Preparation method, product and application of cell-derived exosome subpopulation

Abstract

The invention discloses a preparation method, a product and application of a cell-derived exosome subset, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: filtering a cell culture solution to obtain a cell culture supernatant; concentrating by 5-30 times by using a tangential flow system of a 300-500kDa hollow fiber filter, and washing and filtering by 10-20 times to obtain a concentrated solution; carrying out Sephacryl S-400 HR gel filtration column chromatography, and collecting two pieces of ultraviolet absorption peak eluent with the wavelength of 280 nm, so as to obtain exosome subgroups S1 and S2; the sample protein concentration of the concentrated solution is 20 to 50 mg / mL. According to the method, accurate separation of exosome functional subgroups is achieved for the first time, the S1 subgroup is high in immunomodulatory activity, the S2 subgroup is excellent in cell regeneration promoting effect, the technology universality is high, and large-scale production can be achieved. The subgroup can be used for preparing medicines for regulating immunity or promoting cell regeneration, and a precise technical scheme is provided for treating related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method and products and applications of a cell-derived exosome subpopulation. BACKGROUND

[0002] Exosomes are lipid bilayer membrane structure small vesicles with a diameter of 30-150 nm secreted by cells, widely exist in cell culture medium, blood, urine and other biological fluids, and their interiors are wrapped with proteins, lipids, mRNA, miRNA and other biological active molecules. As an important medium for intercellular signal transmission, exosomes can target the biological active substances they carry to the recipient cells, regulate cell proliferation, differentiation, apoptosis and immune response and other physiological and pathological processes, and show great application potential in the fields of immune regulation, tissue repair, disease diagnosis and treatment.

[0003] In recent years, the research and transformation of exosomes in the field of biological medicine have rapidly advanced, and many breakthroughs have been made in the treatment of immune regulation and cell regeneration related diseases. For example, mesenchymal stem cell-derived exosomes have been confirmed to have significant immune regulation effect by inhibiting the expression of inflammatory factors (such as IL-6, TNFα) and promoting the secretion of anti-inflammatory factors (such as IL-10), providing a new treatment direction for rheumatoid arthritis, inflammatory bowel disease and other autoimmune diseases; at the same time, the active molecules carried by exosomes, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF), can promote the proliferation and repair of damaged tissues, and show excellent application prospects in skin wound healing, myocardial injury repair, osteoarthritis treatment and other scenarios.

[0004] However, the heterogeneity of exosomes is a core technical bottleneck restricting their clinical transformation. Exosomes secreted by the same kind of cells have significant differences in particle size distribution, molecular composition, biological activity and other aspects, forming a mixed system with multiple functions. Existing exosome separation technologies (such as differential centrifugation method, density gradient centrifugation method, ultrafiltration method, Capto Core 700 chromatography method, etc.) mainly focus on the overall purification of exosomes, and cannot realize the precise separation of functional subpopulations. Such mixed exosomes have many defects in clinical application: on the one hand, the effects of different functional subpopulations may be antagonistic, resulting in a significant reduction in treatment effect; on the other hand, the existence of non-targeted subpopulations may increase the risk of potential side effects, reducing the safety and targeting of treatment. For example, in the treatment of inflammation, part of the pro-inflammatory active subpopulation may weaken the treatment effect of the anti-inflammatory subpopulation; in tissue repair, the existence of non-proliferation regulation subpopulation will reduce the repair efficiency under unit dose and increase the clinical use cost.

[0005] In addition, the existing exosome separation technology also has many deficiencies: the differential centrifugation method is complicated, time-consuming, has low exosome recovery rate, and easily causes exosome aggregation and destroys the structural integrity of the exosome; the ultrafiltration method can realize concentration, but it is difficult to effectively remove small molecule impurities and protein pollution, and the product purity is insufficient; the Capto Core 700 chromatography method can improve the purity of exosomes, but can only separate exosomes and impurities, and cannot distinguish the functional subgroups inside the exosomes.

[0006] Therefore, developing a preparation method that can realize the accurate separation of exosome functional subgroups and take into account efficiency, purity and biological activity is of great significance for elucidating the mechanism of exosomes, improving the clinical treatment effect, and promoting the industrialization of exosome drugs. SUMMARY

[0007] Based on the defects of the above-mentioned prior art, the present application aims to provide a preparation method and product and application of cell-derived exosome subgroups, which realizes the efficient separation of exosome functional subgroups (S1 and S2) by optimizing the process route and key parameters, and provides a new technical solution and product for the precise treatment of immune regulation and cell regeneration related diseases.

[0008] Terms: In the present application, the term "exosome subgroup" refers to a subset of exosomes with specific functional targeting, which refers to two types of exosomes (S1 and S2) obtained by the preparation method of the present application, which have clear differences in molecular composition (proteins, nucleic acids, etc.) and biological activity, wherein the S1 subgroup is enriched in immune regulation related active molecules, and the S2 subgroup is rich in cell regeneration related functional factors, both of which maintain the integrity of the lipid bilayer membrane structure.

[0009] In the present application, the term "double-layer composite structure filter membrane" refers to a composite filter material used for clarification filtration of cell culture liquid, which is composed of an upper layer of 1.5 μm pore size (retaining cell clumps and large size impurities) and a lower layer of 0.8 μm pore size (retaining cell fragments and small impurities), and the material is selected from biocompatible materials such as polyether sulfone and polyvinylidene fluoride, which can realize stepwise impurity retention and ensure the purity of the raw material for subsequent exosome separation.

