Small-scale exosome separation and purification method and separation and purification kit
By using a purification column of solid resin combined with PBS buffer formed by a composite matrix of dextran and agarose in exosome purification, high-purity exosome purification of small volume samples is achieved by using gravity, solving the problem of time-consuming and low purity in the prior art, and achieving a fast, efficient and low-cost exosome purification effect.
Patent Information
- Application Number
- CN202411487019.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art has problems such as time-consuming, low purity, and the need for large equipment in the process of exosome purification, which is particularly difficult to meet the high-purity exosome purification needs of small-volume samples.
The separation and purification of exosomes were achieved through gravity using a purification column of solid resin combined with liquid PBS buffer formed by highly cross-linking of dextran and agarose composite matrix. This method does not require complex instrument assistance and is suitable for 0.5mL small volume samples, allowing high-purity exosomes to be obtained quickly and efficiently.
It achieves efficient and rapid exosome purification, with a protein removal rate of more than 90%, maintaining the integrity of exosomes, making it easy to operate, and has low cost. The purification column can be reused 5 times.
Smart Images
Figure CN119565380A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of exosomes, and in particular to a small-scale exosome separation and purification method and a separation and purification kit. Technical Background
[0002] As an important medium for intercellular communication, exosomes play an increasingly important role in biological research and clinical applications. However, efficient, economical and intact purification methods of exosomes have always been the main challenge in this field. Currently, there are many methods in the field of exosome purification, but each method has its inherent limitations, such as long time consumption, low purity, and the need for large equipment.
[0003] Ultracentrifugation is one of the most widely used traditional methods. This method separates exosomes from complex biological samples by high-speed centrifugation. However, this method has several disadvantages: it is time-consuming, usually taking several hours; it requires expensive large ultracentrifuges; the shear force generated by high-speed centrifugation may destroy the exosome structure; and the recovery rate is low, usually less than 30%.
[0004] Density gradient centrifugation is an optimized ultracentrifugation method that improves the purification effect by adding density gradient medium to the sample. Although this method can obtain exosomes with higher purity, it is more complicated and time-consuming (may take more than 24 hours), and also requires an ultracentrifuge. In addition, the purified sample is in a high-concentration medium and needs to be replaced by ultrafiltration or other methods.
[0005] Immunoaffinity captures exosomes using specific antibodies and is highly specific. However, this method is costly and requires high antibody specificity. In addition, since only exosomes carrying specific markers can be captured, this may miss certain subpopulations of exosomes, leading to sample bias.
[0006] Precipitation is a simple method that usually uses polymers (such as polyethylene glycol) to precipitate exosomes. Although this method is easy to operate, its main disadvantage is low purity and easy introduction of interfering substances (such as protein aggregates). Especially for high-throughput research and omics research, the presence of a large amount of proteins and nucleic acids will affect subsequent analysis and research.
[0007] In recent years, microfluidics has shown potential in the field of exosome separation. This method uses the principles of microscale fluid dynamics to separate exosomes. However, microfluidics currently faces problems such as expensive equipment, limited processing capacity, and immature technology, making it difficult to be widely used in laboratories.
[0008] Size exclusion chromatography (SEC) technology has recently attracted widespread attention in the field of exosome purification. Although traditional SEC technology can effectively separate exosomes, it usually requires a large-volume chromatography system. This high-cost chromatography device has made SEC mainly concentrated in industrial-grade applications and also limited its popularity in ordinary laboratories. It is worth noting that the demand for small-volume and high-purity exosome purification has long been ignored. There are many types of SEC fillers, and there are significant differences between industrial-grade large-volume purification and small-volume purification in terms of sample loading, flow-through pressure, and flow-through detection methods.
[0009] Given the limitations of existing technologies, it is of great theoretical significance and practical application value to find a gel filler suitable for purifying exosomes and to develop a fast, efficient and simple method for separating and purifying exosomes, especially one suitable for small-volume samples. This can not only meet the needs of scientific research, but also open up new avenues for clinical diagnosis and treatment. Summary of the invention
[0010] The core of the present invention is to separate and purify exosomes by combining sample concentration and size exclusion method. The specific steps are as follows: The purpose of the present invention is to solve the problems existing in the prior art and to provide a small-scale exosome separation and purification method and a separation and purification kit suitable for small-volume samples.
