CN115710572A

CN115710572A - Preparation method of exosome

Info

Publication number: CN115710572A
Application number: CN202211552943.6A
Authority: CN
Inventors: 翟艳华; 杨诗逸; 王晓煜; 何新军; 许可
Original assignee: Suzhou Weisilkang Technology Co ltd
Current assignee: Suzhou Weisilkang Technology Co ltd
Priority date: 2022-12-05
Filing date: 2022-12-05
Publication date: 2023-02-24

Abstract

The invention relates to a preparation method of exosome, which comprises the following steps: 1) Concentrating the cell supernatant by tangential flow filtration to obtain a concentrated solution; 2) Performing denucleation acid treatment on the concentrated solution by using the omnipotent nuclease, and performing heavy suspension to obtain a crude extract; 3) Performing density gradient centrifugation on the crude extract, and collecting an exosome layer; 4) Centrifuging the exosome layer at low speed under the condition that the centrifugal force is less than or equal to 50000g, and removing precipitates to obtain primary purified liquid; 5) And (3) carrying out ultracentrifugation on the primary purified liquid under the condition that the centrifugal force is more than or equal to 100000g, and obtaining a precipitate, namely the exosome. The exosome obtained by the method has the advantages of complete membrane structure, no deformation, higher purity and particle concentration, better quality compared with the exosome prepared by the existing method, high yield and low cost.

Description

Preparation method of exosome

Technical Field

The invention relates to a preparation method of exosome, in particular to a method for separating and purifying exosome from cell supernatant.

Background

Extracellular Vesicles (EVs) are tiny membrane vesicles secreted by most cells of archaea, bacteria and eukaryotes. The extracellular vesicles are of a nanoscale size, have a double-layer closed membrane structure, and comprise lipid, RNA, metabolites, growth factors, cytokines, and other components. Extracellular vesicles are capable of regulating complex cellular communication, maintaining normal physiology of an organism or causing serious disease. Extracellular vesicles are generally divided into three subtypes, depending on the mechanism of biogenesis and secretion of the extracellular vesicles: exosomes, microvesicles and apoptotic bodies, wherein the biogenesis and secretion mechanism of the exosomes is that cytoplasmic membranes bud inwards and then form multivesicular bodies which are fused with the cytoplasmic membranes and secreted to the outside of cells to form membranous vesicles with the particle size of 30-200 nm.

With the development of the field of targeted drug delivery, nano drug carriers are receiving more and more attention. The exosome is used as a natural-source delivery carrier, and has the advantages of low immunogenicity, small toxic and side effects, certain selectivity on receptor cells, high affinity with nucleic acid molecules, capability of penetrating blood brain barriers and the like, so that a plurality of biological medicine companies are put into the development of an exosome drug-loading platform, and the obtainment of high-quality exosomes is a prerequisite condition for loading exosomes.

The exosome extraction technology mainly comprises an ultracentrifugation method, a density gradient centrifugation method, a chemical precipitation method, a size exclusion method, an immunocapture method and the like. Ultracentrifugation separates the supernatant of the exosome through different centrifugal forces and centrifugal times according to the difference of sedimentation speeds of the exosome, the protein, cell debris, cells, organelles and other substances in a sample; the density gradient centrifugation method utilizes the density difference between exosome and other solutes to realize separation; the chemical precipitation method changes the solubility and the dispersibility of exosome through polyethylene glycol (PEG) and the like, so that components with lower solubility are separated out from the solution; the size exclusion method is a method for separating and extracting by using a chromatographic column according to the size of an exosome. Although various exosome extraction techniques as described above have been developed, in actual preparation, it is difficult to achieve both quality and yield of exosomes. The quality is ensured, for example, the purity and the structural stability of the exosome are improved, so that the yield cannot be improved, the production cost and the use cost of the exosome are high, and the production efficiency is low; the yield is ensured, the quality of the exosome, such as purity, integrity of an exosome membrane structure, natural activity of the exosome and the like, is reduced, for example, vesicles containing residual proteins and nucleic acids of cells or other structures generated by the cells in an exosome extract can not be avoided, so that great influence is brought to the functional performance of the exosome, and the development of an exosome drug-loading system is limited.