[0010] In the present application, the term "tangential flow filtration system" refers to a separation device used for exosome ultrafiltration and concentration, the core component of which is a hollow fiber filter (retaining molecular weight 300-500 kDa), which makes the sample flow parallel to the membrane surface by tangential flow, and realizes the separation of exosomes and small molecule impurities under the action of transmembrane pressure, and cooperates with 15-20 times volume washing to efficiently remove culture medium residues and protein pollution.

[0011] In the present application, the term "wash" refers to the displacement operation of impurities in the tangential flow filtration process, which means that while ultrafiltration is concentrated, buffer is continuously added and an equal volume of dialysate containing impurities is discharged. The wash ratio is defined as the ratio of the total volume of buffer added to the volume of the initial supernatant before concentration. The purpose is to remove impurities such as salts and small molecule metabolites in the sample, and to improve the purity of exosomes.

[0012] In the present application, the term "gel filtration chromatography" is a core step for separating exosome subgroups. Sephacryl S-400HR gel filtration column is used. Based on the difference in elution volume of exosome subgroups in gel particles, the eluate corresponding to two independent absorption peaks is collected by detecting 280 nm ultraviolet signal, and the functional subgroups are accurately separated.

[0013] In the present application, the term "immune regulation" refers to the core biological function of exosome subgroup S1, which means that the balance of immune response of the body is adjusted by inhibiting the expression of inflammatory factors (such as IL-6, TNFα, IL-1β) and regulating the polarization of immune cells. It is suitable for intervention in inflammation-related diseases or autoimmune diseases.

[0014] In the present application, the term "promoting cell regeneration" refers to the core biological function of exosome subgroup S2, which means that the proliferation of target cells such as fibroblasts and epithelial cells (such as increasing the positive rate of Ki67 and BrdU) is promoted, and the repair and regeneration of damaged tissues are accelerated. It is suitable for wound healing and tissue damage repair.

[0015] In the present application, the term "mammal" refers to a group of vertebrates with mammalian characteristics (viviparous, mammalian), and the cells derived therefrom have the ability to stably secrete functional exosomes. It includes but is not limited to human, mouse, rat, rabbit, monkey and other species. The present application preferably uses human mammalian cells (such as mesenchymal stem cells, neural stem cells, etc.), which secrete exosomes with high adaptability to physiological environment in terms of biological functions such as immune regulation and cell regeneration, and have better safety and effectiveness in clinical application.

[0016] In the present application, the term "CV" is the abbreviation of "Column Volume", which refers to the total volume of gel filler inside the gel filtration chromatography column (such as Sephacryl S-400HR chromatography column). It is the core measurement unit of buffer volume, sample volume and washing volume in chromatography operation.

[0017] The technical scheme of the present application comprises: On the one hand, the present application provides a preparation method of cell-derived exosome subgroups, which comprises the following steps: Step (1) filtering the cell culture solution to obtain cell culture supernatant; Step (2) the cell culture supernatant is concentrated 5-30 times by using a tangential flow filtration system of a 300-500 kDa hollow fiber filter, and is washed and filtered 10-20 times to obtain a concentrated solution; Step (3) the concentrated solution is subjected to chromatographic separation by a Sephacryl S-400 HR gel filtration column, and 50-500 mAU interval eluate and 500-200 mAU interval eluate are collected according to the 280 nm ultraviolet absorption peak to obtain a cell-derived exosome subpopulation S1 and a cell-derived exosome subpopulation S2; the protein concentration of the concentrated solution for loading is 20-50 mg / mL.

[0018] Specifically, the cell in step (1) is a mammalian cell.

[0019] Preferably, the mammalian cell is derived from any one or more of a mesenchymal stem cell, a neural stem cell, an embryonic stem cell, an induced pluripotent stem cell, a myocardial cell, an airway epithelial cell, and a fibroblast.

[0020] Further preferably, the mesenchymal stem cell is derived from any one or more of fat, bone marrow, umbilical cord, dental pulp, placenta, amniotic membrane, umbilical cord blood, synovial membrane, muscle tissue, and periodontal membrane.

[0021] Specifically, the culture medium used in the cell culture liquid comprises a serum-free culture medium, a serum substitute culture medium, and an exosome-free serum culture medium.

[0022] Preferably, the culture medium used in the cell culture liquid is a serum-free culture medium.

[0023] Specifically, the loading capacity of the filtration in step (1) is 150-300 L / m 2 , and the flow rate of the filtration is 100-300 LMH.

[0024] Further specifically, the loading capacity of the filtration in step (1) is 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, or 290-300 L / m 2 .

[0025] Preferably, the loading capacity of the filtration in step (1) is 160-170, 170-180, 180-190, or 190-200 L / m 2 .

[0026] Further preferably, the loading capacity of the filtration in step (1) is 183 L / m 2 .

[0027] Further particularly, the flow rate of the filtration in step (1) is 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290 or 290-300 LMH.

[0028] Preferably, the flow rate of the filtration in step (1) is 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190 or 190-200 LMH.

[0029] Further preferably, the flow rate of the filtration in step (1) is 150 LMH.

[0030] Particularly, the filtration in step (1) is filtration using a double-layer composite filter membrane, and the material of the double-layer composite filter membrane comprises any one or more of polyether sulfone, polyvinylidene fluoride, polytetrafluoroethylene, mixed cellulose ester, acetate fiber, glass fiber.

[0031] Preferably, the material of the double-layer composite filter membrane is hydrophilic polyether sulfone.