[0011] An exosome separation and purification kit comprises a columnar box body, wherein an upper sieve plate and a lower sieve plate are arranged in the columnar box body, a purification column is formed between the upper sieve plate and the lower sieve plate, the purification column is filled with solid resin, and the space between the solid resins is filled with liquid PBS buffer; Preferably, the solid resin is formed by highly cross-linking a composite matrix of dextran and agarose.
[0012] Preferably, the volume of the liquid PBS buffer between the upper frit and the lower frit is 25% to 35% of the volume of the purification column.
[0013] Preferably, the volume of the liquid PBS buffer between the upper frit and the lower frit is 30% of the volume of the purification column.
[0014] A method for separating and purifying exosomes comprises the following steps: Pre-treating the sample to be purified; Inject the pretreated sample into the reagent kit so that the sample passes through the upper frit plate, the purification column and the lower frit plate in sequence; Collect samples that pass through the upper frit plate, purification column, and lower frit plate in sequence to obtain exosome fractions; Preferably, the following steps are included: After obtaining the exosome fraction, a purification column regeneration step is also included: NaOH solution is added to the kit to clean and regenerate the filler.
[0015] Preferably, pre-treatment of the sample comprises at least one of ultracentrifugation, ultrafiltration, tangential flow filtration and PEG precipitation.
[0016] Preferably, the sample is a small volume sample of 0.5 mL-5 mL.
[0017] Preferably, the timing of collecting the exosome fraction is: after adding the sample to the kit, collecting 0.5 mL to 1.5 mL of the volume of liquid PBS buffer corresponding to the liquid flowing out between the upper sieve plate and the lower sieve plate.
[0018] Preferably, the filler can be cleaned and regenerated using 0.5M NaOH, and the filler can be reused after regeneration.
[0019] Preferably, the kit further comprises an adapter for carrying a syringe barrel, and the adapter is used to add 5 mL-50 mL of PBS buffer.
[0020] Preferably, the kit achieves separation and purification of exosomes by gravity.
[0021] Preferably, the volume of the purification column is 8 mL-16 mL.
[0022] The present invention discloses a small-scale exosome separation and purification method and a separation and purification kit, which are suitable for the preparation of high-purity exosomes from small-volume samples. The method comprises: pre-treating and concentrating a solution containing exosomes by ultracentrifugation, ultrafiltration or PEG precipitation to obtain a crude extract of exosomes; the crude extract is purified by a column, the purification column is based on the size exclusion principle, combined with the gravity column design, and is composed of a stationary phase formed by a highly cross-linked composite matrix of dextran and agarose and a mobile phase PBS, and the exosomes are separated from small particles such as proteins and nucleic acids during the flow of the crude extract; by collecting the flow-through liquid of a specific fraction, a purified exosome solution is obtained. The purification method of the present invention does not require the assistance of complex instruments, and can achieve efficient separation only by gravity, which is particularly suitable for the preparation of exosomes from a small-volume sample of 0.5 mL. The purification column can be reused through a simple regeneration step, which significantly reduces the cost. The method is not only simple and fast to operate (completed in about 30 minutes), but also can obtain high-purity (protein removal rate> 90%) exosomes, and maintain the integrity of the exosomes.
[0023] The present invention has the following innovations and advantages: 1. Technological innovation: The highly cross-linked composite matrix of dextran and agarose is applied to the purification of small-volume exosomes, filling the technical gap of small-volume high-purity exosome purification; 2. High compatibility: This method is compatible with a variety of sample pretreatment techniques (such as ultracentrifugation, ultrafiltration, tangential flow, PEG precipitation), further improving the purity of exosomes; 3. High purity: effectively remove proteins and free nucleic acids, with a protein removal rate of over 90%; 4. Easy to operate: No need to rely on protein purification instruments and complex chromatography systems, efficient purification can be achieved only by gravity, which greatly simplifies the operation process and equipment requirements; 5. Low shear force: lossless separation is achieved through gravity, preserving the complete structure of exosomes; 6. Reusable: Each purification column can be reused 5 times. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the structure of the exosome purification column of the present invention.