Patent CN111321108A discloses an exosome separation method, which adopts PEG6000 precipitation, dialysis and iodixanol density centrifugation to jointly process exosomes, shortens the processing time, but shows from the result of a transmission electron microscope that the structure of a cup and a tray of exosomes is not obvious, exosomes deform, and only one exosome is contained in the electron microscope image, so that the yield is sacrificed when the purity is improved.

Patent CN1109646694A discloses an exosome extraction method, which improves the purity and yield of exosomes based on density gradient centrifugation and ultracentrifugation, but from a transmission electron microscope image, the structure of a cup and a tray of exosomes is not obvious, the exosomes are deformed, although a plurality of exosomes exist in a visual field, the background is noisy, the impurities in an extract are more, and the exosome quality needs to be improved.

Despite the prior art, a preparation method which can simultaneously consider the quality and yield of exosomes is always lacked. For the research on the medicine-carrying function of the exosome, how to improve the preparation method of the exosome and obtain the exosome with high quality and high yield is an urgent problem to be solved.

Disclosure of Invention

The technical problem solved by the invention is to overcome the defects of the prior art and provide an improved preparation method of exosomes.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for preparing exosomes, comprising: the preparation method comprises the following steps:

1) Concentrating the cell supernatant by adopting a tangential flow filtration method to obtain a concentrated solution, wherein a hollow fiber column with the molecular weight cutoff of 100 kDa-750 kDa is adopted for tangential flow concentration, and the concentration multiple is 10-20 times;

2) Performing denucleation acid treatment on the concentrated solution by using omnipotent nuclease, and performing heavy suspension to obtain a crude extract;

3) Performing density gradient centrifugation on the crude extract, and collecting an exosome layer;

4) Carrying out low-speed centrifugation on the exosome layer under the condition that the centrifugal force is less than or equal to 50000g, and removing precipitates to obtain a primary purified solution;

5) And (3) carrying out ultracentrifugation on the primary purified liquid under the condition that the centrifugal force is more than or equal to 100000g, and obtaining a precipitate, namely the exosome.

Preferably, in step 1), the tangential flow filtration method employs a hollow fiber column with a molecular weight cut-off of 100kDa to 500kDa, and further, a hollow fiber column of 100kDa or 200kDa or 300kDa or 500kDa can be selected.

Preferably, in step 1), the concentration is 10-fold, 12-fold, 14-fold, 16-fold, 18-fold or 20-fold.

Preferably, in the step 2), the totipotent nuclease and the water-soluble metal salt are added into the concentrated solution, the temperature is controlled to be 25-37 ℃, the nucleic acid in the concentrated solution is cracked, then ultracentrifugation is carried out under the condition that the centrifugal force is 100000-200000 g, and the precipitate is collected and dissolved by using a buffer solution, so as to obtain the crude extract.

Further preferably, the water-soluble metal salt used in step 2) includes, but is not limited to, water-soluble magnesium salt, water-soluble calcium salt, water-soluble manganese salt. It is further preferred that the water-soluble metal salt is one or more of magnesium chloride, calcium chloride or manganese chloride.

Specifically, the temperature is controlled by means of a water bath.

Specifically, the buffer used for dissolving the precipitate is PBS buffer, tris buffer or HEPES buffer, preferably PBS buffer.

Preferably, in the step 3), a mixed solution of iodixanol solution and sucrose buffer solution is used as a gradient medium, the mixed solution is firstly added into a centrifuge tube, the crude extract and iodixanol solution are premixed and added to the bottom of the gradient medium, finally, the centrifuge tube is filled with a sealing liquid, centrifugation is carried out under the condition that the centrifugal force is 100000 g-200000 g, and an exosome layer is collected.

According to some embodiments, a buffer solution is added to the centrifuge tube prior to adding the mixture of iodixanol solution and sucrose buffer.

According to some embodiments, the mixed solution of the iodixanol solution and the sucrose buffer solution is formed by mixing the iodixanol solution and the sucrose buffer solution according to a volume ratio of 1 to (4-6), wherein the mass concentration of the iodixanol solution is 55-65%, the pH value of the sucrose buffer solution is 7.0-7.5, the sucrose buffer solution comprises sucrose, tris HCl (Tris HCl) and Ethylene Diamine Tetraacetic Acid (EDTA), and the concentration of the sucrose is 200-300 mM.

