A method for preparing an exosome

Abstract

The present application relates to a preparation method of exosomes, comprising the following steps: 1) concentrating cell supernatant to obtain a concentrated solution by using a tangential flow filtration method; 2) removing nucleic acids from the concentrated solution by using a universal nuclease and resuspending to obtain a crude extract; 3) performing density gradient centrifugation on the crude extract, and collecting the exosome layer; 4) performing low-speed centrifugation on the exosome layer under a centrifugal force less than or equal to 50000g, and removing the precipitate to obtain a preliminary purified solution; and 5) performing ultracentrifugation on the preliminary purified solution under a centrifugal force greater than or equal to 100000g, and the obtained precipitate is the exosomes. The exosomes obtained by the method have a complete and un-deformed membrane structure, higher purity and particle concentration, and better quality compared with exosomes prepared by existing methods. Meanwhile, the method has high yield and low cost, and helps to realize the industrialized preparation of exosomes, and provides strong support for targeted drug delivery research using exosomes as carriers.

Description

Technical Field

[0001] This invention relates to a method for preparing exosomes, specifically a method for separating and purifying exosomes from cell supernatant. Background Technology

[0002] Extracellular vehicles (EVs) are tiny membrane vesicles secreted by most cells of archaea, bacteria, and eukaryotes. EVs are nanometer-sized, possess a double-layered closed membrane structure, and contain components such as lipids, RNA, metabolites, growth factors, and cytokines. EVs regulate complex cell communication, maintain normal physiology, or trigger serious diseases. Based on their biogenesis and secretion mechanisms, EVs are generally classified into three subtypes: exosomes, microvesicles, and apoptotic bodies. The biogenesis and secretion mechanism of exosomes involves inward budding of the cytoplasmic membrane, followed by the formation of multivesicles. These multivesicles fuse with the cytoplasmic membrane and are secreted extracellularly, forming membrane vesicles with a diameter of 30-200 nm.

[0003] With the development of targeted drug delivery, nanomedicine carriers have received increasing attention. Exosomes, as a naturally derived delivery carrier, have advantages such as low immunogenicity, minimal toxicity, selectivity for recipient cells, high affinity for nucleic acid molecules, and the ability to cross the blood-brain barrier. Therefore, many biopharmaceutical companies are investing in the development of exosome drug delivery platforms, and obtaining high-quality exosomes is a prerequisite for exosome drug delivery.

[0004] Exosome extraction techniques mainly include ultracentrifugation, density gradient centrifugation, chemical precipitation, size exclusion, and immunocapture. Ultracentrifugation separates exosome supernatants based on the differences in sedimentation rates of exosomes, proteins, cell debris, cells, and organelles in the sample, using different centrifugal forces and times. Density gradient centrifugation utilizes the density difference between exosomes and other solutes for separation. Chemical precipitation uses polyethylene glycol (PEG) and other substances to alter the solubility and dispersibility of exosomes, causing less soluble components to precipitate from the solution. Size exclusion is a method of separation and extraction based on exosome size using chromatographic columns. Although many of these exosome extraction techniques have been developed, in actual preparation, it is difficult to simultaneously achieve both exosome quality and yield. Ensuring quality, such as improving purity and exosome structural stability, cannot increase yield, leading to high production and usage costs and low production efficiency. Conversely, ensuring yield reduces the purity, membrane integrity, and natural activity of exosomes. For example, exosome extracts often contain residual proteins, nucleic acids, or vesicles from other cell structures, which significantly impacts the functionality of exosomes and limits the development of exosome drug delivery systems.

[0005] Patent CN111321108A discloses a method for separating exosomes, which uses PEG6000 precipitation, dialysis and density centrifugation of iodixanol to process exosomes. Although the processing time is shortened, the transmission electron microscopy results show that the "cup-and-disc" structure of the exosomes is not obvious, the exosomes are deformed, and only one exosome is shown in the electron microscopy image. It can be seen that while improving the purity, the yield is sacrificed.

[0006] Patent CN1109646694A discloses a method for extracting exosomes, which improves the purity and yield of exosomes based on density gradient centrifugation and ultracentrifugation. However, the transmission electron microscopy image shows that the "cup-and-disc" structure of the exosomes is not obvious and the exosomes are deformed. Although multiple exosomes are present in the field of view, the background is noisy, indicating that there are many impurities in the extract and the quality of the exosomes needs to be improved.