[0032] Preferably, the double-layer composite filter membrane is 1.5 μm in the upper layer + 0.8 μm in the lower layer.

[0033] Particularly, the material of the hollow fiber filter in step (2) comprises any one or more of polysulfone, polyether sulfone, polyvinylidene fluoride, sulfonated polysulfone, polyether ether ketone.

[0034] Preferably, the material of the hollow fiber filter in step (2) is polysulfone.

[0035] Particularly, the molecular weight cut-off of the hollow fiber filter in step (2) is 300-350, 350-400, 400-450 or 450-500 kDa.

[0036] Preferably, the molecular weight cut-off of the hollow fiber filter in step (2) is 300 kDa.

[0037] Specifically, the washing in step (2) is washing for 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19 or 19-20 times.

[0038] Further preferably, the washing in step (2) is washing for 20 times.

[0039] Specifically, the buffer used in the washing in step (2) comprises any one or more of PBS buffer, Tris-HCl buffer, HEPES buffer, physiological saline, PBST buffer, MES buffer.

[0040] Preferably, the buffer used in the washing in step (2) is PBS buffer.

[0041] Specifically, the transmembrane pressure of the tangential flow filtration system in step (2) is 0.2-2 bar.

[0042] Further specifically, the transmembrane pressure of the tangential flow filtration system in step (2) is 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1.0, 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9 or 1.9-2.0 bar.

[0043] Preferably, the transmembrane pressure of the tangential flow filtration system in step (2) is 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9 or 0.9-1.0 bar.

[0044] Preferably, the transmembrane pressure of the tangential flow filtration system in step (2) is 0.5 bar.

[0045] Specifically, the ultrafiltration concentration in step (2) is concentration of the cell culture supernatant by 5-30 times.

[0046] Further specifically, the ultrafiltration concentration in step (2) is concentration of the cell culture supernatant by 5-10, 10-15, 15-20, 20-25 or 25-30 times.

[0047] Preferably, the ultrafiltration concentration in step (2) is concentration of the cell culture supernatant by 10-15 or 15-20 times.

[0048] Further preferably, the ultrafiltration concentration in step (2) is to concentrate the cell culture supernatant by 20 times.

[0049] In particular, the chromatography separation process in step (3) comprises: equilibration, sample loading, elution and in situ cleaning.

[0050] Further particularly, the equilibration comprises: equilibrating the Sephacryl S-400 HR gel filtration column with a buffer. Preferably, in the equilibration step, the buffer comprises: any one or more of PBS buffer, Tris-HCl buffer, HEPES buffer, physiological saline, PBST buffer, MES buffer.

[0051] Further preferably, the buffer comprises PBS buffer.

[0052] Preferably, in the equilibration step, the volume of the buffer is 1-3 CV and the flow rate is 20-40 cm / h.

[0053] Further preferably, in the equilibration step, the volume of the buffer is 2 CV and the flow rate is 30 cm / h.

[0054] Further particularly, the sample loading of the concentrated solution comprises: sample loading of the concentrated solution with a protein concentration of 20-50 mg / mL, a sample loading volume of 2%-5% of the volume of the Sephacryl S-400 HR gel filtration column, and a flow rate of 5-15 cm / h. Preferably, in the sample loading of the concentrated solution: the concentrated solution comprises a protein concentration of 20-30, 30-40 or 40-50 mg / mL.

[0055] Further preferably, in the sample loading of the concentrated solution: the concentrated solution comprises a protein concentration of 35 mg / mL.

[0056] Preferably, in the sample loading of the concentrated solution: the sample loading volume of the concentrated solution comprises 2%-3%, 3%-4% or 4%-5% of the volume of the Sephacryl S-400 HR gel filtration column.

[0057] Further preferably, in the sample loading of the concentrated solution: the sample loading volume of the concentrated solution comprises 3% of the volume of the Sephacryl S-400 HR gel filtration column.

[0058] Preferably, during said loading of the concentrated solution: said loading flow rate of the concentrated solution comprises 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 cm / h.

[0059] Further preferably, during said loading of the concentrated solution: said loading flow rate of the concentrated solution comprises 15 cm / h.

[0060] Further specifically, said elution comprises: eluting the Sephacryl S-400 HR gel filtration column using a buffer at a flow rate of 5-15 cm / h, starting collection at the onset of the rise in the UV signal at 280 nm, and stopping collection when close to the baseline level, collecting the 50-500 mAU interval eluate and the 500-200 mAU interval eluate to obtain a cell-derived exosome subpopulation S1 and a cell-derived exosome subpopulation S2.

[0061] Preferably, during said elution: said flow rate comprises 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 cm / h.

[0062] Further preferably, during said elution: said flow rate comprises 15 cm / h.

[0063] Preferably, during said elution: said buffer comprises any one or more of: a PBS buffer, a Tris-HCl buffer, a HEPES buffer, a physiological saline, a PBST buffer, a MES buffer.

[0064] Further preferably, during said elution: said buffer comprises a PBS buffer.

[0065] Further specifically, said in-situ cleaning comprises flushing with a NaOH solution.

[0066] Preferably, said NaOH solution is a 0.25-1 M NaOH solution.

[0067] Further preferably, said NaOH solution is a 0.25-0.5 or 0.5-1 M NaOH solution.

[0068] Still further preferably, said NaOH solution is a 0.5 M NaOH solution.

[0069] Preferably, said NaOH solution has a volume of 2-5 CV.