According to some specific embodiments, in the premixing step, a iodixanol solution with the mass concentration of 55-65% and the crude extract are mixed according to the volume ratio of 1 to (1-3).

According to some embodiments, the blocking solution is PBS buffer, tris buffer or HEPES buffer or any combination thereof.

After centrifugation, the exosome layer is a white interfacial layer that migrates to between the occlusion liquid and the gradient media.

In the invention, by optimized pretreatment, fewer layers are required during density gradient centrifugation, and the operation is more convenient.

According to some embodiments, the sucrose buffer comprises 200mM to 300mM sucrose, 5mM to 15mM Tris HCl, 0.5mM to 1.5mM EDTA.

Preferably, the centrifugal force used in step 4) is between 20000g and 50000g. By adopting proper centrifugal force, residual protein in the exosome gradient layer can be effectively removed, and exosome precipitation can be avoided.

Preferably, the centrifugal force used in step 5) is 100000g to 200000g, and more preferably, it is centrifuged under conditions of a centrifugal force of 100000g to 150000 g.

In the invention, the collected exosome precipitate is stored after being resuspended by PBS buffer solution, tris buffer solution, HEPES buffer solution or sterile enzyme-free water, and can be directly used at the later stage.

In the invention, all the centrifugation in the preparation method is carried out at 0-8 ℃.

According to some embodiments, the preparation method is as follows:

(1) Concentrating the cell supernatant by 10-20 times by adopting a tangential flow filtration method with the molecular weight cutoff of 100-750 kDa to obtain concentrated solution;

(2) Adding totipotent nuclease and water-soluble metal salt into the concentrated solution, controlling the temperature to be 25-37 ℃ to crack nucleic acid in the concentrated solution, then carrying out ultracentrifugation under the condition that the centrifugal force is 100000-200000 g, collecting precipitates and dissolving the precipitates by using buffer solution to obtain the crude extract;

(3) Adding a sealing liquid into the centrifuge tube, adding a mixed solution of iodixanol solution and sucrose buffer solution into the centrifuge tube from the bottom of the sealing liquid by taking the mixed solution as a gradient medium, adding premixed crude extract and iodixanol solution into the bottom of the gradient medium, filling the centrifuge tube with the sealing liquid, centrifuging under the condition that the centrifugal force is 100000-200000 g, and collecting white color between a sealing liquid layer and the gradient medium layer after the centrifugation is finished to obtain an exosome layer;

(4) Carrying out low-speed centrifugation on the exosome layer under the condition that the centrifugal force is 20000 g-50000 g, and removing precipitates to obtain primary purified liquid;

(5) And (3) ultracentrifuging the primary purified liquid under the condition that the centrifugal force is 100000-200000 g, and obtaining a precipitate, namely the exosome.

Preferably, the water-soluble metal salt used in step 2) includes, but is not limited to, water-soluble magnesium salt, water-soluble calcium salt, water-soluble manganese salt.

More specifically, in the step (2), the water-soluble metal salt is one or more of magnesium chloride, calcium chloride or manganese chloride, and the concentration thereof in the system is 0.5 mM-2 mM, such as 0.5mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM, 2mM.

More specifically, in the step (2), the final concentration of the totipotent nuclease in the system is 15U/mL-25U/mL, such as 15U/mL, 18U/mL, 20U/mL, 22U/mL, 24U/mL, 25U/mL.

More specifically, in the step (2), the time for cracking the nucleic acid is 3-16 h. According to some embodiments, the temperature is 25 ℃ to 32 ℃ and the time for nucleic acid cleavage is 8 to 16 hours. According to other embodiments, the temperature is 32 ℃ to 37 ℃ and the time for nucleic acid cleavage is 3h to 8h.

More specifically, in the step (3), the density gradient centrifugation time is 15-20 h;

more specifically, in the step (4), the low-speed centrifugation is carried out for 20-40 min;

more specifically, in the step (5), the ultracentrifugation time is 2 to 5 hours.

More specifically, the pellet collected in step (6) is resuspended using PBS buffer, tris buffer, HEPES buffer, or sterile, enzyme-free water and then stored.

Preferably, before the concentration, the cell supernatant is subjected to two-stage microfiltration treatment, wherein the pore size of a filter membrane used for the first stage of microfiltration is 0.3-0.5 μm, and the pore size of a filter membrane used for the second stage of microfiltration is 0.2-0.25 μm.