[0007] Currently, there is a lack of preparation methods that can simultaneously ensure both the quality and yield of exosomes. For research on the drug-carrying function of exosomes, improving the preparation methods to obtain high-quality, high-yield exosomes is an urgent problem to be solved. Summary of the Invention

[0008] The technical problem solved by the present invention is to overcome the shortcomings of the prior art and provide an improved method for preparing exosomes.

[0009] To solve the above technical problems, the present invention adopts the following technical solution:

[0010] A method for preparing exosomes, characterized in that the preparation method includes the following steps:

[0011] 1) The cell supernatant was concentrated using a tangential flow filtration method to obtain a concentrate, wherein a hollow fiber column with a molecular weight cutoff of 100kDa to 750kDa was used for tangential flow concentration, and the concentration factor was 10 to 20 times.

[0012] 2) The concentrate was subjected to nucleic acid removal treatment using a totipotent nuclease and then resuspended to obtain a crude extract;

[0013] 3) Perform density gradient centrifugation on the crude extract and collect the exosome layer;

[0014] 4) The exosome layer was centrifuged at low speed under a centrifugal force of less than or equal to 50,000 g to remove the precipitate and obtain a preliminary purified solution;

[0015] 5) The preliminary purified solution is subjected to ultracentrifugation at a centrifugal force of 100,000 g or more, and the resulting precipitate is the exosome.

[0016] Preferably, in step 1), the tangential flow filtration method uses a hollow fiber column with a molecular weight cutoff of 100kDa to 500kDa. Further, a hollow fiber column of 100kDa, 200kDa, 300kDa, or 500kDa can be selected.

[0017] Preferably, in step 1), the concentration factor is 10 times, 12 times, 14 times, 16 times, 18 times, or 20 times.

[0018] Preferably, in step 2), a totipotent nuclease and a water-soluble metal salt are added to the concentrate, and the temperature is controlled at 25°C to 37°C to lyse the nucleic acid in the concentrate. Then, the concentrate is subjected to ultracentrifugation at a centrifugal force of 100,000g to 200,000g, the precipitate is collected and dissolved in buffer solution to obtain the crude extract.

[0019] More preferably, the water-soluble metal salt used in step 2) includes, but is not limited to, water-soluble magnesium salt, water-soluble calcium salt, and water-soluble manganese salt. Even more preferably, the water-soluble metal salt is one or more of magnesium chloride, calcium chloride, or manganese chloride.

[0020] Specifically, a water bath is used to control the temperature.

[0021] Specifically, the buffer solution used to dissolve the precipitate is PBS buffer, Tris buffer, or HEPES buffer, preferably PBS buffer.

[0022] Preferably, in step 3), a mixture of iodixanol solution and sucrose buffer is used as a gradient medium. The mixture is first added to a centrifuge tube, and the crude extract is premixed with iodixanol solution and added to the bottom of the gradient medium. Finally, the centrifuge tube is filled with sealing solution and centrifuged at a centrifugal force of 100,000 g to 200,000 g to collect the exosome layer.

[0023] According to some specific embodiments, a sealing solution is added before adding the mixture of iodixanol solution and sucrose buffer to the centrifuge tube.

[0024] According to some specific embodiments, the mixture of iodixanol solution and sucrose buffer is prepared by mixing iodixanol solution and sucrose buffer at a volume ratio of 1:(4-6), wherein the mass concentration of iodixanol solution is 55%-65%, the pH value of sucrose buffer is 7.0-7.5 and contains sucrose, tris(hydroxymethyl)aminomethane hydrochloride (TrisHCl) and ethylenediaminetetraacetic acid (EDTA), wherein the concentration of sucrose is 200mM-300mM.

[0025] According to some specific embodiments, during the premixing process, an iodixanol solution with a mass concentration of 55% to 65% is mixed with the crude extract at a volume ratio of 1:(1 to 3).

[0026] According to some specific embodiments, the sealing solution is PBS buffer, Tris buffer, or HEPES buffer, or any combination thereof.

[0027] After centrifugation, the exosome layer was a white interface layer that migrated to the spacer medium and the gradient medium.

