Exosome purification method

Abstract

The invention relates to the technical field of biology, in particular to an exosome purification method. The exosome purification method provided by the invention is used for solving the problems in the prior art that the exosome purification is low in yield, long in time consumption and short in scale, the morphological structure and physiological function of the exosome are easily damaged, the structural integrity of the exosome is damaged, and the industrial production of the exosome is not facilitated. On the basis of the ultrafiltration and chromatography separation technology, the components of the buffer solution in the purification process are optimized, the total separation yield reaches 90% or above, the technology has the advantages of being easy to amplify, low in equipment requirement and the like, the separation operation can be completed within 4 hours, and a high-purity product is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for purifying exosomes. BACKGROUND

[0002] Exosomes are nanoscale vesicles (30-150nm in diameter) secreted by cells, with complex structure and high organization. The outer layer of exosomes is composed of a lipid bilayer, and the membrane is rich in various proteins, including four transmembrane proteins (such as CD63, CD81), heat shock proteins (such as Hsp70, Hsp90), cytoskeletal proteins, membrane transport and fusion proteins, etc. These proteins play an important role in cell communication and exosome function. The inside of the exosome contains various nucleic acids, such as mRNA, miRNA, non-coding RNA and DNA, which can be delivered to recipient cells to regulate gene expression and cell function. The structure and composition of exosomes make them an important mediator of intercellular information transmission, with functions such as promoting tissue regeneration, anti-aging and immune regulation. In recent years, exosomes have shown great potential in the fields of disease treatment, drug carriers and regenerative medicine due to their natural delivery ability, low immunogenicity and the ability to cross biological barriers.

[0003] The common exosome separation methods on the market at present include ultra-centrifugation, ultrafiltration, molecular exclusion chromatography, polymer precipitation and immunoaffinity. Exosomes have biological activity, but their membrane structure is fragile and easily inactivated under normal temperature and light conditions. They are greatly affected by temperature, humidity, shear force and pH. The current separation scheme has low recovery rate of exosomes and the membrane structure is easily damaged. Some separation methods are time-consuming, low-yield, and rely on expensive precision instruments and equipment, which is not conducive to commercial application.

[0004] According to the "Preparation and Testing Specifications for Human Mesenchymal Stem Cell Exosomes" issued by FDSA, exosomes need to be produced under GMP standards to ensure traceability from cell source, culture expansion to purification and cryopreservation, stable process and small batch difference. However, most researches are still in the laboratory stage, and there is a lack of mature process system for large-scale production. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a new method for purifying exosomes, which is used to solve the problems in the prior art that the yield of exosomes is low, the time is long, the scale is lacking, and the morphology and physiological function of exosomes are easily damaged, the integrity of the exosome structure is damaged, and the industrial production of exosomes is not conducive. Based on the separation technology of ultrafiltration + chromatography, the composition of the buffer in the purification process is optimized, so that the total yield of separation is more than 90%, and the process is easy to scale up, the equipment requirement is low, and the like. And the separation operation can be completed within 4 hours, and high-purity products are obtained.

[0006] To this end, the first aspect of the present application provides a buffer for purifying exosomes. According to an embodiment of the present application, the buffer comprises:

[0007] Hepes, a binary sugar, a non-ionic surfactant, and sodium chloride.

[0008] The inventors have optimized the composition of the buffer for purifying exosomes, and found that the use of the buffer of the present application containing Hepes, a binary sugar, a non-ionic surfactant, and sodium chloride can make the total yield of exosome separation more than 90%, and the process is easy to scale up, the equipment requirement is low, the separation operation can be completed within 4 hours, and high-purity products are obtained.

[0009] According to an embodiment of the present application, the concentration of Hepes is 10-50mM.

[0010] According to an embodiment of the present application, the binary sugar comprises at least one selected from trehalose, sucrose, glucose, maltose, and fructose.

[0011] According to an embodiment of the present application, the binary sugar is trehalose, and the concentration of the trehalose is 10-50mM.

[0012] According to an embodiment of the present application, the concentration of sodium chloride is 100-200mM.