Further preferably, a deep-layer filtration membrane can be used to remove part of the residual cells, cell debris and other impurities with larger particles before the two-stage microfiltration treatment, so that the pressure of the two-stage filtration can be reduced, and the blockage phenomenon in the two-stage microfiltration treatment can be prevented.

Preferably, the cell supernatant is composed of cells at a density greater than 9X 10 ⁶ The cell culture solution with cell viability of more than 90% is obtained by multi-stage centrifugation, wherein the multi-stage centrifugation comprises first-stage centrifugation and second-stage centrifugation, the centrifugal force of the first-stage centrifugation is less than 10000g, and the centrifugal force of the second-stage centrifugation is 10000 g-50000 g.

In the present invention, the cell is not particularly limited, and may be one or more of human embryonic kidney cell, mesenchymal stem cell, induced pluripotent stem cell, liver cell, immune cell, stromal cell, fibroblast, amniotic cell, erythrocyte, chondrocyte, endothelial cell, and epithelial cell.

In a preferred embodiment, the cell is a human embryonic kidney cell.

The invention also provides the exosome prepared by the preparation method of the exosome and application of the exosome in a drug delivery system.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:

the exosome obtained by the method has the advantages of complete membrane structure, no deformation, higher purity, particle concentration and exosome marker protein positive rate, better quality compared with the exosome prepared by the existing method, simple steps, high yield and low cost.

Drawings

FIG. 1 is a transmission electron micrograph of exosomes prepared in example 1;

FIG. 2 is a transmission electron micrograph of exosomes prepared in comparative example 1;

FIG. 3 is a particle size distribution diagram of the exosomes prepared in example 1;

FIG. 4 is a particle size distribution diagram of exosomes prepared in comparative example 1;

FIG. 5 is a graph showing the results of positive rate detection of the surface proteins CD9, CD63 and CD81 of the exosomes prepared in example 1;

FIG. 6 is a graph showing the results of positive rate detection of surface proteins CD9, CD63 and CD81 of exosomes prepared in comparative example 1;

FIG. 7 is a gel electrophoresis image of protein expression of exosomes prepared in example 1 and comparative example 1.

Detailed Description

Exosomes as drug in vivo delivery systems are one of the major points of targeted drug research at present. Although there are many methods for extracting exosomes from cell culture fluid, how to obtain exosomes with high quality and high yield has been a difficulty in the art. It is difficult to achieve both exosome quality and exosome yield in both small-scale laboratory preparations and large-scale industrial preparations. The reason for this analysis is that mainly because the cell culture solution contains proteins and nucleic acids remaining in the host cells and other vesicle structures produced by the cells, these substances need to be removed as much as possible during the extraction process of exosomes, which are vesicle structures, are deformed and broken during the preparation process, the natural activity is greatly reduced, and unreasonable preparation flows or inappropriate step parameters all affect the quality of exosomes. In the preparation process, experimenters cannot confirm the direct influence of each step of operation on the quality and yield of exosomes in the extract, and cannot adjust at any time, and after the preparation is finished, experimenters cannot confirm details of the preparation method to be adjusted and how to adjust according to the quality analysis of the exosomes, which is also the reason of difficult research on exosomes. Therefore, a production method capable of obtaining exosomes of high quality and high yield has not been developed. The present invention aims to solve the aforementioned problems.

The invention has unexpected positive effect on ensuring the purity and yield of the exosome prepared subsequently and the natural bioactivity by carrying out overall design on the process, including organic combination among different processes and step flow design. The invention further optimizes the preparation condition parameters, and the prepared exosome has the advantages of complete structure, no deformation, high purity, high natural activity and high yield. The method is beneficial to realizing the industrial preparation of the exosome, and provides a powerful support for the research of targeted drug delivery with the exosome as a carrier.

Definition of terms

The terms "exosome" and "EVs" are interchangeable, including vesicles derived from endosomal, lysosomal and/or endolysosomal pathways, involving native exosomes, genetically modified/engineered exosomes without any modification.

When describing the term "exosome", it is usually intended not to refer to one exosome, but to a population of multiple exosomes, the concentration of which is usually expressed in terms of the number of exosomes (number of particles) per unit volume, e.g., per milliliter.