[0070] Further preferably, said NaOH solution has a volume of 2-3, 3-4, or 4-5 CV.

[0071] Further preferably, the volume of the NaOH solution is 3 CV.

[0072] Preferably, the flow rate of the NaOH solution comprises 10-30 cm / h.

[0073] Further preferably, the flow rate of the NaOH solution comprises 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24, 24-25, 25-26, 26-27, 27-28, 28-29 or 29-30 cm / h.

[0074] Further preferably, the flow rate of the NaOH solution is 26 cm / h.

[0075] In particular, the concentration of the cell-derived exosome subpopulation in step (3) is 1 x 10 7 - 1 x 10 11 particles / mL.

[0076] Further in particular, the concentration of the cell-derived exosome subpopulation in step (3) is 1 x 10 7 - 1 x 10 8 , 1 x 10 8 - 1 x 10 9 , 1 x 10 9 - 1 x 10 10 or 1 x 10 10 - 1 x 10 11 particles / mL.

[0077] Preferably, the concentration of the cell-derived exosome subpopulation in step (3) is 1 x 10 10 particles / mL.

[0078] In another aspect, the present application provides a cell-derived exosome subpopulation obtained by the preparation method of any one of the above.

[0079] In another aspect, the present application provides use of the cell-derived exosome subpopulation of any one of the above in preparation of an immunomodulatory drug or a drug for promoting cell regeneration.

[0080] In another aspect, the present application provides an immunomodulatory or cell-regeneration-promoting drug, wherein the drug comprises the cell-derived exosome subpopulation of any one of the above.

[0081] In particular, the drug further comprises a pharmaceutically acceptable excipient.

[0082] Preferably, the excipients include, but are not limited to, any one or more of fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, bacteriostatic agents, isotonicity adjusting agents, suspending agents, solubilizers, co-solvents, preservatives, flavoring agents.

[0083] In another aspect, the present application provides a method for immunomodulation or promoting cell regeneration, the method comprising using the cell-derived exosome subpopulation or the medicament as described in any one of the above.

[0084] In particular, the method comprises administering an effective amount of the cell-derived exosome subpopulation or the medicament.

[0085] Preferably, the effective amount comprises 1 x 10 7 -1 x 10 8 , 1 x 10 8 -1 x 10 9 , 1 x 10 9 -1 x 10 10 , 1 x 10 10 -1 x 10 11 particles / mL of the cell-derived exosome subpopulation.

[0086] In another aspect, the present application provides the use of the cell-derived exosome subpopulation in immunomodulation, the method comprising the following steps: a. adding LPS to stimulate mouse macrophages M0 to polarize to M1 to produce an inflammatory response; b. adding LPS while adding the cell-derived exosome subpopulation; c. evaluating the expression of inflammatory factors.

[0087] In particular, the inflammatory factors include, but are not limited to, IL-6, TNFα, IL-1β.

[0088] In particular, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 7 -1 x 10 11 particles / mL.

[0089] Further in particular, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 7 -1 x 10 8 , 1 x 10 8 -1 x 10 9 , 1 x 10 9 -1 x 10 10 , 1 x 10 10 -1 x 10 11 particles / mL.

[0090] Preferably, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 10 particles / mL.

[0091] In another aspect, the present application provides the use of the above-mentioned cell-derived exosome subpopulation in cell regeneration, and the cell regeneration application method comprises the following steps: a. After the fibroblasts are cultured to a certain degree of confluence, the complete culture medium is removed and replaced with a serum-free basic culture medium; b. Adding a cell-derived exosome subpopulation to the basic culture medium; c. Evaluating cell proliferation.

[0092] Specifically, the cell proliferation evaluation indicators include but are not limited to CCK8, Ki67, and BrdU.

[0093] Preferably, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 7 -1 x 10 11 particles / mL.

[0094] Further specifically, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 7 -1 x 10 8 , 1 x 10 8 -1 x 10 9 , 1 x 10 9 -1 x 10 10 , or 1 x 10 10 -1 x 10 11 particles / mL.

[0095] Preferably, the concentration of the cell-derived exosome subpopulation in step b is 1 x 10 10 particles / mL.

[0096] The beneficial effects of the present application are: 1. In the prior art, exosome separation is mostly mixed system purification (such as differential centrifugation and CaptoCore700 chromatography), which cannot distinguish functional specific subpopulations, resulting in limited efficacy or even antagonism. The present application realizes the functional subpopulation separation (S1 and S2) of exosomes for the first time: the S1 subpopulation is enriched with immune-regulatory active molecules, and the IL-6 inhibition rate is increased by more than 40% compared with traditional mixed exosomes; the S2 subpopulation is rich in cell regeneration-related factors, and the fibroblast proliferation rate is increased by more than 35% compared with traditional methods, which fundamentally solves the clinical translation bottleneck caused by exosome heterogeneity.

[0097] 2、The preparation method of the application is suitable for mesenchymal stem cells (fat, bone marrow, umbilical cord and the like), neural stem cells, myocardial cells and the like of various mammalian cells, and it is verified through experiments that the exosomes of different cell sources can all realize subpopulation separation through the process, and the immunomodulatory activity of the S1 subpopulation and the proliferation promoting activity of the S2 subpopulation are consistent, and the universality is significantly better than that of the existing targeted separation technology. Meanwhile, the tangential flow filtration system in the process supports large-scale continuous operation, and the gel filtration chromatography can realize mass production through column volume amplification.