[0028] In this invention, optimized pretreatment reduces the number of layers required for density gradient centrifugation, making the operation more convenient.

[0029] According to some specific embodiments, the components of the sucrose buffer include 200mM to 300mM sucrose, 5mM to 15mM Tris HCl, and 0.5mM to 1.5mM EDTA.

[0030] Preferably, the centrifugal force used in step 4) is 20,000 g to 50,000 g. By using an appropriate centrifugal force, residual proteins in the exosome gradient layer can be effectively removed, while exosome precipitation can be avoided.

[0031] Preferably, the centrifugal force used in step 5) is 100,000g to 200,000g, and more preferably, centrifugation is performed under a centrifugal force of 100,000g to 150,000g.

[0032] In this invention, the collected exosome precipitates are resuspended in PBS buffer, Tris buffer, HEPES buffer, or sterile enzyme-free water and then stored for later use.

[0033] In this invention, all centrifugation in the preparation method is carried out at 0℃ to 8℃.

[0034] According to some implementation methods, the preparation method is as follows:

[0035] (1) The cell supernatant was concentrated 10 to 20 times by tangential flow filtration with a molecular weight cutoff of 100 kDa to 750 kDa to obtain a concentrated solution;

[0036] (2) Add to the concentrate a universal nuclease and water-soluble metal salt, control the temperature at 25℃~37℃ to lyse the nucleic acid in the concentrate, and then perform ultracentrifugation at a centrifugal force of 100000g~200000g, collect the precipitate and dissolve it with buffer solution to obtain the crude extract.

[0037] (3) Add sealing solution to the centrifuge tube, using a mixture of iodixanol solution and sucrose buffer as a gradient medium. Add the sealing solution to the centrifuge tube from the bottom. Then add the premixed crude extract and iodixanol solution to the bottom of the gradient medium. Finally, fill the centrifuge tube with sealing solution and centrifuge at a centrifugal force of 100,000 g to 200,000 g. After centrifugation, collect the white interface layer between the sealing solution layer and the gradient medium layer to obtain the exosome layer.

[0038] (4) The exosome layer was centrifuged at low speed under a centrifugal force of 20,000 g to 50,000 g to remove the precipitate and obtain a preliminary purified solution.

[0039] (5) The preliminary purified solution is subjected to ultracentrifugation at a centrifugal force of 100,000g to 200,000g, and the resulting precipitate is the exosome.

[0040] Preferably, the water-soluble metal salts used in step 2) include, but are not limited to, water-soluble magnesium salts, water-soluble calcium salts, and water-soluble manganese salts.

[0041] More specifically, in step (2), the water-soluble metal salt is one or more of magnesium chloride, calcium chloride or manganese chloride, and its concentration in the system is 0.5mM to 2mM, for example 0.5mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM or 2mM.

[0042] More specifically, in step (2), the final concentration of the pluripotent nuclease in the system is 15 U / mL to 25 U / mL, for example, 15 U / mL, 18 U / mL, 20 U / mL, 22 U / mL, 24 U / mL, or 25 U / mL.

[0043] More specifically, in step (2), the nucleic acid lysis time is 3h to 16h. According to some specific embodiments, the temperature is 25℃ to 32℃, and the nucleic acid lysis time is 8h to 16h. According to other specific embodiments, the temperature is 32℃ to 37℃, and the nucleic acid lysis time is 3h to 8h.

[0044] More specifically, in step (3), the density gradient centrifugation time is 15h to 20h;

[0045] More specifically, in step (4), the low-speed centrifugation time is 20 min to 40 min;

[0046] More specifically, in step (5), the ultracentrifugation time is 2h to 5h.

[0047] More specifically, the precipitate collected in step (6) is stored after being resuspended in PBS buffer, Tris buffer, HEPES buffer or sterile enzyme-free water.

[0048] Preferably, before concentration, the cell supernatant is subjected to two-stage microfiltration, wherein the first-stage microfiltration uses a filter membrane with a pore size of 0.3μm to 0.5μm, and the second-stage microfiltration uses a filter membrane with a pore size of 0.2μm to 0.25μm.

[0049] More preferably, before the two-stage microfiltration process, a deep filtration membrane can be used to remove some residual cells, cell debris and other larger impurities, which can reduce the pressure of the two-stage filtration and prevent clogging during the two-stage microfiltration process.

