Exosome preserving fluid as well as preparation method and application thereof

Abstract

The invention discloses an exosome preserving fluid as well as a preparation method and application thereof. The exosome preserving fluid comprises Tween, albumin and a buffer solution. The exosome preserving fluid which is simple in formula and capable of effectively maintaining the activity and quantity of exosomes is developed, and the exosome preserving fluid can be used for short-term preservation, long-term cryopreservation and freeze-drying preservation of the exosomes through synergistic cooperation of the Tween, the albumin and the buffer solution.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an exosome preservation solution and a preparation method and application thereof. Background Art

[0002] Exosomes are extracellular vesicles with a diameter ranging from about 40 to 160 nm (average of about 100 nm), which can contain many components of cells, including DNA, RNA, lipids, metabolites, and cytoplasmic and cell surface proteins. Exosomes participate in the transport of substances and also carry and transmit important biological information. They play an important role in mediating immune antigen presentation, dendritic cell activation, neurodegenerative diseases, tumor cell antigen transfer, and tumor diagnosis and treatment.

[0003] Exosomes have a double-layer lipid membrane vesicle structure with good stability, which can protect the internal biological molecules from the influence of various enzymes in body fluids, thereby maintaining their integrity and biological activity. However, the integrity and biological activity of the extracted exosomes may be affected by factors such as the storage medium, storage temperature and time. At present, exosomes are generally suspended in PBS buffer after extraction, and the most commonly used storage method is cryopreservation. However, cryopreservation may cause changes in the shape and physical properties of exosomes, and may also lead to the formation and aggregation of multilayer vesicles. Repeated freezing and thawing will cause changes in the biological characteristics, content and marker composition of molecules on the surface of exosomes. Although -80℃ is recognized as the most suitable storage temperature for various biological specimens, the freezing and thawing process can easily cause vesicle destruction, resulting in a large number of "contaminants" in the exosomes, and it is sometimes difficult to maintain such low temperature conditions during processing or transportation. Although 4℃ storage can keep the vesicles intact, it is easy to cause the loss of proteins and nucleic acids in the exosomes. Therefore, how to effectively preserve exosomes has always been a difficult problem in the field of exosome research. The development of preservation solutions and preservation methods is one of the hot spots in the field of exosome research.

[0004] For example, CN111226902A discloses an exosome preservation solution and a method for preserving exosomes, wherein the exosome preservation solution includes bovine serum albumin, trehalose, glycerol, dimethyl sulfoxide and phosphate buffer, and the exosome preservation solution is heated at 85°C to 95°C for 10 to 30 minutes to form a colloidal solution, and then the exosomes are preserved in the colloidal solution. However, the formula of the exosome preservation solution is complicated, and the preservation method is complicated to operate.

[0005] In summary, the development of exosome preservation fluid with simple formula and easy operation is of great significance to the field of exosome research. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides an exosome preservation solution and a preparation method and application thereof, and designs a new exosome preservation solution formula with a simple formula that can stably preserve exosomes for a long time.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an exosome preservation solution, comprising Tween, albumin and a buffer.

[0009] In the present invention, a new formula of exosome preservation solution is designed, Tween increases the solubility of other effective ingredients, inhibits the automatic aggregation of macromolecular proteins, and improves their stability; albumin regulates the dynamic balance of intracellular water and maintains the relative stability of cell osmotic pressure; the buffer solution provides a suitable liquid environment; the three components work together to preserve exosomes for a long time at low temperature without affecting their activity.

[0010] Preferably, the Tween includes any one of Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Tween 81 or Tween 85, or a combination of at least two of them, and preferably includes a combination of Tween 20 and Tween 40.

[0011] Preferably, the albumin comprises human serum albumin and / or bovine serum albumin.

[0012] Preferably, the buffer comprises PBS buffer and / or sodium acetate buffer.

[0013] Preferably, the concentration of Tween in the exosome preservation solution is 0.001% to 0.1% (v / v), including but not limited to 0.002% (v / v), 0.003% (v / v), 0.005% (v / v), 0.006% (v / v), 0.007% (v / v), 0.01% (v / v), 0.05% (v / v), 0.06% (v / v), 0.07% (v / v), 0.08% (v / v) or 0.09% (v / v).

[0014] Preferably, the concentration of albumin in the exosome preservation solution is 0.01% to 1% (v / v), including but not limited to 0.02% (v / v), 0.03% (v / v), 0.05% (v / v), 0.06% (v / v), 0.1% (v / v), 0.2% (v / v), 0.5% (v / v), 0.6% (v / v), 0.7% (v / v), 0.8% (v / v) or 0.9% (v / v).