[0013] According to an embodiment of the present application, the non-ionic surfactant comprises at least one selected from Tween 80, Tween 20, and poloxamer.

[0014] According to an embodiment of the present application, the non-ionic surfactant is Tween 80, and the mass concentration is 0.001-0.01%.

[0015] According to an embodiment of the present application, the pH of the buffer is 6.5-7.5.

[0016] The second aspect of the present application provides the use of the buffer of the first aspect in the preparation of exosomes.

[0017] According to an embodiment of the present application, the exosome is derived from stem cells.

[0018] According to an embodiment of the present application, the stem cells include at least one selected from umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, iPSCs, and embryonic stem cells.

[0019] A third aspect of the present application provides a method for preparing exosomes. According to an embodiment of the present application, the method includes purifying exosomes using the buffer of the first aspect.

[0020] A fourth aspect of the present application provides a method for preparing exosomes. According to an embodiment of the present application, the method includes:

[0021] (1) obtaining cell culture supernatant containing exosomes;

[0022] (2) clarifying the cell culture supernatant using hollow fibers to obtain a clarified system;

[0023] (3) subjecting the clarified system to ultrafiltration to obtain a concentrated solution, and washing the concentrated solution using the buffer of the first aspect to obtain an ultrafiltration-treated system;

[0024] (4) subjecting the ultrafiltration-treated system to chromatography to obtain the exosomes.

[0025] According to an embodiment of the present application, the cells from which the exosomes in step (1) are derived are stem cells.

[0026] According to an embodiment of the present application, the stem cells include at least one selected from umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal iPSCs, and embryonic stem cells.

[0027] According to an embodiment of the present application, the cell culture supernatant is collected when the cell culture confluency is 85-100%.

[0028] According to an embodiment of the present application, the hollow fiber pore size in step (2) is 0.22-0.65 μm.

[0029] According to an embodiment of the present application, the hollow fiber used in step (3) for ultrafiltration is 100-750 KD.

[0030] According to an embodiment of the present application, the buffer of the first aspect is used to flush the chromatography column in step (4).

[0031] According to an embodiment of the present application, the filler used in the chromatography process is selected from at least one of Capto Core 700, Capto Core 400, NW Rose Viral M, NW Rose Viral L, and MaXtar COLL700.

[0032] The buffer for purifying exosomes and the method for preparing exosomes based on the buffer provided by the present application have the following beneficial effects:

[0033] 1) The formula of the buffer for purifying exosomes provided by the present application can make the exosome recovery rate reach more than 90%;

[0034] 2) In the method for preparing exosomes provided by the present application by using ultrafiltration + chromatography, the steps of clarification, ultrafiltration, chromatography, etc. can be linearly scaled up, the process can be large-scale commercialized, and the requirement for equipment is low;

[0035] 3) The method for preparing exosomes provided by the present application can complete the separation process within 4 hours.

[0036] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0037] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0038] Figure 1 A flow chart of exosome purification of one embodiment of the present application is shown;

[0039] Figure 2 Contour plots showing DOE of the formula of the purification buffer in Example 2 of the present application are shown. DETAILED DESCRIPTION

[0040] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not intended to limit the present application, which can be embodied in various forms.

[0041] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0042] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not considered critical for the invention. Any numeric range recited is intended to include all values from the lower value to the upper value, inclusive of both values, and to also include any value approximately or roughly between the lower value and the upper value. Numeric ranges can be expressed as "from X to Y," where X and Y are numbers. The range can also be expressed as "from X to Y," where X is a number and Y is infinity. The range can also be expressed as "from X to Y," where X is a number and Y is not specified. In each case, the range includes the endpoints.

[0043] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly described otherwise, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] In this document, the terms "comprising" or "comprise" are open-ended, that is, the inclusion of a stated specification of elements does not exclude other elements.

[0045] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0046] The common exosome separation methods on the market at present include ultracentrifugation, ultrafiltration, molecular exclusion chromatography, polymer precipitation, and immunoaffinity. Exosomes have biological activity, but their membrane structure is fragile and can be easily inactivated under normal temperature and light conditions, and is greatly affected by temperature, humidity, shear force, and pH. The current separation scheme has low recovery rate of exosomes and the membrane structure is easily damaged. Some separation methods are time-consuming, low-yield, and dependent on expensive and precise instruments and equipment, which is not conducive to commercial application.