[0050] Preferably, the cell supernatant has a cell density greater than 9 × 10⁻⁶ cells. 6 Cell culture medium with cells / mL and cell viability greater than 90% is obtained by multi-stage centrifugation, wherein the multi-stage centrifugation includes a first-stage centrifugation and a second-stage centrifugation, wherein the centrifugal force of the first-stage centrifugation is less than 10,000g and the centrifugal force of the second-stage centrifugation is 10,000g to 50,000g.

[0051] In this invention, the cells are not particularly limited and can be one or more of human embryonic kidney cells, mesenchymal stem cells, induced pluripotent stem cells, hepatocytes, immune cells, stromal cells, fibroblasts, amniotic cells, erythrocytes, chondrocytes, endothelial cells, and epithelial cells.

[0052] In a preferred embodiment, the cells are human embryonic kidney cells.

[0053] The present invention also provides exosomes prepared by the above-described method and the application of the above-described exosomes in a drug delivery system.

[0054] By adopting the above technical solution, the present invention has the following advantages compared with the prior art:

[0055] The exosomes obtained by the method of this invention have intact and undeformed membrane structures, higher purity, particle concentration, and higher positive rate of exosome marker proteins. Compared with exosomes prepared by existing methods, the quality of exosomes is better. At the same time, the method of this invention is simple, has a high yield, and is low in cost. Attached Figure Description

[0056] Figure 1 Transmission electron microscopy image of exosomes prepared in Example 1;

[0057] Figure 2 Transmission electron microscopy image of exosomes prepared in Comparative Example 1;

[0058] Figure 3The particle size distribution diagram of the exosomes prepared in Example 1;

[0059] Figure 4 The particle size distribution of exosomes prepared in Comparative Example 1 is shown.

[0060] Figure 5 The graph shows the positive rate detection results of the surface proteins CD9, CD63, and CD81 of the exosomes prepared in Example 1;

[0061] Figure 6 The positive rate detection results of surface proteins CD9, CD63 and CD81 of exosomes prepared for Comparative Example 1 are shown in the figure.

[0062] Figure 7 Gel electrophoresis images of protein expression in exosomes prepared in Example 1 and Comparative Example 1. Detailed Implementation

[0063] Using exosomes as an in vivo drug delivery system is currently a key focus of targeted drug research. Although various methods exist for extracting exosomes from cell culture media, obtaining high-quality and high-yield exosomes remains a challenge in this field. Whether in small-scale laboratory preparations or large-scale industrial production, it is difficult to simultaneously achieve high quality and high yield of exosomes. This is primarily because cell culture media contain residual proteins and nucleic acids from host cells, as well as other vesicle structures produced by the cells. During exosome extraction, these substances need to be removed as much as possible. However, exosomes, being vesicles, are deformed and broken during the preparation process, resulting in a significant reduction in their natural activity. Improper preparation procedures or inappropriate step parameters can negatively impact the quality of exosomes. During preparation, researchers cannot directly confirm the impact of each step on the quality and yield of exosomes in the extract, making adjustments impossible. After preparation, researchers also cannot use exosome quality analysis to identify which details of the preparation method need adjustment and how. This is another reason for the difficulties in exosome research. Therefore, a method for preparing exosomes with both high quality and high yield has yet to be developed. The present invention aims to solve the aforementioned problems.

[0064] This invention, through holistic process design, including the organic combination of different processes and step-by-step flow design, has played an unexpectedly positive role in ensuring the purity, yield, and natural bioactivity of the subsequently prepared exosomes. Furthermore, this invention optimizes the preparation conditions and parameters, resulting in exosomes with intact and undeformed structures, high purity, high natural activity, and high yield. This method facilitates the industrial-scale preparation of exosomes and provides strong support for research on targeted drug delivery using exosomes as carriers.

[0065] Terminology Definition

[0066] The terms “exosomes” and “EVs” are used interchangeably and include vesicles derived from the endosome, lysosome and / or endosomal pathways, including unmodified natural exosomes and genetically modified / engineered exosomes.

[0067] When describing the term "exosome," it usually refers to a group of multiple exosomes rather than a single exosome, and its concentration is usually expressed as the number of exosomes (particles) per unit volume (e.g., per milliliter).