[0025] Figure 2 Flow chart of the exosome isolation and purification process.
[0026] Figure 3 The morphology of exosomes before and after passing through the column was observed using transmission electron microscopy.
[0027] Figure 4 The particle size distribution of exosomes before and after passing through the column.
[0028] Figure 5 This is the development image of exosome marker proteins of samples before and after passing through the column by Western blotting.
[0029] Figure 6 Protein concentration of each fraction measured by the biuret method after the purification column was reused 5 times DETAILED DESCRIPTION
[0030] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.
[0031] A small-scale exosome separation and purification method: Sample pretreatment: Clarification and concentration of biological fluid samples by ultracentrifugation, ultrafiltration, tangential flow filtration and PEG precipitation; Loading the sample into the column: Load 0.5 mL of the prepared sample into the purification column; Collect exosome fractions: collect 1.5 mL of 0.3 times the volume of the column as the purified exosome fraction; Column regeneration: Add more than 1 column volume of 0.5M NaOH solution to clean and regenerate the filler.
[0032] The exosome separation and purification kit of the present invention mainly includes the following key components: Gravity column empty column: used to contain the stationary phase and mobile phase, and is the core component for achieving size exclusion separation.
[0033] Stationary phase: A gel filler based on size exclusion method is used. The lipid is formed by a highly cross-linked composite matrix of dextran and agarose.
[0034] Liquid mobile phase: Use PBS (phosphate buffered saline) as the mobile phase.
[0035] The preparation method of PBS is as follows: dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4 in 1000mL distilled water; adjust the pH to 7.4 c; make up to 1000mL with distilled water; sterilize by high pressure or filtration.
[0036] Purification column: A complete purification column assembled from a gravity column, stationary phase and mobile phase.
[0037] Adapter: Designed to carry a syringe barrel, it can add 5-50mL of solution at a time for cleaning and regeneration of purification columns.
[0038] The method according to the embodiment of the present invention can meet the needs of small-scale high-purity exosome extraction and scientific research needs. It does not require multiple chromatography, has simple operation steps, does not rely on large-scale instruments and equipment, and only uses gravity for low shear. Therefore, it has no effect on the structure and activity of exosomes, and a single chromatography can obtain high-purity and high-activity exosomes.
[0039] When the extract is concentrated, the concentration method used is one or more of ultracentrifugation, ultrafiltration, and PEG precipitation. The above concentration is beneficial to increase the concentration of exosomes in the solution.
[0040] The crude extract is ultrafiltered and concentrated using a hollow fiber column or an ultrafiltration membrane package to obtain a concentrated solution, comprising: vacuum filtering the crude extract; ultrafiltering and concentrating the vacuum filtered crude extract using a hollow fiber column or an ultrafiltration membrane package to obtain a concentrated solution, wherein the molecular interception rate of the hollow fiber column or the ultrafiltration membrane package is 100kd-300kd.
[0041] The liquid to be extracted includes body fluid and / or culture fluid.
[0042] The body fluid includes a mixture of one or more of serum, plasma, semen, ascites, amniotic fluid, cerebrospinal fluid, urine, milk and breast milk.
[0043] The culture solution includes one or both of cell culture supernatant and bacterial culture supernatant.
[0044] The treated crude extract is subjected to molecular sieve-based chromatography to obtain a flow-through solution, which includes: adding the crude extract to the upper chamber of the column, gradually adding a buffer solution PBS, and collecting the obtained exosome flow-through fraction after the void volume.
[0045] The single loading volume of the crude extract is 0.5 mL, the void volume is 30% of the column volume, and the exudate flow-through fraction is the 1-1.5 mL fraction after the void volume.
[0046] The particle size of the molecular sieve filler is 45-165 μm, and the conjugate is formed by highly cross-linking a composite matrix of dextran and agarose.
[0047] The present invention also provides an exosome purification kit, which consists of a gravity column, a stationary phase, a liquid mobile phase, a purification column, and an adapter. Embodiment 1:
[0048] Validation and optimization of purification column void volume and exosome collection fractions This example describes in detail the optimization process of the purification column void volume and exosome collection fraction to determine the optimal exosome isolation conditions.