[0047] For example, ultracentrifugation is the most common method for separating exosomes in laboratories at present and is considered as the "gold standard" for exosome extraction. The sample is centrifuged at 300-400 x g for 10 minutes to precipitate the main part of the cells, centrifuged at 2000 x g to remove cell debris, and centrifuged at 10,000 x g to remove aggregates of biological polymers, apoptotic bodies, and other structures with higher buoyant density than exosomes. Exosomes contained in the supernatant are precipitated by ultracentrifugation at >100,000 x g (100,000-200,000 x g). However, this method has high requirements for equipment, complicated operation, low recovery rate of exosomes, poor process repeatability, and small processing capacity, and is not easy to be scaled up industrially.

[0048] Based on this, the inventors of the present application have obtained a new buffer for purifying exosomes and a method for preparing exosomes based on the buffer through a large number of experimental explorations. Specifically as follows:

[0049] According to one specific embodiment of the present application, the present application provides a buffer for purifying exosomes, comprising:

[0050] Hepes, a binary sugar, a non-ionic surfactant, and sodium chloride.

[0051] The concentration of Hepes in the buffer for purifying exosomes provided by the present application is not particularly limited, preferably, a concentration of 10-50 mM of Hepes is used, which can obtain a higher exosome recovery rate. For example, in the buffer for purifying exosomes, the concentration of Hepes can be 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, etc.

[0052] According to one specific embodiment of the present application, the binary sugar includes but is not limited to at least one of trehalose, sucrose, glucose, maltose, fructose, and other binary sugars known in the art, all of which are encompassed in the composition of the buffer of the present application.

[0053] According to one preferred embodiment of the present application, the binary sugar is trehalose, and the concentration of trehalose is 10-50 mM. For example, in the buffer for purifying exosomes, the concentration of trehalose can be 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, etc.

[0054] It should be noted that the concentration of sodium chloride in the buffer provided by the present application is not particularly limited, preferably, the concentration of sodium chloride is 100-200 mM. For example, the concentration of sodium chloride is 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, etc. Thus, the exosome recovery rate can be further improved, and the activity of the obtained exosomes can be maintained.

[0055] It should be noted that the non-ionic surfactant includes but is not limited to at least one selected from Tween 80, Tween 20, and poloxamer.

[0056] According to one preferred embodiment of the present application, the non-ionic surfactant is Tween 80, and the mass concentration is 0.001-0.01%. For example, in the buffer for purifying exosomes, the mass concentration of the non-ionic surfactant Tween 80 is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, etc.

[0057] According to a specific embodiment of the present application, the pH of the buffer is 6.5-7.5. For example, the pH of the buffer is 7.0±0.1.

[0058] The present application also provides use of the aforementioned buffer in preparation of exosomes.

[0059] According to a specific embodiment of the present application, when the aforementioned buffer is used to prepare exosomes, the cell from which the exosomes are derived is not particularly limited, and preferably, the exosomes are derived from stem cells.

[0060] According to a specific embodiment of the present application, the stem cells include, but are not limited to, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, iPSCs, embryonic stem cells, etc., and can also be any one of the unlisted cells or stem cells that secrete exosomes. When the exosomes are derived from embryonic stem cells, commercially available embryonic stem cells are used.

[0061] According to a specific embodiment of the present application, the present application provides a method for preparing exosomes, which comprises purifying exosomes using the aforementioned buffer.

[0062] According to a more specific embodiment of the present application, the present application provides a method for preparing exosomes, which comprises the following steps as shown in Figure 1

[0063] (1) obtaining cell culture supernatant containing exosomes;

[0064] (2) clarifying the cell culture supernatant using hollow fibers to obtain a clarified system;

[0065] (3) subjecting the clarified system to ultrafiltration to obtain a concentrated solution, and washing the concentrated solution using the aforementioned buffer to obtain an ultrafiltration-treated system;

[0066] (4) subjecting the ultrafiltration-treated system to chromatography to obtain the exosomes.