[0098] 3、The separated exosome subpopulation of the application has strong functional specificity: the S1 subpopulation can significantly inhibit the expression of LPS-induced macrophage inflammatory factors (IL-6, TNF alpha and IL-1 beta), and is suitable for the treatment of autoimmune diseases and inflammation-related diseases; the S2 subpopulation can efficiently promote the proliferation of fibroblasts, epithelial cells and the like, and can be used in wound repair, tissue regeneration and the like. Compared with traditional mixed exosomes, the treatment target of the subpopulation product is stronger, and the side effect is lower, which provides a new precise treatment strategy for the fields of immune regulation and cell regeneration, and has a broad clinical conversion prospect. BRIEF DESCRIPTION OF DRAWINGS

[0099] Figure 1 It is a tangential flow filtration system condition optimization effect diagram; A in the figure is an effect diagram of the molecular weight cut-off of different hollow fiber filters; B is an effect diagram of different washing filtration multiples.

[0100] Figure 2 It is a chromatography column purification spectrum.

[0101] Figure 3 It is a chromatography system condition optimization effect diagram; A in the figure is the IL-6 inhibition rate of component F1-component F10; B is the proliferation promoting rate of component F1-component F10; C is the IL-6 inhibition rate of different chromatography purification methods; D is the proliferation promoting rate of different chromatography purification methods; ns in the figure represents no significant difference between groups; **** represents significant difference p<0.0001.

[0102] Figure 4 It is an effect diagram of different elution flow rates on subpopulation separation.

[0103] Figure 5 It is an effect diagram of the preparation method of the cell source exosome subpopulation in different cells; A in the figure is the IL-6 inhibition rate; B is the proliferation promoting rate.

[0104] Figure 6 It is an exosome purity comparison diagram of Comparative Example 1 and Example 3; * in the figure represents significant difference p<0.05.

[0105] Figure 7Figure 1 is a comparison chart of immunomodulatory experiments and cell proliferation experiments of Comparative Example 2 and Example 3; A in the figure is IL-6 inhibition rate; B is proliferation rate; ns represents no significant difference between groups; **** represents significant difference p<0.0001. DETAILED DESCRIPTION

[0106] The following non-limiting examples can make those of ordinary skill in the art more fully understand the present application, but in no way limit the present application. The following content is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application according to the disclosed content, which should also belong to the scope of the present application claimed.

[0107] The present application is further described below in the form of specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the present application are obtained through conventional commercial channels unless otherwise specified.

[0108] Example 1 Preparation of cell culture medium 1. Preparation of human umbilical cord mesenchymal stem cell culture medium: Human umbilical cord mesenchymal stem cells were purchased from Plnomer (CAT: CP-CL11) and seeded at a density of 10,000 cells / cm 2 The cells were cultured in complete medium (CAT: CM-CL11) and when the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium for 48 h. The cell culture medium was then collected for exosome purification.

[0109] 2. Preparation of human bone marrow mesenchymal stem cell culture medium: Human bone marrow mesenchymal stem cells were purchased from Plnomer (CAT: CP-H166) and seeded at a density of 10,000 cells / cm 2 The cells were cultured in complete medium (CAT: CM-H166) and when the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium for 48 h. The cell culture medium was then collected for exosome purification.

[0110] 3. Preparation of human dental pulp mesenchymal stem cell culture medium: Human dental pulp mesenchymal stem cells were purchased from Saiye Biotechnology (CAT: HUXDP-01001) and seeded at a density of 10,000 cells / cm 2 The cells were cultured in complete medium (CAT: HUXDP-90011) and when the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium for 48 h. The cell culture medium was then collected for exosome purification.

[0111] 4. Preparation of human adipose tissue mesenchymal stem cell culture medium: Human adipose-derived mesenchymal stem cells were purchased from Plnomics (CAT: CP-H202) at 10,000 cells / cm 2 Density inoculation culture in complete medium (CAT: CM-H202), after the cells grow to 80% confluence, remove the complete medium, replace with serum-free basal medium continue to culture for 48h after collection of cell culture fluid for exosome purification.

[0112] 5, human neural stem cell culture fluid preparation: Human neural stem cells were purchased from Shengnianbio (CAT: SNP-H235) at 20,000 cells / cm 2 Density inoculation culture in complete medium (CAT: SNPM-H235), after the cells grow to 80% confluence, remove the complete medium, replace with serum-free basal medium continue to culture for 48h after collection of cell culture fluid for exosome purification.

[0113] 6, human myocardial cell culture fluid preparation: Human myocardial cells were purchased from Plnomics (CAT: CP-H076) at 20,000 cells / cm 2 Density inoculation culture in complete medium (CAT: CM-H076), after the cells grow to 80% confluence, remove the complete medium, replace with serum-free basal medium continue to culture for 48h after collection of cell culture fluid for exosome purification.

[0114] 7, human small airway epithelial cell culture fluid preparation: Human small airway epithelial cells were purchased from Plnomics (CAT: CP-H010) at 10,000 cells / cm 2 Density inoculation culture in complete medium (CAT: CM-H010), after the cells grow to 80% confluence, remove the complete medium, replace with serum-free basal medium continue to culture for 48h after collection of cell culture fluid for exosome purification.

[0115] 8, human embryonic stem cell culture fluid preparation: Human embryonic stem cells were purchased from Plnomics (CAT: CL-0890) at 100,000 cells / cm 2 Density inoculation culture in complete medium (CAT: CM-0890), after the cells grow to 80% confluence, replace with fresh medium continue to culture for 48h after collection of cell culture fluid for exosome purification.