[0015] Preferably, the concentration of the buffer in the exosome preservation solution is 1 to 100 mmol / L, including but not limited to 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 30 mmol / L, 50 mmol / L, 60 mmol / L, 80 mmol / L, 90 mmol / L, 95 mmol / L, 96 mmol / L, 97 mmol / L, 98 mmol / L or 99 mmol / L.

[0016] Preferably, the exosome preservation solution comprises 0.001% to 0.1% (v / v) Tween, 0.01% to 1% (v / v) albumin and 1 to 100 mmol / L buffer.

[0017] In a second aspect, the present invention provides a method for preparing the exosome preservation solution according to the first aspect, the preparation method comprising:

[0018] Tween, albumin and buffer are mixed to obtain the exosome preservation solution.

[0019] In a third aspect, the present invention provides use of the exosome preservation solution described in the first aspect in the preservation of exosomes.

[0020] In a fourth aspect, the present invention provides a method for preserving exosomes, the method comprising:

[0021] The exosomes are mixed with the exosome storage solution described in the first aspect.

[0022] Preferably, the method for preserving exosomes further comprises the step of cryopreservation.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention develops an exosome preservation solution with a simple formula that can effectively maintain the activity and quantity of exosomes. The solution is composed of three components, namely, Tween, albumin and buffer, and can be used for short-term preservation, long-term freezing and freeze-dried preservation of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Save the effect map for exosomes;

[0026] Figure 2 This is a picture of the storage effect of exosomes after freeze-drying;

[0027] Figure 3 This is a picture of the effect of exosomes stored at 37°C;

[0028] Figure 4 This is a diagram showing the effects of exosomes stored at 4°C and 25°C. DETAILED DESCRIPTION

[0029] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] Example 1

[0032] In this example, exosomes were prepared.

[0033] (1) Place the supernatant of HEK293 cell culture medium in a centrifuge tube, centrifuge at 10,000 g for 30 min at 4°C, and collect the supernatant.

[0034] (2) Filter the supernatant collected in step (1) through a 0.22 μm filter membrane.

[0035] (3) placing the 0.22 μm supernatant collected in step (2) in a centrifuge tube, centrifuging at 130,000 g for 70 min at 4°C, and resuspending the precipitate with 1 mL of PBS (phosphate buffered saline) to obtain a crude exosome extract;

[0036] (4) The crude exosome extract obtained in step (3) was placed in a centrifuge tube, centrifuged at 130,000 g for 70 min at 4°C, and the precipitate was resuspended in 1 mL of PBS to obtain exosomes.

[0037] The prepared HEK293 exosomes were measured by particle tracking analysis (NTA) technology.

[0038] Example 2

[0039] This embodiment provides an exosome preservation solution and a preservation method.

[0040] The storage solution contains 0.05% (v / v) Tween-20 (polysorbate 20), 0.2% (v / v) HSA (human serum albumin, purchased from Aladdin, product number: R283928) and 10 mM PBS buffer.

[0041] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0042] Example 3

[0043] This embodiment provides an exosome preservation solution and a preservation method.

[0044] The storage solution contains 0.001% (v / v) Tween-20, 0.01% (v / v) HSA and 1 mM sodium acetate buffer.

[0045] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0046] Example 4

[0047] This embodiment provides an exosome preservation solution and a preservation method.

[0048] The storage solution contains 0.1% (v / v) Tween-20, 1% (v / v) BSA (bovine serum albumin, purchased from Biofroxx, catalog number: 4240GR100) and 100 mM sodium acetate buffer.

[0049] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0050] Example 5

[0051] This embodiment provides an exosome preservation solution and a preservation method.

[0052] The storage solution contains 0.05% (v / v) Tween-80, 0.2% (v / v) BSA and 10 mM PBS buffer.

[0053] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0054] Example 6

[0055] This embodiment provides an exosome preservation solution and a preservation method.

[0056] The storage solution contains 0.03% (v / v) Tween-20, 0.02% (v / v) Tween-40, 0.2% (v / v) HSA and 10 mM PBS buffer.

[0057] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0058] Comparative Example 1

[0059] This comparative example provides an exosome preservation solution and a preservation method.

[0060] The storage solution is PBS buffer.

[0061] The PBS buffer was mixed with the exosomes prepared in Example 1, 10^11 exosomes were mixed in every 200 μL of the preservation solution, and the mixture was stored at -80°C.