[0067] According to a specific embodiment of the present application, the method further comprises sterilization.

[0068] According to a specific embodiment of the present application, the cell from which the exosomes are derived in step (1) is not particularly limited, and preferably, the exosomes are derived from stem cells.

[0069] ​According to a specific embodiment of the present application, the stem cells include, but are not limited to, umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, iPSCs, embryonic stem cells, etc., and can also be any one of the unlisted cells or stem cells that secrete exosomes.

[0070] According to a specific embodiment of the present application, the cell culture supernatant is collected when the cell culture confluency is 85-100%. Preferably, the cell culture confluency is 90%.

[0071] According to a specific embodiment of the present application, the hollow fiber pore size in step (2) is 0.22-0.65 μm. For example, the hollow fiber pore size can be 0.22 μm, 0.45 μm, 0.65 μm, etc.

[0072] According to a specific embodiment of the present application, the hollow fiber used in step (3) for ultrafiltration treatment is 100-750 KD. For example, the hollow fiber used in ultrafiltration treatment can be ultrafiltration hollow fiber MWCO, with specifications of 100 KD, 300 KD, 500 KD, or 750 KD, etc.

[0073] According to a specific embodiment of the present application, there is no particular limitation on the concentration multiple of the clarified system in step (3) for ultrafiltration treatment. For example, the clarified system is concentrated by 20-30 times for ultrafiltration treatment.

[0074] According to a specific embodiment of the present application, there is no particular limitation on the washing multiple in step (3). For example, the washing multiple is 6-10 times.

[0075] According to a specific embodiment of the present application, the buffer described above is used to flush the chromatography column in step (4) for chromatography. There is no particular limitation on the filler used for chromatography.

[0076] According to a preferred embodiment of the present application, the filler used for chromatography treatment is selected from at least one of Capto Core 700, Capto Core 400, NW Rose Viral M, NW Rose Viral L, and MaXtar COLL 700. Preferably, Capto Core 700 or Capto Core 400.

[0077] According to a specific embodiment of the present application, the sterilization treatment can be filtration sterilization or other sterilization methods known in the art. When it is filtration sterilization, the pore size of the filter used can be, for example, 0.22 μm.

[0078] According to one specific embodiment of the present application, the present application provides a method for purifying exosomes, comprising:

[0079] 1) Cell culture and harvest of supernatant: umbilical cord mesenchymal stem cells are inoculated in stem cell culture medium at 5000-8000 Cells / cm 2 at 37°C, 5.0% CO2 culture. The cell confluence is about 90%, and the supernatant is collected, wherein the buffer formula is: 25Mm Hepes 25mM trehalose 100-200mM sodium chloride 0.005% Tween 80 pH 7.0±0.1;

[0080] 2) Clarification: clarification is performed using hollow fibers with a pore size of 0.65 μm, and the filter is washed with buffer after filtration is completed;

[0081] 3) Ultrafiltration: after clarification, 500KD hollow fibers are used to concentrate 20-30 times, and 6-10 times of washing filtration is performed using buffer;

[0082] 4) Chromatography: after ultrafiltration, the sample is loaded onto an equilibrated Core 700 multi-mode filler for chromatography and rinsing with buffer, and the flow-through is collected. The filler height is 10-20 cm, and the chromatography flow rate is 100-200 cm / h;

[0083] 5) Sterilization: sterilization filtration is performed using a 0.22 μm filter.

[0084] The solutions of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase on the market.

[0085] Example 1

[0086] The following method is used to prepare MSC cell-derived exosomes:

[0087] (1) MSC cell separation and culture: umbilical cords are provided by healthy donors, and after treatment, tissue block adhesion culture method is used, and the cells are cultured for 14 days, and P0 generation cells are obtained, which are primary umbilical cord mesenchymal stem cells. And through digestion and subculture, the main cell bank (P2) and the working cell bank (P4) are established, and the cell bank related tests and tests are carried out, and the starting material of the exosomes in this application is the working bank P4 cells.