[0116] 9, human induced pluripotent stem cell culture fluid preparation: Human induced pluripotent stem cells were purchased from Plnomics (CAT: CL-1059) at 100,000 cells / cm 2Density seeding culture in complete medium (CAT: CM-1059), after the cells grow to 80% confluence, replace fresh medium to continue culture for 48h, then collect the cell culture fluid for exosome purification.

[0117] 10. Preparation of human dermal fibroblast cell culture fluid: Human dermal fibroblasts were purchased from Promocell (CAT: CP-H103), seeded at 10000 cells / cm 2 Density seeding culture in complete medium (CAT: CM-H103), after the cells grow to 80% confluence, remove the complete medium, replace with serum-free basal medium to continue culture for 48h, then collect the cell culture fluid for exosome purification.

[0118] Example 1 Optimization of tangential flow filtration system conditions 1. Preparation of cell culture supernatant The cell culture supernatant was obtained by clarifying the human umbilical cord mesenchymal stem cell culture fluid collected in Basic Example 1. The cell culture fluid was filtered with a double-layer composite structure (upper layer 1.5 μm + lower layer 0.8 μm) precision Sartopore clarifying filter membrane package (part number U33CPPLELA1P) with a load of 183 L / m 2 and a filtration flow rate of 150 LMH.

[0119] 2. Optimization of tangential flow filtration system conditions 2.1 Hollow fiber filter condition optimization The tangential flow concentration system used 100 kDa, 300 kDa, 500 kDa, or 750 kDa hollow fiber filters (polysulfone material) to concentrate the cell culture supernatant 20-fold in volume, and PBS buffer (0.0067M PO4, pH 7.0-7.2) was used to wash the filter 20-fold, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH, to obtain concentrated solutions, respectively.

[0120] 2.2 Wash multiple optimization The tangential flow concentration system used 300 kDa hollow fiber filters (polysulfone material) to concentrate the cell culture supernatant 20-fold in volume, and PBS buffer (0.0067M PO4, pH 7.0-7.2) was used to wash the filter 5, 10, 15, 20, or 25 times, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH, to obtain concentrated solutions, respectively.

[0121] 3. Differential centrifugation system control The collected human umbilical cord mesenchymal stem cell culture solution was centrifuged at 300xg for 10 minutes, and the supernatant was taken; centrifuged at 2000xg for 10 minutes, and the supernatant was taken; centrifuged at 10000xg for 30 minutes, and the supernatant was taken; centrifuged at 100000xg at 4°C for 90 minutes, and the supernatant was removed. The remaining precipitate was resuspended in PBS and centrifuged again at 100000xg for 90 minutes. Finally, 1 mL of PBS was added to resuspend the precipitate to obtain exosomes.

[0122] 4. Determination of exosome recovery rate and exosome purity Different concentrated solutions obtained by tangential flow filtration systems under different conditions, and cell suspensions obtained by differential centrifugation system control, were analyzed by nanoparticle tracking technology (NTA) for particle size distribution in exosome subgroups and exosome concentration (particales / μL), and by BCA for protein concentration (μg / μL) in exosome subgroups, to calculate the purity of exosome subgroups.

[0123] Exosome recovery rate (%) = total particle number of finally harvested exosomes / total particle number of exosomes in human umbilical cord mesenchymal stem cell culture solution x 100%.

[0124] Exosome purity (particales / μg) = exosome concentration (particales / μL) / protein content (μg / μL).

[0125] 5. Determination results In the tangential flow ultrafiltration concentration process, this embodiment considered hollow fiber filters with different interception molecular weights (100kDa-750kDa) and different washing filtration multiples. The differential centrifugation (UC) method was used as a control for the classic purification method of exosomes, and the recovery rate of exosome particles and the purity were used as evaluation indexes. The results are as follows Figure 1 .

[0126] For exosome recovery rate, the differential centrifugation method had the lowest exosome recovery rate, less than 20%; the 100kDa and 300kDa filters had the highest recovery rate, both above 95%; followed by 500kDa and 750kDa. For exosome purity, 100kDa was the lowest, and the purity of exosomes from 300kDa to 750kDa was all higher than 3x10 8 particales / μg, close to the exosome purity of the differential centrifugation method. Therefore, the hollow fiber filters with interception molecular weights of 300kDa and 500kDa were selected, and the hollow fiber filter with an interception molecular weight of 300kDa was preferred.

[0127] In a tangential flow filtration system, the washing process also affects the purity of the product. In this embodiment, the effects of 5-25 times washing on the recovery rate and purity of exosomes were considered. The recovery rate was normal, and the purity increased with the increase of the washing ratio, reaching the highest value at 20 times. Therefore, 15 or 20 times is more appropriate, with 20 times being preferred.

[0128] Example 2: Optimization of Chromatography System Conditions 1. Preparation of cell culture supernatant The human umbilical cord mesenchymal stem cell culture medium collected in Basic Example 1 was clarified and filtered to obtain the cell culture supernatant. Clarification and filtration involved filtering the cell culture medium using a Quadrate clarification filter membrane pack (catalog number U33CPPLELA1P) with a double-layer composite structure (upper layer 1.5μm + lower layer 0.8μm) and a flow rate of 183 L / m³. 2 The filtration flow rate is 150 LMH.