The term "genetically modified/engineered exosome" refers to exosomes derived from genetically modified/engineered cells, which typically comprise recombinant or exogenous DNA or protein products thereof.

The term "culturing cells" refers to expanding the number or concentration of any cells that can produce EV under appropriate conditions, for example, in suspension or adherent culture or any other type of culture system.

The term "any cell that can produce EV" includes, but is not limited to, one or more of human embryonic kidney cells, mesenchymal stem cells, stromal cells, fibroblasts, amniotic cells, erythrocytes, chondrocytes, endothelial cells, epithelial cells, and the like.

The term "drug delivery system" refers to a formulation that delivers a pharmaceutically active ingredient to a desired body site and/or provides for the timely release of a therapeutic agent. Herein, a pharmaceutically active ingredient deliverable by a cell-derived exosome includes a small molecule drug or a biotherapeutic agent, which is not naturally present in the cell-derived exosome, said biotherapeutic agent being selected from a peptide, protein, polysaccharide or nucleic acid selected from a single-or double-stranded DNA, iRNA, siRNA, shRNA, mRNA, non-coding RNA (ncRNA), antisense RNA, LNA, morpholino oligonucleotide or analogues or conjugates thereof.

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 can be used as long as it does not contain serum.

The term "isolated and purified" means that the test sample containing the analyte of interest and the interfering substance are physically isolated or separated from each other.

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

The term "membrane" means a semi-permeable material that can be used to separate components in a feed fluid into a permeate that passes through the material and a retentate that is retained by the material.

The term "density gradient centrifugation" refers to the formation of a continuous or discontinuous density gradient in a centrifuge tube with a certain medium, mixing a test sample containing an analyte of interest with a bottom layer, and layering and separating the analyte of interest by the action of a centrifugal force field.

The terms "tangential flow concentration (TFF) system" and "cross-flow filtration" are used interchangeably and refer to the separation of suspended particles from a fluid mixture, including the separation of particles having defined characteristics (e.g., a desired particle size range) from a heterogeneous mixture of particles in a fluid mixture, and the concentration of the fluid.

The terms "totipotent nucleic acid" and "broad-spectrum nuclease" are interchangeable, and are a nonspecific endonuclease derived from Serratia marcescens (Serratia marcocen), which can completely digest nucleic acid (containing single-stranded, double-stranded, linear, circular, natural or denatured DNA and RNA) into 5' -monophosphate oligonucleotide of 2-5 bases in length by cutting any nucleotide in a strand under very wide conditions.

The technical solution of the present invention is further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not noted are generally those in routine experiments. The following examples and comparative examples are only examples of human embryonic kidney cells, and other cells such as fibroblasts, mesenchymal stem cells or stromal cells are also applicable to the above-mentioned production method, and parameters in some steps can be appropriately adjusted for different cells.

In the present invention, unless otherwise specified, the instruments, raw materials and reagents used are commercially available, and the experimental techniques and detection methods are conventional in the art. In the present invention, the centrifuge used is a low temperature centrifuge. The membrane package used for the depth filtration can be selected from commercially available products of Merck, pall, cytiva, sidoris, germany, 3M, and the like. The following examples and comparative examples used a membrane package of CSCCD1070PCP from huntington, corbert, which was effective in intercepting a portion of cells, cell debris and other large particles of impurities.

Example 1

The embodiment provides a preparation method of exosome, which comprises the following specific steps:

[1]37℃、8％CO ₂ culturing HEK293F cells with a serum-free medium under 120rpm horizontal shaking table (amplitude of 19 mm) until the viable cell density is higher than 9 x 10 ⁶ Per mL, the cell viability is more than 90%.

[2] 1000mL of the culture medium (cells and supernatant) was collected, 6000g was centrifuged at 4 ℃ for 20min, and supernatant A was collected.

[3] The supernatant A16000g was centrifuged at 4 ℃ for 30min, and the supernatant B was collected.

[4] Supernatant C was obtained by depth filtration.

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

[6] And (4) performing tangential flow filtration and concentration on the supernatant D by 15 times through a 300kDa hollow fiber column to obtain a concentrated solution.

[7]Adding MgCl into the concentrated solution ₂ The solution (final concentration is 1 mM) and the totipotent nuclease (final concentration is 20U/mL), and the water bath is carried out for 16h at 25 ℃ or 3h at 37 ℃.