[0088] (2) Cell culture and harvest of supernatant: MSC cells are taken out from the MSC cell working bank in liquid nitrogen tank, and thawed in 37°C water bath. Umbilical cord mesenchymal stem cells are inoculated in stem cell culture medium at 5000-8000 Cells / cm2 Inoculate in stem cell culture medium, 37℃, 5.0% CO2 culture. The cell confluence is about 90%, and the MSC supernatant is collected;

[0089] (3) Clarification: MSC supernatant is clarified by hollow fiber, and the pore size of hollow fiber (hollow fiber manufacturer: REPLIGEN, model: D02-E65U-07-N) is 0.65 μm;

[0090] (4) Ultrafiltration: After clarification, 500KD hollow fiber (hollow fiber manufacturer: REPLIGEN, model: D04-E500-05-N) is used for 25 times concentration, and 6 times washing is performed with buffer. The buffer formula is: 25mM Hepes, 25mM trehalose, 150mM sodium chloride and 0.005% Tween 80, pH 7.0;

[0091] (5) Chromatography: After ultrafiltration, the sample is loaded into the equilibrated Capto Core 700 multi-mode filler (manufacturer: Cytiva) for chromatography, and the buffer is used for washing, and the flow-through is collected. The buffer used is consistent with the ultrafiltration buffer. The filler height is 10 cm, and the chromatography flow rate is 100 cm / h.

[0092] (6) Sterilization: 0.22 μm filter is used for sterilization filtration.

[0093] Table 1 below shows the volume of the liquid system obtained at each step in the preparation of exosomes, the total number of exosome particles and the recovery rate.

[0094] Table 1

[0095]

[0096] Example 2:

[0097] A 5-factor, 2-level DOE is performed on each parameter of the exosome purification buffer, as shown in Table 2. The contour plot of the DOE of the purification buffer formula is shown in Figure 2 .

[0098] Table 2

[0099] Factor High Low HEPES 10 50 Trehalose 10 50 NaCl 100 200 Tween 80 0.001 0.01 pH 6.5 7.5

[0100] The supernatant harvested from MSC culture is purified, and the exosome recovery rate after purification is calculated for each group. It can be seen that the end point of each component concentration has good yield, and reducing the component yield will decrease. The experimental results are as follows in Table 3:

[0101] Table 3

[0102] Run HEPES Trehalose NaCl Tween 80 pH Yield 1 10 50 200 0.01 6.5 94.2％ 2 10 10 100 0.01 7.5 93.5％ 3 50 10 100 0.001 7.5 92.1％ 4 10 50 200 0.001 6.5 91.2％ 5 50 10 200 0.001 6.5 90.5％ 6 50 50 100 0.01 6.5 96.8％ 7 10 10 200 0.001 7.5 94.7％ 8 10 10 200 0.01 6.5 95％ 9 50 50 200 0.001 7.5 97.3％ 10 50 10 200 0.01 7.5 98.1％ 11 10 50 100 0.01 7.5 93.9％ 12 50 50 100 0.001 6.5 91.4％ 13 10 50 100 0.001 7.5 92％ 14 50 10 100 0.01 6.5 94.2％ 15 10 10 100 0.001 6.5 90.0％ 16 50 50 200 0.01 6.5 97.5％ 17 30 30 150 0.0055 7 92.8％ 18 30 30 150 0.0055 7 92.5％ 19 30 30 150 0.0055 7 92.9％ 20 30 30 150 0.0055 7 93.1％ 21 30 30 150 0.0055 7 92.6％ 22 30 30 150 0.0055 7 93％ 23 30 30 150 0.0055 6 85.1％ 24 30 0 150 0.0055 7 54.6％ 25 30 30 150 0 7 70.4％ 26 0 30 150 0.0055 7 69.7％ 27 30 30 250 0.0055 7 87.9％

[0103] Comparative Example 1

[0104] (1) Cell culture and harvest supernatant steps were the same as Example 1, and 200 ml of MSC supernatant was obtained;

[0105] (2) Clarification: the MSC supernatant was clarified with 0.65 pm hollow fiber;

[0106] (3) Ultrafiltration: after clarification, 20-fold concentration was performed with 500KD hollow fiber, and 6-fold washing was performed with 10 mM PBS;

[0107] (4) Chromatography: after ultrafiltration, the sample was loaded onto Capto Core 700 equilibrated with 10 mM PBS for chromatography (flow rate was 10 cm / h);

[0108] (5) Sterilization: sterilization filtration was performed with a 0.22 pm filter.