[0129] 2. Concentration of cell culture supernatant The tangential flow concentration system uses a 300 kDa hollow fiber filter (polysulfone material) to concentrate the cell culture supernatant by 20 times, wash it 20 times with PBS buffer (0.0067 M PO4, pH 7.0-7.2), transmembrane pressure (TMP): 0.5 bar, flow rate 385 LMH to obtain the concentrate.

[0130] 3. Optimization of chromatography system conditions 3.1 Purification using Sephacryl S-400 HR chromatography column Equilibration: Equilibrate the Sephacryl S-400 HR gel filter column (purchased from Cytiva, catalog number 28935605) with PBS buffer (0.0067M PO4, pH 7.0-7.2), 2CV, flow rate 30cm / h.

[0131] Sample loading: The concentrated protein concentration is 35 mg / mL, the loading volume is 3% of the column volume, and the flow rate is 15 cm / h.

[0132] Elution: Wash with PBS buffer (0.0067M PO4, pH 7.0-7.2) at a flow rate of 15 cm / h. Start collecting when the UV signal rises at 280 nm and stop collecting when it approaches the baseline level. Collect the elution buffer in the UV absorption peak range of 50-500 mAU as S1 and the elution buffer in the UV absorption peak range of 500-200 mAU as S2.

[0133] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution at a flow rate of 10cm / h.

[0134] 3.2 Capto Core 700 chromatography column purification Equilibration: Equilibrate Capto Core 700 gel filtration column (purchased from Cytiva, part number 17548115) with PBS buffer (0.0067M PO4, pH 7.0-7.2), 10 CV, flow rate 155 cm / h.

[0135] Loading: Load the concentrated solution with protein concentration 35 mg / mL, loading volume 5 CV, start collection when the 280 nm UV signal rises during the loading process, stop collection when it approaches the baseline level, and obtain the exosome product.

[0136] Regeneration: Rinse with PBS buffer, 3 CV, flow rate 155 cm / h.

[0137] Cleaning in place (CIP): Rinse with 0.5M NaOH solution, 3 CV, flow rate 26 cm / h.

[0138] 4. Effect verification 4.1 Immune regulation experiment (1) Day 1: Mouse macrophage RAW264.7 cells were seeded in a 6-well plate at a density of 2×10 5 cells / well and cultured overnight to adhere to the wall; (2) Day 2: Remove the old culture medium and add fresh culture medium containing 100 ng / mL lipopolysaccharide (LPS) and continue to culture for 24 h, and add 1×10 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2) to the experimental group, and add an equal amount of PBS to the blank group; (3) Day 3: Collect the cell supernatant, centrifuge at 2000×g for 5 minutes, and then take the supernatant for enzyme-linked immunosorbent assay to detect IL-6 expression.

[0139] 4.2 Cell proliferation experiment (1) Day 1: Human dermal fibroblasts HSF were seeded in a 96-well cell culture plate at a density of 4×10 4 / mL and cultured overnight to adhere to the wall; (2) Day 2: Remove the old culture medium and add fresh culture medium and continue to culture for 72 h, and add 1×10 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2) to the experimental group, and add an equal amount of PBS to the blank group; (3) Day 5: Remove the old culture medium, add 100 μL DMEM / F12 basic culture medium and 10 μL CCK8 solution to each well of the cell culture plate, and continue to incubate for 30 minutes, and measure the absorbance value (A) at 450 nm, and the proliferation rate%=OD experimental group / OD blank group×100%.

[0140] 5. Determination results In the chromatography purification step, Capto Core 700 chromatography column is increasingly used in the field of exosomes for purification, but this chromatography method cannot achieve the purpose of subpopulation separation, therefore, Sephacryl S-400 HR chromatography column is used for purification in the present application, the atlas is as shown in Figure 2 , two absorption peaks appear in the elution process, the components in the elution process are collected, and the activity is detected by immune regulation and fibroblast proliferation experiment, the results are as shown in Figure 3 A-B, the immune regulation activity of components 4-6 is strong, and components 7-10 are biased to promote proliferation activity, components 4-6 are combined and named as S1, and components 7-10 are combined and named as S2, and the activity is compared with the concentrated liquid (TFF) and Capto Core 700 purified sample (TFF+core700), the results are as shown in Figure 2 C-D, the immune regulation activity, S1 subpopulation is significantly better than TFF, TFF+core700 and S2 subpopulation, and S2 subpopulation is significantly better than other samples in promoting proliferation activity.

[0141] Example 3 Preparation method of cell-derived exosome subpopulation of the present application 1. Preparation of cell culture supernatant The human umbilical cord mesenchymal stem cell culture solution collected in the basic example 1 is clarified and filtered to obtain the cell culture supernatant. The cell culture solution is filtered with a double-layer composite structure (upper layer 1.5 μm + lower layer 0.8 μm) precision Sartopore clarifying filter membrane bag (model number U33CPPLELA1P) with a load of 183 L / m 2 , and a filtration flow rate of 150 LMH.

[0142] 2. Concentration of cell culture supernatant The tangential flow concentration system uses a 300 kDa hollow fiber filter (polysulfone material) to concentrate the cell culture supernatant by 20 times, and PBS buffer (0.0067M PO4, pH 7.0-7.2) is washed and filtered by 20 times, the transmembrane pressure (TMP) is 0.5 bar, and the flow rate is 385 LMH, to obtain the concentrated liquid.

[0143] 3. Purification of Sephacryl S-400 HR chromatography column Equilibrium: Sephacryl S-400 HR gel filtration column (purchased from Cytiva, model number 28935605) is equilibrated with PBS buffer (0.0067M PO4, pH 7.0-7.2) at 2 CV and a flow rate of 30 cm / h.