[0049] like Figure 1 As shown in the figure, the purification column consists of a solid phase and a liquid phase, where the volume of the liquid phase is the volume of water outside the filler, also known as the void volume. During the exosome purification process, the exosome fraction flows out first after the void volume. However, for small-volume samples, the optimal ratio of the void volume to the filler volume and the optimal collection volume have not been studied in depth.
[0050] The present invention designed the experimental scheme in proportion, with the filler volume ranging from 8-16mL and the sample loading amount being 0.5mL. The following key parameters were explored using the exosome electron microscopic morphology, exosome particle number concentration and exosome protein markers as evaluation indicators: 1. Void volume ratio: The void volume to column volume ratio is divided into four gradients of 10%, 20%, 30% and 40%.
[0051] 2. Exosome collection fractions: 500 μL / fraction was collected, and fractions 1, 1-2, 1-3, and 1-4 after the void volume were studied respectively.
[0052] The specific steps are as follows: 1. Optimization of the void volume of the purification column: 1.1 Prepare 4 sets of purification columns and receive the flow-through at the ratio of void volume to column volume of 10%, 20%, 30% and 40% respectively.
[0053] 1.2 Equilibrate the purification column with PBS buffer.
[0054] 2. Sample loading: 2.1 For each set of purification columns, load 0.5 mL of crude exosome solution from cell supernatant.
[0055] 3. Exosome Collection: 3.1 Collect the effluent at 500 μL / fraction.
[0056] 3.2 For each group of purification columns, collect fractions 1, 1-2, 1-3, and 1-4 after the void volume, respectively.
[0057] 4Exosome analysis: 4.1 Observation of exosome morphology by transmission electron microscopy (TEM).
[0058] 4.2 Nanoparticle tracking analysis (NTA) was used to detect the number concentration of exosomes in each fraction.
[0059] 4.3 Western Blot was used to detect exosome marker proteins (CD63, CD9, TSG101).
[0060] 5. Data Analysis: 5.1 Evaluation of exosome morphology by electron microscopy.
[0061] 5.2 Calculate the recovery rate of exosome particles.
[0062] 5.3 Analysis of the abundance of exosomal protein markers.
[0063] 6 Overall rating: 6.1 Comprehensive score under each group of conditions. The comprehensive score is the sum and average of the exosome electron microscopy morphology score, the exosome particle number recovery rate score and the exosome protein marker detection score. The exosome particle number recovery rate score is the exosome particle number recovery rate x10.
[0064] The results showed that in terms of void volume optimization, when the void volume accounted for 30% of the column volume, the comprehensive score of exosomes was significantly higher than other proportions (10%, 20% and 40%). This finding provides key parameters for purification column design, which helps to maximize the separation efficiency and purity of exosomes.
[0065] In terms of optimization of exosome collection fractions, experimental results show that under the optimal condition of 30% void volume, the optimal exosome separation effect can be achieved when the 1-2 or 1-3 fractions after the void volume are collected, which is characterized by high purity, high recovery rate and good morphological integrity. This precise collection strategy not only improves the purity of exosomes, but also ensures maximum sample recovery.
[0066] Table 1
[0067] *The comprehensive score is the sum of the exosome electron microscopy morphology score, the exosome particle number recovery rate score and the exosome protein marker detection score. The exosome particle number recovery rate score is the exosome particle number recovery rate x10.
[0068] Example 2: Isolation of exosomes from cell culture supernatant using PEG precipitation combined with purification columns according to Figure 2 The separation and purification process shown is carried out, and the specific steps are as follows: 1. Sample preparation: 1.1 Concentrate the cell culture supernatant 100-fold using PEG precipitation.
[0069] 1.2 Filter the sample using a 0.22 μm filter membrane to remove large particles.
[0070] 2. Column preparation and balance: 2.1 Equilibrate the purification column to room temperature (18-25°C).
[0071] 2.2 Remove the covers at the top and bottom of the column and fix it vertically on the laboratory bench.
[0072] 2.3 Wash the column with 20 mL PBS and wait for the PBS to flow through completely.
[0073] 3. Sample loading: 3.1 After PBS has drained away, add 0.5 mL of pretreated sample immediately.