[0062] Comparative Example 2

[0063] This comparative example provides an exosome preservation solution and a preservation method.

[0064] The preservation solution is SAGM preservation solution: 0.877 g / 100 mL of physiological saline, 0.0169 / mL of adenine, 0.9 g / mL of glucose, 0.525 g / 100 mL of mannitol, and 10 mM of PBS buffer.

[0065] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0066] Comparative Example 3

[0067] This comparative example provides an exosome preservation solution and a preservation method.

[0068] The storage solution is HAT storage solution: 25 mM (0.299 g / 50 mL) HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 0.2% (v / v) HSA, 25 mM (0.428 g / 50 mL) trehalose, and 10 mM PBS buffer.

[0069] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0070] Comparative Example 4

[0071] This comparative example provides an exosome preservation solution and a preservation method.

[0072] The storage solution is TD storage solution: 25 mM trehalose, 1% (v / v) DMSO (dimethyl sulfoxide), and 10 mM PBS buffer.

[0073] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0074] Comparative Example 5

[0075] This comparative example provides an exosome preservation solution and a preservation method.

[0076] The storage solution is T storage solution: 25 mM trehalose, 10 mM PBS buffer.

[0077] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0078] Comparative Example 6

[0079] This comparative example provides an exosome preservation solution and a preservation method.

[0080] The difference between the storage solution and Example 2 is that Tween is replaced by an equal amount of BSA.

[0081] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0082] Comparative Example 7

[0083] This comparative example provides an exosome preservation solution and a preservation method.

[0084] The only difference between the storage solution and Example 2 is that albumin is replaced with an equal amount of Tween 20.

[0085] The preservation solution was mixed with the exosomes prepared in Example 1, with 10^11 exosomes mixed in every 200 μL of preservation solution, and stored at -80°C.

[0086] Test Example 1

[0087] This test example uses ELISA (enzyme-linked immunosorbent assay) to detect the storage capacity of exosomes in different preservation solutions. This detection method uses an ELISA plate coated with CD9 antibodies to capture exosomes, and HRP-labeled CD81 detection antibodies are used for detection. Exosomes quantified by NTA are used as standards to draw a standard curve. This method can simultaneously detect two proteins in exosomes and detect the content of complete exosomes in the solution.

[0088] The above samples were placed in -80°C freezer, and the samples of each embodiment and comparative example were taken out every week for enzyme-linked immunosorbent assay. The test was conducted for 10 weeks in total. The test results were compared weekly. The results of the changes in the number of exosomes are shown in Table 1. Figure 1It is the average value (pieces / mL) of the exosome concentration preserved for 10 weeks, and the exosome concentration is proportional to the preservation effect. Because of the batch effect of the test kit, there is a lack of comparability between groups, but the trend within each group is consistent. From the above results, it can be seen that the present invention uses Tween, albumin and buffer to synergistically prepare exosome preservation solution, which is compared with the prior art commonly used PBS as a preservation solution (Comparative Example 1) and the reported effect of using HEPES, trehalose and HSA with PBS buffer (Comparative Example 3) as a preservation solution, etc. The preservation effect is significantly improved. In addition, by comparing Example 2 with Example 6, it can be seen that controlling a specific Tween combination in the present invention can further improve the preservation effect.

[0089] Table 1

[0090]

[0091]

[0092] Table 2

[0093]

[0094] Test Example 2

[0095] This test example compares the storage effects of freeze-drying at different temperatures after mixing the preservation solution and exosomes in Example 2.

[0096] The experimental group was mixed with 2×10^9 exosomes and 20μL of the preservation solution in Example 2, the control group 1 was 20μL of PBS buffer (common exosome preservation solution in the prior art), the control group 2 was 20μL of the preservation solution in Example 2, and the control group 3 was 2×10^9 exosomes mixed with 20μL of PBS buffer. The above samples were freeze-dried and concentrated, and the experimental group and control group 3 were stored at 4°C and 25°C for 3 weeks, respectively, and the exosome concentration was detected by ELISA every week. Figure 2 The test results show that the exosomes can be stored for 3 weeks or more at 4°C and 25°C after being freeze-dried with the preservation solution of the present invention, while the exosomes are seriously lost after freeze-drying with PBS as the exosome preservation solution. This shows that the preservation solution designed by the present invention can be effectively used for room temperature storage after freeze-drying, which can improve the preservation effect, maintain the stability of exosomes, and avoid changes in the particle size, concentration and activity of exosomes.