[0068] The term "genetically modified / engineered exosomes" refers to exosomes derived from genetically modified / engineered cells, which typically contain recombinant or exogenous DNA or its protein products.

[0069] The term "cultured cells" refers to the expansion of the number or concentration of any cells that can produce EVs under appropriate conditions, such as in suspension culture, adherent culture, or any other type of culture system.

[0070] The term "any cell capable of producing EVs" includes, but is not limited to, one or more of the following: human embryonic kidney cells, mesenchymal stem cells, stromal cells, fibroblasts, amniotic cells, erythrocytes, chondrocytes, endothelial cells, and epithelial cells.

[0071] The term "drug delivery system" refers to a formulation that delivers a drug active ingredient to a desired body site and / or releases a therapeutic agent at the appropriate time. In this application, the drug active ingredients that can be delivered by cell-derived exosomes include small molecule drugs or biological therapeutic agents, wherein the biological therapeutic agents are not naturally present in cell-derived exosomes, and are selected from peptides, proteins, polysaccharides, or nucleic acids, wherein the nucleic acids are selected from single-stranded or double-stranded DNA, iRNA, siRNA, shRNA, mRNA, non-coding RNA (ncRNA), antisense RNA, LNA, morpholino oligonucleotides, or their analogues or conjugates.

[0072] The term "serum-free medium" refers to any cell culture medium that does not contain heterologous or homologous serum, and any cell culture medium known in the art may be used as long as it does not contain serum.

[0073] The term "separation and purification" refers to the physical isolation or separation of a test sample containing the analyte of interest from interfering substances.

[0074] The term "centrifugal force" refers to the apparent outward force that pulls a rotating body away from its center of rotation. The method is preferably a mechanical method, and more preferably carried out by applying centrifugal force in a rotating device such as a centrifuge.

[0075] The term "membrane" refers to a semi-permeable material, which can be used to separate components in a supply fluid into permeates that pass through the material and retainers that are trapped by the material.

[0076] The term "density gradient centrifugation" refers to the process of creating a continuous or discontinuous density gradient in a centrifuge tube using a specific medium. The test sample containing the analyte of interest is mixed with the bottom layer, and the analyte of interest is separated into layers by the centrifugal force field.

[0077] The terms “tangential flow concentration (TFF) system” and “cross-flow filtration” are used interchangeably to refer to the separation of suspended particles from a fluid mixture, including the separation of particles with defined characteristics (e.g., a required particle size range) from a non-homogeneous particle mixture of fluid mixtures and the concentration of the fluid.

[0078] The terms "totipotent nucleic acid" and "broad-spectrum nuclease" are interchangeable. A totipotent nuclease is a non-specific endonuclease derived from Serratia Marcescens that can cleave any nucleotide within the strand under very broad conditions, completely digesting nucleic acids (including single-stranded, double-stranded, linear, circular, native or denatured DNA and RNA) into 5'-monophosphate oligonucleotides of 2 to 5 bases in length.

[0079] The technical solution of the present invention is further described below with reference to specific implementation examples, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. The following embodiments and comparative examples only use human embryonic kidney cells as examples. Other cells, such as fibroblasts, mesenchymal stem cells or stromal cells, are also applicable to the above preparation method. For different cells, the parameters in some steps can be appropriately adjusted.

[0080] Unless otherwise specified, all instruments, raw materials, and reagents used in this invention are commercially available, and the experimental techniques and detection methods are conventional techniques in the field. The centrifuge used in this invention is a low-temperature centrifuge. The membrane packs used for deep filtration can be commercially available products from companies such as Merck (Germany), Pall (USA), Cytiva (USA), Sartorius (Germany), and 3M (USA). The following examples and comparative examples use membrane packs with the product number CSCCD1070PCP from Hangzhou Kebote Co., Ltd., which can effectively intercept some cells, cell debris, and other larger particulate impurities.

[0081] Example 1

[0082] This embodiment provides a method for preparing exosomes, the specific steps of which are as follows:

[0083] [1] HEK293F cells were cultured in serum-free medium at 37℃, 8% CO2, and 120 rpm on a horizontal shaker (amplitude 19 mm) until the viable cell density was greater than 9 × 10⁻⁶ cells / year. 6 Cells / mL, cell viability greater than 90%.