[0074] 3.2 After the sample has completely entered the column bed, add 0.3 column volumes of PBS for elution.
[0075] 3.3 Wait for the PBS buffer to flow out completely.
[0076] 4. Exosome collection: 4.1 Add 0.5 mL PBS in portions, collect three 0.5 mL fractions of eluate and combine them.
[0077] 4.2 The collected fractions are the purified exosome samples.
[0078] 5. Exosome analysis: 5.1 Determination of protein concentration by biuret assay (BCA) 5.2 Observation of exosome morphology by transmission electron microscopy (TEM).
[0079] 5.3 Nanoparticle tracking analysis (NTA) was used to detect the number concentration of exosomes in each fraction.
[0080] 5.3 Use Western Blot to detect exosome marker proteins (CD63, CD9, TSG101).
[0081] Table 2
[0082] Results: PEG precipitation was used to initially enrich the cell culture supernatant. Although it can effectively concentrate exosomes, Figure 3 As shown in the figure, transmission electron microscopy observation of crude samples extracted by PEG precipitation method (i.e., samples before column extraction) showed that there were a large number of non-specific impurities such as proteins and nucleic acids in the samples. Protein quantification by biuret method (BCA) (Table 2) showed that the total protein content in the samples obtained by PEG precipitation was 3052.64 ng / μL, and the protein content of 0.5 mL sample was 1526.32 μg.
[0083] The crude exosomes after PEG precipitation were purified by column, and protein quantitative analysis showed that the total protein content of the purified sample dropped to 114.11μg, indicating that the purification column can effectively remove about 92.53% of protein impurities. Transmission electron microscopy observations further confirmed the significant improvement in the purification effect: compared with the blurred background and a large number of impurity particles in the PEG precipitation sample, the purified sample background was clear, the exosomes showed a typical round or cup-shaped structure, the particle size distribution was uniform (30-150nm), and there were no obvious protein aggregates and other impurities.
[0084] like Figure 4 As shown in Figure 2, the concentration of purified exosomes was 1.64E+10 particles / mL as determined by nanoparticle tracking analysis (NTA), the particle size distribution was concentrated, the particle size uniformity was good, and the particle recovery rate reached 60.74% (Table 2). Figure 5 As shown, Western blot analysis showed that the bands of marker proteins such as CD63, CD9 and TSG101 in the purified exosome samples were clear, further confirming the high purity and specificity of the samples.
[0085] The above results fully demonstrate that the combination of PEG precipitation and purification column can effectively overcome the disadvantage of high impurity content of single PEG precipitation method. This method not only ensures the efficient recovery of exosomes, but also significantly improves the purity of the product. The obtained exosomes have ideal morphological characteristics and biological properties. The purified exosomes can be directly used in a variety of subsequent experiments, such as transmission electron microscopy observation, proteomics analysis, RNA sequencing, etc.
[0086] This example fully demonstrates the application advantages of this product in exosome purification and verifies the effectiveness and practicality of the method of the present invention. This method not only meets the needs of scientific research, but also provides reliable technical support for clinical applications.
[0087] Example 3: Regeneration and reuse performance evaluation of purification columns The specific steps are as follows: 1. Regeneration and reuse of purification columns 1.1 Complete the isolation and purification of exosomes according to standard procedures.
[0088] 1.2 Use the adapter to carry a 20 mL syringe and add 1 column volume of 0.5 M NaOH to the purification column to clean and regenerate the packing.
[0089] 1.3 After regeneration, wash with at least 2 column volumes of PBS for pH neutralization.
[0090] 1.4 Repeat the above steps for 5 consecutive times of exosome isolation and purification.
[0091] 2. Performance evaluation indicators: 2.1 Purification efficiency: The protein concentration was determined by the biuret assay (BCA) and the protein removal rate was calculated.
[0092] 2.2 Exosome recovery rate: Nanoparticle tracking analysis (NTA) was used to determine the number of recovered exosome particles after each purification.
[0093] 2.3 Morphological integrity: The morphological structure of exosomes was observed by transmission electron microscopy (TEM).
[0094] 2.4 Marker protein expression: Western blot was used to detect marker proteins such as CD63, CD9 and TSG101.