[8] After completion of the water bath, the resulting concentrate was centrifuged at 133900g at 4 ℃ for 60min, and the precipitate was resuspended in 3.25mL of PBS, and then repeatedly flushed with a 1mL syringe until the precipitate was completely dissolved, to obtain 3.25mL of a crude extract.

[9] Sucrose (Sucrose) buffer (250 mM Sucrose, 10mM Tris HCl, 1mM EDTA, pH 7.4) was prepared, and a first mixed solution of Sucrose buffer and Iodixanol (Iodixanol) solution with a mass concentration of 60% (W/V) was prepared as a gradient medium, wherein 17.5% (V/V) (Iodixanol solution/(Iodixanol solution + Sucrose buffer)). Premixing the crude extract with 60% Iodixanol (Iodixanol) solution to prepare 45% (V/V) (Iodixanol solution/(Iodixanol solution + crude extract)) second mixed solution, and using PBS as an encapsulation solution. Adding 6mL of PBS to the bottom of a centrifuge tube with the volume of 39mL by using a sample injection needle, adding the first mixed solution and the second mixed solution from the bottommost part of the centrifuge tube in sequence by using the sample injection needle, and filling the tube with the PBS from the upper part of the liquid surface. Meanwhile, a blank control group without adding the crude extraction solution is arranged.

[10] And (3) centrifuging at the temperature of 150000g and 4 ℃ for 16h, and collecting a white interface layer which is transferred between the PBS and the first layering liquid, namely an exosome layer.

[11] Transferring the exosome layer into a 39mL new tube, filling the tube with PBS, centrifuging at 20000g and 4 ℃ for 30min, removing precipitate which is mainly residual protein, and collecting about 39mL of supernatant E, namely a primary purification solution.

[12] Transferring the supernatant E into a new tube, centrifuging for 3h at 135000g and 4 ℃, and obtaining the precipitate as the exosome.

[13] The pellet was resuspended in 200. Mu.L PBS and stored at 4 ℃.

Example 2

This example provides a method for preparing exosomes, which is essentially the same as example 1, except that a 100kDa hollow fiber column was used for concentrating the cell supernatant.

Example 3

This example provides a method for the preparation of exosomes, which is essentially the same as example 1, except that a 500kDa hollow fiber column was used for concentration of cell supernatants.

Comparative example 1

This comparative example provides a method for producing exosomes, which is substantially the same as example 1, except that steps [4] to [6] were not performed, unlike example 1.

Exosome identification experiment

Observing whether the structure of the extracted exosome membrane is complete and the size of the extracted exosome membrane accords with expectation through a Transmission Electron Microscope (TEM); identifying the extracted exosomes through different detection indexes, and analyzing the particle size and particle number concentration of the exosomes through a nanoparticle tracking technology (NTA), wherein the lower the particle number concentration is, the lower the exosome yield is; detecting the positive rates of surface proteins CD9, CD63 and CD81 of the exosome marked by the FITC antibody by a NanoFCM nano-flow detector, and determining the purity of the exosome; determining the expression condition of the marker protein of the exosome by using a Western blot experiment; and detecting the protein concentration before and after the tangential flow concentration by using the BCA method, and calculating the removal rate of impurity protein in the concentrated solution according to the protein concentration and the volume before and after the tangential flow concentration.

The conditions of the exosomes finally obtained in the examples and the comparative examples are observed by a Transmission Electron Microscope (TEM), and under the same magnification and the same visual field, the number of typical vesicles which are complete in membrane structure, have no deformation and are in a cup-tray structure in the exosomes in the examples is larger, and the background impurities are less. Comparing fig. 1 and fig. 2, it can be seen that the exosome membrane prepared in example 1 has a complete structure, no deformation, a size according with expectation, more typical vesicles and obviously less background impurities; the exosomes prepared in comparative example 1 had a significantly reduced number of typical vesicles, with significantly more background impurities. In all three examples, namely example 1 to example 3, the background impurities are few, but the typical vesicle number of the exosomes prepared in example 1 and example 2 is obviously more than that of example 3.