[0084] [2] Collect 1000 mL of culture medium (cells and supernatant), centrifuge at 6000 g and 4 °C for 20 min, and collect supernatant A.

[0085] [3] Collect 16000g of supernatant A, centrifuge at 4℃ for 30min, and collect supernatant B.

[0086] [4] Obtain the supernatant C by deep filtration.

[0087] [5] The supernatant C was filtered through a 0.45 μm filter membrane and a 0.22 μm filter membrane in sequence, and the supernatant D was collected.

[0088] [6] The supernatant D was concentrated 15 times by tangential flow filtration through a 300kDa hollow fiber column to obtain the concentrate.

[0089] [7] Add MgCl2 solution (final concentration 1mM) and pluripotent nuclease (final concentration 20U / mL) to the concentrate, and incubate in a water bath at 25°C for 16h or at 37°C for 3h.

[0090] [8] After the water bath, 133900g of the concentrate was centrifuged at 4℃ for 60min. The precipitate was resuspended with 3.25mL PBS and repeatedly pipetted with a 1mL syringe until the precipitate was completely dissolved, yielding 3.25mL of crude extract.

[0091] [9] Prepare sucrose buffer (250 mM Sucrose, 10 mM Tris HCl, 1 mM EDTA, pH 7.4). Prepare a first mixture of 17.5% (V / V) (Iodixanol solution / (Iodixanol solution + Sucrose buffer)) using sucrose buffer and 60% (w / v) iodixanol solution as a gradient medium. Premix the crude extract and 60% (w / v) iodixanol solution to prepare a second mixture of 45% (V / V) (Iodixanol solution / (Iodixanol solution + crude extract)), using PBS as the sealing solution. Add about 6 mL of PBS to the bottom of a centrifuge tube with a syringe. Then, add the first and second mixtures sequentially from the bottom of the centrifuge tube using the syringe. Finally, fill the tube with PBS from the top. A blank control group without the crude extract solution was also set up.

[0092]

[10] Centrifuge at 150000g and 4℃ for 16h, and collect the white interfacial layer that migrates between PBS and the first layering solution, which is the exosome layer.

[0093]

[11] The exosome layer was transferred to a new 39 mL tube, filled with PBS, and centrifuged at 20000 g and 4 °C for 30 min to remove the precipitate. The precipitate was mainly residual protein. The supernatant E was collected in about 39 mL, which was the preliminary purification solution.

[0094]

[12] Transfer the supernatant E to a new tube, centrifuge at 135000g and 4℃ for 3h, and the precipitate is the exosome.

[0095]

[13] The precipitate was resuspended in 200 μL PBS and stored at 4 °C.

[0096] Example 2

[0097] This embodiment provides a method for preparing exosomes, which is basically the same as in Embodiment 1, except that a 100kDa hollow fiber column is used to concentrate the cell supernatant.

[0098] Example 3

[0099] This embodiment provides a method for preparing exosomes, which is basically the same as in Embodiment 1, except that a 500kDa hollow fiber column is used to concentrate the cell supernatant.

[0100] Comparative Example 1

[0101] This comparative example provides a method for preparing exosomes, which is largely the same as that in Example 1, except that steps [4] to [6] are not performed.

[0102] Exosome identification experiment

[0103] The integrity and size of the extracted exosome membrane structure were observed using transmission electron microscopy (TEM). The extracted exosomes were identified using different detection indicators. Nanoparticle tracking (NTA) was used to analyze the particle size and number concentration of exosomes; a lower number concentration indicated a lower exosome yield. The purity of the exosomes was determined by detecting the positivity rates of FITC antibody-labeled surface proteins CD9, CD63, and CD81 using NanoFCM nanoflow cytometry. Western blot experiments were used to determine the expression of exosome marker proteins. The protein concentration before and after tangential flow concentration was detected using the BCA method. Based on the protein concentration and the volume before and after tangential flow concentration, the removal rate of impurity proteins in the concentrate was calculated.