[0095] Table 3
[0096] *The comprehensive score is the sum of the protein removal rate score, exosome particle number recovery rate score, exosome electron microscopy morphology score and exosome protein marker detection score. The protein removal rate score is protein removal rate x 10, and the exosome particle number recovery rate score is exosome particle number recovery rate x 10.
[0097] Experimental results: As shown in Table 3, in the evaluation of the purification column for 5 consecutive uses, for the same sample, the recovery rate of exosomes purified each time remained stable, with a fluctuation range of ±1%. Through transmission electron microscopy, it was found that the exosomes obtained from the 5 purifications all showed a typical cup-shaped or round structure, with a diameter distribution between 30-150nm and good morphological integrity. Western blot analysis showed that the expression levels of marker proteins such as CD63, CD9 and TSG101 in the exosome samples after each purification were basically the same, indicating that the purification column has a stable separation effect.
[0098] In terms of purification efficiency, the protein removal rates of the five purifications were maintained above 90%. Figure 6 As shown in the figure, the fraction protein concentration remains stable, which indicates that the purification column still maintains good separation performance after multiple uses. It is worth noting that the standardized regeneration process, including PBS washing and 20% ethanol disinfection, can effectively prevent cross-contamination between samples and ensure the reliability of purification results.
[0099] In summary, this experiment confirmed that the purification column has excellent reusability. After standardized regeneration treatment, the purification column can maintain a stable separation effect, which not only reduces the experimental cost but also improves the practicality of the method. It is recommended to strictly follow the regeneration process in daily use to ensure that the purification column can be reused 5 times.
[0100] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.
Claims
1. A kit for separation and purification of exosomes, characterized in that: The invention comprises a columnar box body, wherein an upper sieve plate and a lower sieve plate are arranged in the columnar box body, a purification column is formed between the upper sieve plate and the lower sieve plate, the purification column is filled with solid resin, and the space between the solid resins is filled with liquid PBS buffer solution.
2. The exosome separation and purification kit according to claim 1, characterized in that: The solid resin is a highly cross-linked composite matrix of dextran and agarose.
3. The exosome separation and purification kit according to claim 1, characterized in that: The volume of the liquid PBS buffer between the upper and lower frits is 25% to 35% of the volume of the purification column.
4. The exosome separation and purification kit according to claim 3, characterized in that: The volume of the liquid PBS buffer between the upper frit and the lower frit is 30% of the volume of the purification column.
5. A method for separating and purifying exosomes, using the kit according to any one of claims 1 to 4, characterized in that: The following steps are included: Pre-treating the sample to be purified; Inject the pretreated sample into the reagent kit so that the sample passes through the upper frit plate, the purification column and the lower frit plate in sequence; The samples that passed through the upper frit, purification column and lower frit were collected in sequence to obtain the exosome fraction.
6. The method for separating and purifying exosomes according to claim 5, characterized in that: The following steps are included: After obtaining the exosome fraction, a purification column regeneration step is also included: NaOH solution is added to the kit to clean and regenerate the filler.
7. The method for separating and purifying exosomes according to claim 5, characterized in that: The pretreatment of the sample comprises at least one of ultracentrifugation, ultrafiltration, tangential flow filtration and PEG precipitation.
8. The method for separating and purifying exosomes according to claim 5, characterized in that: The sample is a small volume sample of 0.5mL-5mL.
9. The method for separating and purifying exosomes according to claim 5, characterized in that: The timing of collecting the exosome fraction is: after adding the sample to the kit, collect the volume of liquid PBS buffer corresponding to the liquid flowing out between the upper and lower sieve plates, and then collect 0.5mL-1.5mL of the liquid that flows out.
10. The method for separating and purifying exosomes according to claim 5, characterized in that: The filler can be cleaned and regenerated using 0.5M NaOH and can be reused after regeneration.
11. The exosome separation and purification kit according to claim 5, characterized in that: The kit also includes an adapter for carrying a syringe barrel, which is used to add 5mL-50mL of PBS buffer.
12. The exosome separation and purification kit according to claim 5, characterized in that: The kit achieves separation and purification of exosomes through gravity.
13. The exosome separation and purification kit according to claim 5, characterized in that: The volume of the purification column is 8mL-16mL.