Particle size and particle number concentration of exosomes of example and comparative example were analyzed by nanoparticle tracking technique (NTA), and the results are shown in fig. 3 and 4: the particle size distribution of the exosome in example 1 is narrow, the peak particle size is 142.4nm, the average particle size is 173.1nm, the uniformity is good, the particle number concentration of the exosome is 1.01E +12 (particle number/mL), the particle number concentration of the exosome in comparative example 1 is 1.41E +11 (particle number/mL), and the exosome yield of comparative example 1 is obviously lower than that of example 1.

According to the positive rates of the exosome surface proteins CD9, CD63 and CD81 of the comparative examples and comparative examples of the NanoFCM, the positive rates of the exosome surface proteins CD9, CD63 and CD81 prepared in the examples were all higher than those of the comparative examples. According to fig. 5 and 6, it is shown that the exosomes of example 1 have positive rates for the surface proteins CD9, CD63 and CD81, respectively: 26.2%, 31.2% and 42.9%, the positivity rates of the surface proteins CD9, CD63 and CD81 of the exosomes of comparative example 1 were: 22.1%, 22.7% and 35.5%, the exosome purity of example 1 was significantly higher than comparative example 1.

The exosomes of the examples and the comparative examples are further determined and compared according to the expression of marker proteins of the exosomes of the experimental examples and the comparative examples by Western blot. Fig. 7 shows that the expression level of the marker protein CD9 of the exosome of example 1 is significantly higher than that of the exosome of comparative example 1, and the expression levels of the other marker proteins are not much different (the expression levels of the TSG101 and CD81 proteins of example 1 are slightly higher than that of comparative example 1), which indicates that the vesicle particles obtained in example 1 and comparative example 1 are indeed exosomes. The expression of CD63 was diffuse, whereas the expression of CD63 in comparative example 1 was more diffuse than in example 1, indicating a lower purity than in example 1. The number concentration of particles before (supernatant D) passing through the membrane and the number concentration of particles after (concentrated solution) passing through the membrane in the tangential flow concentration stage of example 1, example 2 and example 3 are respectively detected by using a nanoparticle tracking technology (NTA), and the vesicle recovery rate in the tangential flow concentration stage is calculated by combining the volume before (supernatant D) passing through the membrane and the volume after (concentrated solution) passing through the membrane, wherein the vesicle recovery rate (%) = (the number concentration of particles after passing through the membrane is multiplied by the volume after passing through the membrane) ÷ (the number concentration of particles before passing through the membrane is multiplied by the volume before passing through the membrane) multiplied by 100%; example 1 example 2 and example 3 the protein concentration before (supernatant D) and the protein concentration after (concentrate) the membrane were measured at the tangential flow concentration stage according to the BCA method, and the impurity protein removal rate (%) of = (protein concentration after membrane × volume after membrane) ÷ (protein concentration before membrane × volume before membrane) × 100% was calculated by combining the volume before (supernatant D) and the volume after (concentrate) the membrane. The results of vesicle recovery (%) and impurity protein removal (%) are shown in Table 1, respectively.

TABLE 1

Before and after tangential flow concentration	Vesicle recovery (%)	Removal rate of impurity protein (%)
			100kDa hollow fiber column	116.67	98.67
300kDa hollow fiber column	57.21	99.84
			500kDa hollow fiber column	57.66	100

Table 1 shows that the removal rates of proteins by the 500kDa hollow fiber column and the 300kDa hollow fiber column reach 100% and 99.84%, respectively, and it is known that the impurity proteins in the supernatant D can be effectively removed by the hollow fiber column with the molecular weight cut-off less than or equal to 500 kDa. The recovery efficiency of the 100kDa hollow fiber column and the 300kDa hollow fiber column for the vesicles was 78.26% and 85.71%, respectively. Along with the reduction of the molecular weight cut-off, the content of other impurity molecules in the concentrated solution after passing through the membrane is increased, pressure is brought to the subsequent extraction and purification steps, along with the increase of the molecular weight cut-off, the recovery rate of vesicles is reduced, and the final yield of exosomes is reduced, so that a hollow fiber column with the molecular weight cut-off of 100kDa-500kDa is preferably selected in the tangential flow concentration step by combining various factors.

The present invention is described in detail in order to make those skilled in the art understand the content and practice the present invention, and the present invention is not limited to the above embodiments, and all equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing exosomes, characterized in that: the preparation method comprises the following steps:

2) Performing denucleation acid treatment on the concentrated solution by using an omnipotent nuclease, and performing heavy suspension to obtain a crude extract;

2. The method of claim 1, wherein: in step 1), the tangential flow filtration method adopts a hollow fiber column with the molecular weight cutoff of 100kDa or 200kDa or 300kDa or 500 kDa.