[0097] Test Example 3

[0098] This test example verifies the storage effect of the mixture of the preservation solution and exosomes in Example 2 at 37°C.

[0099] The experimental group was a mixture of 2×10^10 exosomes and 200μL of the preservation solution in Example 2, the control group 1 was a mixture of 2×10^10 exosomes and 200μL of PBS buffer, the control group 2 was a mixture of 2×10^10 exosomes and 200μL of the preservation solution in Example 2, and the control group 3 was a mixture of 2×10^10 exosomes and 200μL of PBS buffer. The control group 3 was stored at 37°C for one day; the experimental groups 1, 2, 3, and 4 indicated that the samples of the experimental groups were stored at 37°C for 1, 2, 3, and 4 weeks, respectively, and the exosome concentration was detected by ELISA every week. The ELISA test was performed immediately after the sample preparation of the control group 1 and the control group 2 was completed, and the test results were used as the starting value of the exosome concentration. Figure 3 is the concentration of exosomes stored at 37°C for 4 weeks, and the ordinate is the exosome concentration (units / mL). The exosome concentration is proportional to the preservation effect. The test results show that after the exosomes are added to the preservation solution of the present invention, they can be stored at 37°C for at least 2 weeks without losing more than 50%, while when PBS is used as the exosome preservation solution, the exosomes are severely lost after being stored at 37°C for one day. Therefore, this stability experiment shows that the exosome preservation solution can improve the preservation effect, maintain the stability of the exosomes, and avoid changes in the particle size, concentration and activity of the exosomes.

[0100] Test Example 4

[0101] This test example compares the storage effect of the mixture of the preservation solution and exosomes in Example 2 at different temperatures.

[0102] The experimental group was mixed with 2×10^9 exosomes and 20 μL of the preservation solution in Example 2, and the control group was mixed with 2×10^9 exosomes and 20 μL of PBS buffer. The experimental group and the control group were stored at 4°C and 25°C for 4 weeks, respectively, and the exosome concentration was detected by ELISA every week. Figure 4 For the preservation of exosome concentration (units / mL) for 4 weeks, the test results show that the exosomes can be preserved for 4 weeks or more at 4°C and 25°C after being freeze-dried by adding the preservation solution of the present invention, while the exosomes are seriously lost after using PBS as the exosome preservation solution. This shows that the preservation solution designed by the present invention can be effectively applied to 4°C and room temperature storage, which can improve the preservation effect, maintain the stability of exosomes, and avoid changes in the particle size, concentration and activity of exosomes.

[0103] In summary, the present invention develops an exosome preservation solution with a simple formula that can effectively maintain the activity and quantity of exosomes. The three components of Tween, albumin and buffer are coordinated and can be used for short-term preservation, long-term freezing and freeze-dried preservation of exosomes.

[0104] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An exosome preservation solution, characterized in that: The exosome preservation solution comprises Tween, albumin and buffer.

2. The exosome preservation solution according to claim 1, characterized in that The Tween includes any one of Tween 20, Tween 21, Tween 40, Tween 60, Tween 61, Tween 80, Tween 81 or Tween 85, or a combination of at least two of them.

3. The exosome preservation solution according to claim 1 or 2, characterized in that The albumin includes human serum albumin and / or bovine serum albumin.

4. The exosome preservation solution according to any one of claims 1 to 3, characterized in that The buffer comprises PBS buffer and / or sodium acetate buffer.

5. The exosome preservation solution according to any one of claims 1 to 4, characterized in that The concentration of Tween in the exosome preservation solution is 0.001% to 0.1% (v / v).

6. The exosome preservation solution according to any one of claims 1 to 5, characterized in that The concentration of albumin in the exosome preservation solution is 0.01% to 1% (v / v); Preferably, the concentration of the buffer in the exosome preservation solution is 1 to 100 mmol / L.

7. The exosome preservation solution according to any one of claims 1 to 6, characterized in that: The exosome preservation solution comprises 0.001% to 0.1% (v / v) Tween, 0.01% to 1% (v / v) albumin and 1 to 100 mmol / L buffer.

8. A method for preparing the exosome preservation solution according to any one of claims 1 to 7, characterized in that: The preparation method comprises: Tween, albumin and buffer are mixed to obtain the exosome preservation solution.

9. Use of the exosome preservation solution according to any one of claims 1 to 7 in the preservation of exosomes.

10. A method for preserving exosomes, characterized in that: The method comprises: mixing exosomes with the exosome preservation solution according to any one of claims 1 to 7.

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