[0104] The exosomes obtained in the examples and comparative examples were observed by transmission electron microscopy (TEM). At the same magnification and within the same field of view, the exosomes in the examples showed a greater number of typical "cup-and-disc" vesicles with intact and undeformed membrane structures, and fewer background impurities. (Comparison) Figure 1 and Figure 2 As can be seen, the exosome membrane structure prepared in Example 1 was intact and undeformed, with the expected size, a greater number of typical vesicles, and significantly less background impurities. In contrast, the exosomes prepared in Comparative Example 1 showed a significantly reduced number of typical vesicles and significantly more background impurities. In Examples 1 to 3, background impurities were very low, but the number of typical vesicles in the exosomes prepared in Examples 1 and 2 was significantly greater than that in Example 3.

[0105] The particle size and number concentration of exosomes in the examples and comparative examples were analyzed using nanoparticle tracking (NTA) technology. The results are as follows: Figure 3 and Figure 4 As shown: The exosomes of Example 1 have a narrow particle size distribution, with a peak particle size of 142.4 nm and an average particle size of 173.1 nm, exhibiting good uniformity. The particle number concentration of the exosomes is 1.01E+12 (particles / mL), while the particle number concentration of the exosomes of Comparative Example 1 is 1.41E+11 (particles / mL). The exosome yield of Comparative Example 1 is significantly lower than that of Example 1.

[0106] According to the NanoFCM comparison of the positive rates of exosome surface proteins CD9, CD63, and CD81 between the examples and the comparative examples, the positive rates of exosomes prepared in the examples for CD9, CD63, and CD81 were all higher than those in the comparative examples. Figure 5 and Figure 6 The results showed that the positive rates of surface proteins CD9, CD63, and CD81 of exosomes in Example 1 were 26.2%, 31.2%, and 42.9%, respectively, while the positive rates of surface proteins CD9, CD63, and CD81 of exosomes in Comparative Example 1 were 22.1%, 22.7%, and 35.5%, respectively. The purity of exosomes in Example 1 was significantly higher than that in Comparative Example 1.

[0107] Based on the expression of marker proteins in the exosomes of the experimental examples and the comparative examples, Western blot analysis was conducted to further identify and compare the exosomes of the experimental examples and the comparative examples. Figure 7The results showed that the expression level of the marker protein CD9 in the exosomes of Example 1 was significantly higher than that in the exosomes of Comparative Example 1. The expression levels of other marker proteins were not significantly different (the expression levels of TSG101 and CD81 in Example 1 were slightly higher than those in Comparative Example 1), indicating that the vesicle particles obtained in Example 1 and Comparative Example 1 were indeed exosomes. The expression of CD63 was diffuse, while the CD63 expression in Comparative Example 1 was more diffuse than that in Example 1, indicating that its purity was lower than that in Example 1. Nanoparticle tracking (NTA) technology was used to detect the particle number concentration before membrane transfer (supernatant D) and after membrane transfer (concentrate) in Examples 1, 2, and 3 during the tangential flow concentration stage. The vesicle recovery rate during the tangential flow concentration stage was calculated by combining the volumes of the supernatant before membrane transfer (supernatant D) and the concentrate after membrane transfer (concentrate): Vesicle recovery rate (%) = (particle number concentration after membrane transfer × volume after membrane transfer) ÷ (particle number concentration before membrane transfer × volume before membrane transfer) × 100%. The protein concentration before membrane transfer (supernatant D) and after membrane transfer (concentrate) in Examples 1, 2, and 3 during the tangential flow concentration stage was detected by the BCA method. The impurity protein removal rate was calculated by combining the volumes of the supernatant before membrane transfer (supernatant D) and the concentrate after membrane transfer (concentrate): Impurity protein removal rate (%) = (protein concentration after membrane transfer × volume after membrane transfer) ÷ (protein concentration before membrane transfer × volume before membrane transfer) × 100%. The results of vesicle recovery rate (%) and impurity protein removal rate (%) are shown in Table 1.

[0108] Table 1

[0109] Before and after tangential flow concentration Vesicle recovery rate (%) Impurity protein removal rate (%) 100kDa hollow fiber column 78.26 98.67 300kDa hollow fiber column 85.71 99.84 500kDa hollow fiber column 57.66 100

[0110] Table 1 shows that the 500kDa hollow fiber column and the 300kDa hollow fiber column achieved protein removal rates of 100% and 99.84%, respectively, indicating that hollow fiber columns with a molecular weight cutoff of 500kDa or less can effectively remove impurity proteins from the supernatant D. The vesicle recovery efficiencies of the 100kDa and 300kDa hollow fiber columns were 78.26% and 85.71%, respectively. With decreasing molecular weight cutoff, the content of other impurity molecules in the concentrate after membrane treatment increases, putting pressure on subsequent extraction and purification steps. With increasing molecular weight cutoff, the vesicle recovery rate decreases, leading to a decrease in the final exosome yield. Therefore, considering various factors, hollow fiber columns with a molecular weight cutoff of 100kDa-500kDa are preferred for the tangential flow concentration step.