3. The method of claim 1, wherein: in the step 2), adding totipotent nuclease and water-soluble magnesium salt into the concentrated solution, controlling the temperature to be 25-37 ℃ to crack nucleic acid in the concentrated solution, then performing ultracentrifugation under the condition that the centrifugal force is 100000-200000 g, collecting precipitate and dissolving by using buffer solution to obtain the crude extract.

4. The method of claim 1, wherein: step 3) is carried out as follows: the mixed solution of iodixanol solution and sucrose buffer solution is used as a gradient medium and is added into a centrifugal tube; premixing the crude extract with iodixanol solution, adding the premixed extract to the bottom of a gradient medium, and filling a centrifugal tube with a sealing liquid; centrifuging under the condition that the centrifugal force is 100000 g-200000 g, and collecting an exosome layer.

5. The method of claim 4, wherein: adding a sealing solution before adding the mixed solution of the iodixanol solution and the sucrose buffer solution into the centrifuge tube;

the mixed solution of the iodixanol solution and the sucrose buffer solution is prepared by mixing the iodixanol solution and the sucrose buffer solution according to the volume ratio of 1: (4-6), wherein the mass concentration of the iodixanol solution is 55-65%, the pH value of the sucrose buffer solution is 7.0-7.5, the sucrose buffer solution contains sucrose, tris (hydroxymethyl) aminomethane hydrochloride and ethylenediamine tetraacetic acid, and the concentration of the sucrose is 200-300 mM;

during premixing, mixing iodixanol solution with mass concentration of 55-65% and the crude extract according to the volume ratio of 1: 1-3;

the sealing solution is PBS buffer solution, tris buffer solution or HEPES buffer solution or any combination thereof.

6. The method of claim 1, wherein: the centrifugal force adopted in the step 4) is 20000 g-50000 g; and/or the centrifugal force adopted in the step 5) is 100000 g-200000 g.

7. The method of claim 1, wherein: the preparation method specifically comprises the following steps:

(1) Concentrating the cell supernatant by 10-20 times by adopting a tangential flow filtration method with the molecular weight cutoff of 100-750 kDa to obtain a concentrated solution;

8. The method of claim 7, wherein: in the step (2), the water-soluble metal salt is one or more of magnesium chloride, calcium chloride or manganese chloride, the concentration of the water-soluble metal salt in a system is 0.5 mM-2 mM, the concentration of the totipotent nuclease in the system is 15U/mL-25U/mL, and the time for carrying out the nucleic acid cleavage is 3 h-16 h;

and/or in the step (3), the centrifugation time of the density gradient centrifugation is 15-20 h;

and/or in the step (4), the centrifugation time of the low-speed centrifugation is 20 min-40 min;

and/or in the step (5), the centrifugal time of the ultracentrifugation is 2-5 h;

and/or, the precipitate collected in the step (5) is stored after being resuspended by using PBS buffer, tris buffer, HEPES buffer or sterile and enzyme-free water.

9. The production method according to claim 1 or 7, characterized in that: before the concentration, the cell supernatant is firstly subjected to two-stage microfiltration treatment, wherein the aperture of a filter membrane adopted by the first-stage microfiltration is 0.3-0.5 μm, and the aperture of a filter membrane adopted by the second-stage microfiltration is 0.2-0.25 μm.

10. The production method according to claim 1 or 7, characterized in that: the cell supernatant has a cell density of greater than 9 × 10 ⁶ The cell culture solution with cell viability of more than 90% is obtained by multi-stage centrifugation, wherein the multi-stage centrifugation comprises first-stage centrifugation and second-stage centrifugation, the centrifugal force of the first-stage centrifugation is less than 10000g, and the centrifugal force of the second-stage centrifugation is 10000 g-50000 g.

11. The method of claim 1, wherein: the cells are one or more of human embryonic kidney cells, mesenchymal stem cells, induced pluripotent stem cells, liver cells, immune cells, stromal cells, fibroblasts, amniotic cells, red blood cells, chondrocytes, endothelial cells and epithelial cells.

12. An exosome produced by the method for producing an exosome according to any one of claims 1 to 11.