiting protein denaturation. Secondly, it maintains the stability of osmotic pressure and provides a physiological environment for exosomes.

[0010] A method for preserving exosomes includes the following steps: S1. Mix the formulated amounts of glycerol, trehalose, albumin, aseptic protectant, vitamin E, ethylenediaminetetraacetic acid, and protease inhibitor, and then add them to phosphate buffer solution for dissolution to obtain an exosome preservation solution; S2. Adjust the pH of the above exosome preservation solution to 7.2 - 7.6, and mix the exosome stock solution and the preservation solution in a certain proportion to obtain a suspension; S3. Aliquot and sterilize the suspension obtained in step S2, and then perform programmed cooling to -80°C for cryopreservation; S4. When needed, thaw the cryopreserved suspension.

[0011] Furthermore, steps S1, S2, and S3 need to be carried out at 4°C.

[0012] Furthermore, the certain ratio in step S2 is 1:5 - 1:20 (V / V).

[0013] Furthermore, the programmed cooling rate in step S3 is 1 - 2°C / min.

[0014] In summary, the present application has the following beneficial effects: 1. An exosome preservation solution and an exosome preservation method provided by the present application can effectively improve the stability of exosomes, maintain the biological activity of exosomes, and extend the preservation time of exosomes through the synergistic effect of its components.

[0015] 2. The raw materials of the exosome preservation solution of the present application all meet international standards and domestic regulations. The preservation solution of the present invention causes extremely little damage to human cells, tissues, and organs, and has the effects of safety, non-toxicity, and high efficiency. Specific Embodiments

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] The raw materials involved in the specific embodiments of the present application are of pharmaceutical grade. In addition, bovine serum albumin is heat-denatured (heated at 80°C for 30 minutes) to reduce its immunogenicity. Bovine serum albumin is selected from Sigma-Aldrich, product number A7906; phenylmethylsulfonyl fluoride is highly purified, specifically selected from Sigma-Aldrich, product number P8111; the source of the exosome stock solution is purified mesenchymal stem cell exosomes.

[0018] Example 1 A method for preserving exosomes, comprising the following steps: S1. Mix 4 mL (V / V) glycerol, 0.5 g (W / V) trehalose, 0.1 g (W / V) bovine serum albumin, 0.02 g (W / V) sodium azide, 0.1 g (W / V) vitamin E, 0.1 mL of 100 mM ethylenediaminetetraacetic acid solution, and 0.1 mL of 100 mM phenylmethylsulfonyl fluoride solution at 4°C, and then add 0.1 M phosphate buffer solution to make up to 100 mL for dissolution to obtain the exosome preservation solution; S2. At 4°C, finely adjust the pH of the above exosome preservation solution to 7.4 with 0.1 mol / L NaOH / HCl, and mix the exosome stock solution (5 mg / mL) and the preservation solution at a ratio of 1:10 (V / V) to obtain a suspension; S3. Let the suspension obtained in step S2 stand at 4°C for 30 min, aliquot it into 0.5 mL sterile PCR tubes with a pipette, then perform gamma-ray sterilization (20 kGy, 1 h), and then place it at 4°C for 1 h and then perform programmed cooling to -80°C (cooling rate -1°C / min) for freezing; S4. When needed, resuscitate the frozen suspension.

[0019] Example 2 A method for preserving exosomes, comprising the following steps: S1. Mix 4 mL (V / V) glycerol, 0.5 g (W / V) trehalose, 0.1 g (W / V) bovine serum albumin, 0.02 g (W / V) sodium azide, 0.1 g (W / V) vitamin E, 0.1 mL of 100 mM ethylenediaminetetraacetic acid solution, and 0.1 mL of 100 mM phenylmethylsulfonyl fluoride solution at 4°C, and then add 0.1 M phosphate buffer solution to make up to 100 mL for dissolution to obtain the exosome preservation solution; S2. At 4°C, finely adjust the pH of the above exosome preservation solution to 7.4 with 0.1 mol / L NaOH / HCl, and mix the exosome stock solution (5 mg / mL) and the preservation solution at a ratio of 1:10 (V / V) to obtain a suspension; S3. Let the suspension obtained in step S2 stand at 4°C for 30 min, aliquot it into 0.5 mL sterile PCR tubes with a pipette, then perform gamma-ray sterilization (20 kGy, 1 h), and then place it at 4°C for 1 h and then perform programmed cooling to -80°C (cooling rate -1°C / min) for freezing; S4. When needed, resuscitate the frozen suspension.

[0020] Example 3 A method for preserving exosomes, comprising the following steps: S1. Mix 4 mL (V / V) glycerol, 0.5 g (W / V) trehalose, 0.1 g (W / V) bovine serum albumin, 0.02 g (W / V) sodium azide, 0.1 g (W / V) vitamin E, 0.1 mL of 100 mM ethylenediaminetetraacetic acid solution, and 0.1 mL of 100 mM phenylmethylsulfonyl fluoride solution at 4°C, and then add 0.1 M phosphate buffer solution to make up to 100 mL for dissolution to obtain the exosome preservation solution; S2. At 4°C, finely adjust the pH of the above exosome preservation solution to 7.4 with 0.1 mol / L NaOH / HCl, and mix the exosome stock solution (5 mg / mL) and the preservation solution in a ratio of 1:10 (V / V) to obtain a suspension; S3. Let the suspension obtained in step S2 stand at 4°C for 30 min, aliquot it into 0.5 mL sterile PCR tubes with a pipette, then perform gamma-ray sterilization (20 kGy, 1 h), and then place it at 4°C for 1 h and then perform programmed cooling to -80°C (cooling rate -1°C / min) for freezing; S4. When needed, resuscitate the frozen suspension.