[0111] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand the content of the present invention and implement it. However, this description should not be construed as limiting the scope of protection of the present invention. Furthermore, the present invention is not limited to the above-described embodiments. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes, characterized in that: The preparation method includes the following steps: 1) The cell supernatant was concentrated using a tangential flow filtration method to obtain a concentrate, wherein a hollow fiber column with a molecular weight cutoff of 100kDa~300kDa was used for tangential flow concentration, and the concentration factor was 10~20 times, wherein the cells were human embryonic kidney cells; 2) Add 15 U / mL to 25 U / mL to the concentrate and 0.5 mM to 2 mM of water-soluble metal salt, and control the temperature at 25℃ to 37℃ to lyse the nucleic acid in the concentrate. Then, perform ultracentrifugation at a centrifugation force of 100,000 g to 200,000 g, collect the precipitate and dissolve it in buffer to obtain a crude extract. The water-soluble metal salt is magnesium chloride. 3) Add sealing buffer to the centrifuge tube. Use a mixture of 55%–65% iodixanol solution and sucrose buffer at a volume ratio of 1:(4–6) as a gradient medium. Add this mixture from the bottom of the sealing buffer into the centrifuge tube. Then add the premixed crude extract and iodixanol solution to the bottom of the gradient medium. Finally, fill the centrifuge tube with the sealing buffer and centrifuge at a centrifugal force of 100,000 g–200,000 g. After centrifugation, collect the white interface layer between the sealing buffer layer and the gradient medium layer to obtain the exosome layer. The sucrose buffer solution has a pH of 7.0-7.5 and contains sucrose, tris(hydroxymethyl)aminomethane hydrochloride, and ethylenediaminetetraacetic acid, with a sucrose concentration of 200 mM-300 mM. During the premixing process, a 55%–65% (w / w) solution of iodixanol is mixed with the crude extract at a volume ratio of 1:(1–3). The sealing solution is PBS buffer, Tris buffer, or HEPES buffer, or any combination thereof. 4) The exosome layer was centrifuged at low speed under a centrifugal force of 20,000 g to 50,000 g to remove the precipitate and obtain a preliminary purified solution; 5) The preliminary purified solution is subjected to ultracentrifugation at a centrifugal force of 100,000g to 200,000g, and the resulting precipitate is the exosome.

2. The preparation method according to claim 1, characterized in that: In step 1), the tangential flow filtration method uses hollow fiber columns with a molecular weight cutoff of 100kDa, 200kDa, or 300kDa.

3. The preparation method according to claim 1, characterized in that: In step (2), the nucleic acid lysis time is 3h~16h; And / or, in step (4), the centrifugation time of the low-speed centrifugation is 20 min to 40 min; And / or, in step (5), the centrifugation time of the ultracentrifugation is 2h~5h; And / or, the precipitate collected in step (5) is stored after resuspending in PBS buffer, Tris buffer, HEPES buffer or sterile enzyme-free water.

4. The preparation method according to claim 1, characterized in that: Before concentration, the cell supernatant is subjected to two-stage microfiltration. The first stage of microfiltration uses a filter membrane with a pore size of 0.3 μm to 0.5 μm, and the second stage of microfiltration uses a filter membrane with a pore size of 0.2 μm to 0.25 μm.

5. The preparation method according to claim 1, characterized in that: The cell supernatant contains cells with a cell density greater than 9 × 10⁻⁶. 6 Cell culture medium with cell count / mL and cell viability greater than 90% is obtained by multi-stage centrifugation, wherein the multi-stage centrifugation includes a first-stage centrifugation and a second-stage centrifugation. The centrifugal force of the first-stage centrifugation is less than 10,000 g, and the centrifugal force of the second-stage centrifugation is 10,000 g to 50,000 g.

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