[0109] Comparative Example 2

[0110] (1) Cell culture and harvest supernatant steps were the same as Example 1, and 200 ml of MSC supernatant was obtained;

[0111] (2) Clarification: the MSC supernatant was clarified with 0.65 pm hollow fiber;

[0112] (3) Ultrafiltration: after clarification, 20-fold concentration was performed with 500KD hollow fiber, and 6-fold washing was performed with 25 mM Hepes, 150 mM NaCl;

[0113] (4) Chromatography: after ultrafiltration, the sample was loaded onto Capto Core 700 equilibrated with 25 mM Hepes, 150 mM NaCl for chromatography (flow rate was 10 cm / h);

[0114] (5) Sterilization: sterilization filtration was performed with a 0.22 pm filter.

[0115] Recovery rate statistics

[0116] Table 4 below shows the volume of supernatant and liquid system after sterilization, the total particle number of exosomes, and the recovery rate in the exosome preparation method of Example 1, Comparative Example 1 and Comparative Example 2.

[0117] Table 4

[0118]

[0119] The above results show that only the special buffer system of the present application is used for exosome purification, a higher exosome recovery rate can be obtained.

[0120] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some implementations" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples, without contradiction.

[0121] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A buffer for purifying exosomes, characterized in that, Comprising: Hepes, a binary sugar, a non-ionic surfactant, and sodium chloride.

2. The buffer of claim 1, wherein, The concentration of the Hepes is 10-50 mM; Optionally, the binary sugar comprises at least one selected from the group consisting of trehalose, sucrose, glucose, maltose, and fructose; Optionally, the binary sugar is trehalose, and the concentration of the trehalose is 10-50 mM; Optionally, the concentration of the sodium chloride is 100-200 mM.

3. The buffer of claim 1, wherein, The non-ionic surfactant comprises at least one selected from the group consisting of Tween 80, Tween 20, and poloxamer; Optionally, the non-ionic surfactant is Tween 80, and the mass concentration is 0.001-0.01%.

4. The buffer of claim 1, wherein, The pH of the buffer is 6.5-7.

5.

5. Use of the buffer of any one of claims 1-4 in the preparation of exosomes.

6. Use according to claim 5, characterized in that, The exosomes are derived from stem cells; Optionally, the stem cells comprise at least one selected from the group consisting of umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, iPSCs, and embryonic stem cells.

7. A method of preparing an exosome, characterized by, The method comprises exosome purification using the buffer of any one of claims 1-4.

8. A method of preparing an exosome, characterized by, The method comprises: (1) obtaining an exosome-containing cell culture supernatant; (2) clarifying the cell culture supernatant using a hollow fiber to obtain a clarified system; (3) performing ultrafiltration on the clarified system to obtain a concentrated solution, and washing the concentrated solution using the buffer of any one of claims 1-4 to obtain an ultrafiltration-treated system; (4) performing chromatography on the ultrafiltration-treated system to obtain the exosomes.

9. The method of claim 8, wherein, The cells from which the exosomes in step (1) are derived are stem cells; Optionally, the stem cells comprise at least one selected from the group consisting of umbilical cord mesenchymal stem cells, adipose tissue mesenchymal stem cells, bone marrow mesenchymal stem cells, iPSCs, and embryonic stem cells. Optionally, the cell culture supernatant is collected when the cell culture confluency is 85-100%; The pore size of the hollow fiber in step (2) is 0.22-0.65 μm; Optionally, the hollow fiber used in step (3) for ultrafiltration treatment is 100-750 KD.

10. The method of claim 8, wherein, The buffer of any one of claims 1-4 is used to flush the chromatography column in step (4); Optionally, the filler used for chromatography treatment is at least one selected from the group consisting of Capto Core 700, Capto Core 400, NW Rose Viral M, NW Rose Viral L, and MaXtar COLL 700.

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