[0021] Control Example 1 The difference between this control example and Example 1 is that the exosome stock solution (5 mg / mL) and the preservation solution are mixed in a ratio of 1:4 (V / V).

[0022] Control Example 2 The difference between this control example and Example 2 is that direct freezing at -80°C is performed.

[0023] Control Example 3 The difference between this control example and Example 3 is that vitamin E is used instead of ethylenediaminetetraacetic acid.

[0024] Performance Test Perform performance tests on the frozen solutions obtained in Examples 1 - 3 and Control Examples 1 - 3 of this application. Take the exosome preservation solutions prepared in each test group, three replicates for each group, and respectively test the exosome concentration (using the NTA method), particle size distribution PDI (using DLS), RNA integrity RIN (using Agilent Bioanalyzer), cell proliferation induction rate (measuring H9c2 cardiomyocytes, using the CCK-8 method), and surface protein marker CD9 / CD63 / CD81 positive rate (using Western Blot) after resuscitation at 0 month, 1 month, 3 months, and 6 months of freezing. Take 20 μL for each index and calculate the average value.

[0025] Table 1: 0 month Table 2: Resuscitation after 1 month of freezing Table 3: Recovery after cryopreservation for 3 months Table 4: Recovery after cryopreservation for 6 months As can be seen from the above tables, compared with Table 1, the concentrations, RIN, cell proliferation induction rates, and surface protein markers (positive rates) in Tables 2, 3, and 4 all decreased more or less, while the PDI increased. It can be seen from Tables 2, 3, and 4 that compared with Example 1, the concentration of exosomes in Control Example 1 increased, but the actual detection results of each index, including concentration, RIN, surface protein marker (positive rate), and PDI, were far less than those in Example 1; compared with Example 2, Control Example 2 changed the cryopreservation method, and there was a large gap in the index detection results, including concentration, RIN, cell proliferation induction rate, surface protein marker (positive rate), and PDI, compared with Example 2, and its cryopreservation effect was far inferior to that of Example 2; compared with Example 3, in Control Example 3, the concentrations of all actual indexes, RIN, and surface protein markers (positive rates) decreased to a large extent, and the PDI increased, and the result was inferior to that of Example 3.

[0026] The above content is only an example and illustration of the present invention. Those skilled in the art of this technology can make various modifications, supplements, or use similar methods to replace the specific embodiments described, as long as they do not deviate from the invention or exceed the scope defined by this claim book, they shall fall within the protection scope of the present invention.

Claims

1. An exosome preservation solution, characterized in that, The preservation solution comprises glycerol at a concentration of 4 - 6% (V / V), trehalose at 0.5 - 1.5% (W / V), albumin at 0.05 - 0.2% (W / V), a sterile protectant at 0.01 - 0.03% (W / V), vitamin E at 0.05 - 0.1% (W / V), ethylenediaminetetraacetic acid at 0.05 - 0.2 mM, a protease inhibitor at 0.05 - 0.1 mM, and a basic solvent of 0.1 M phosphate buffer solution.

2. The exosome preservation solution according to claim 1, wherein The albumin is at least one of bovine serum albumin and human serum albumin.

3. The exosome preservation solution according to claim 1, wherein, The sterile protectant is at least one of gentamicin, clavulanic acid, and sodium azide.

4. The exosome preservation solution according to claim 1, characterized in that, The protease inhibitor is at least one of trypsin inhibitor, leucine aminopeptidase inhibitor, and phenylmethylsulfonyl fluoride.

5. A method for preserving exosomes with the exosome preservation solution according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Mix the formula amounts of glycerol, trehalose, albumin, sterile protectant, vitamin E, ethylenediaminetetraacetic acid, and protease inhibitor, and then add them to the phosphate buffer solution for dissolution to obtain the exosome preservation solution. S2. Adjust the pH of the above exosome preservation solution to 7.2 - 7.6, and mix the exosome stock solution and the preservation solution in a certain ratio to obtain a suspension. S3. Aliquot, sterilize, and cool the suspension obtained in step S2 at a rate of 1 - 2 °C / min to -80 °C for freezing. S4. During use, resuscitate the frozen suspension.

6. The method for preserving exosomes with the exosome preservation solution according to claim 5, characterized in that, Steps S1, S2, and S3 need to be carried out at 4 °C.

7. A method for preserving exosomes using the exosome preservation solution according to claim 5, characterized in that, For the mixing in step S2, the volume ratio is 1:5 - 1:

20.

8. A method for preserving exosomes using the exosome preservation solution according to claim 5, characterized in that, For the programmed cooling in step S3, the rate is 1 - 2 °C / min.

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