[0144] Loading: The protein concentration of the concentrated solution was 35 mg / mL, and the loading volume was 3% of the column volume at a flow rate of 15 cm / h.

[0145] Elution: The column was washed with PBS buffer (0.0067 M PO4, pH 7.0-7.2) at a flow rate of 15 cm / h, and the collection started when the UV signal at 280 nm began to rise, and stopped when it approached the baseline level. The eluate in the peak range of 50-500 mAU UV absorption was collected as S1, and the eluate in the peak range of 500-200 mAU UV absorption was collected as S2.

[0146] Cleaning in place (CIP): The column was washed with 0.5 M NaOH solution at a flow rate of 10 cm / h for 2 CV.

[0147] Example 4 Verification of the preparation method of the cell-derived exosome subpopulation 1. Scale-up verification (1) Preparation of cell culture supernatant Refer to Example 3 (2) Concentration of cell culture supernatant Refer to Example 3 (3) Purification by Sephacryl S-400 HR column Equilibration: The Sephacryl S-400 HR gel filtration column (purchased from Cytiva, item number 28935605) was equilibrated with PBS buffer (0.0067 M PO4, pH 7.0-7.2) at a flow rate of 30 cm / h for 2 CV.

[0148] Loading: The protein concentration of the concentrated solution was 35 mg / mL, and the loading volume was 3% of the column volume at a flow rate of 15 cm / h.

[0149] Elution: The column was washed with PBS buffer (0.0067 M PO4, pH 7.0-7.2) at a flow rate of 15 cm / h, and the collection started when the UV signal at 280 nm began to rise, and stopped when it approached the baseline level. The eluate in the peak range of 50-500 mAU UV absorption was collected as S1, and the eluate in the peak range of 500-200 mAU UV absorption was collected as S2.

[0150] Cleaning in place (CIP): The column was washed with 0.5 M NaOH solution at a flow rate of 10 cm / h for 2 CV.

[0151] (4) Effect verification The effects of different elution flow rates on the separation of subpopulations were verified by the immunomodulatory experiment and cell proliferation experiment described in Example 2. The results are shown in Figure 4 There was no significant change from 5 cm / h to 15 cm / h, indicating that the surface exosome subpopulation separation was stable and had the possibility of scale-up.

[0152] 2. Universal verification (1) Preparation of cell culture supernatant The cell culture supernatant was obtained by clarifying the cell culture supernatant obtained from different tissue-derived mesenchymal stem cells and different cell types. The clarifying filtration was performed by filtering the cell culture supernatant with a double-layer composite structure (upper layer 1.5 μm + lower layer 0.8 μm) precision Sartopore clarifying filter membrane package (product number U33CPPLELA1P) with a load of 183 L / m 2 and a filtration flow rate of 150 LMH.

[0153] (2) Concentration of cell culture supernatant Reference Example 3 (3) Purification by Sephacryl S-400 HR column Reference Example 3 (4) Effect verification In order to verify the universality of the subpopulation separation method, the exosomes prepared by the cell-derived exosome subpopulation preparation method of the present application were used to verify the immune regulation experiment and cell proliferation experiment described in Reference Example 2 for a variety of different tissue-derived mesenchymal stem cells and different cell types.

[0154] The results are shown in Figure 5 Although the activities of exosome subpopulations from different cells differ, the subpopulation separation method of the present application can successfully separate the subpopulations, and the activities of the S1 subpopulation are consistent with those of the umbilical cord-derived mesenchymal stem cell-derived exosomes. The S2 subpopulation has significant cell proliferation activity, indicating that the subpopulation separation method has good universality.

[0155] Comparative Example 1 The difference between Comparative Example 1 and Example 3 is only in the step "2, concentration of cell culture supernatant". The specific steps are as follows: The tangential flow concentration system uses a 100 kDa hollow fiber filter to concentrate the cell culture supernatant 20 times, and the filter is washed with PBS buffer 25 times. The transmembrane pressure (TMP) is 0.5 bar, the flow rate is 385 LMH, and the concentrated solution is obtained.

[0156] Experimental Example 1: Verification of the purity of the exosomes of Comparative Example 1 The concentrated solution prepared in Comparative Example 1 was analyzed by nanoparticle tracking technology (NTA) to analyze the particle size distribution and exosome concentration (particales / μL) in the exosome subpopulation, and BCA was used to analyze the protein concentration (μg / μL) in the exosome subpopulation, and the purity of the exosome subpopulation was calculated.

[0157] Exosome recovery rate (%) = final harvest exosome total particle number / total particle number of exosomes in human umbilical cord mesenchymal stem cell culture solution x 100%.

[0158] Exosome purity (particales / μg) = exosome concentration (particales / μL) / protein content (μg / μL).

[0159] Results as shown in Table 1, in the tangential flow concentration link, the average purity of exosomes in the concentrated solution obtained by Comparative Example 1 was 5.47E+07 particles / μg, while the average purity of exosomes in the concentrated solution obtained by Example 3 was 3.53E+08 particles / μg, which was one order of magnitude different, and the higher the purity, the better the quality. Figure 6

[0160] Comparative Example 2 The difference between Comparative Example 2 and Example 3 is only in the step of "3, Sephacryl S-400 HR column purification". The specific steps are as follows: Equilibrium: equilibrate Sephacryl S-400 HR gel filtration column (purchased from Cytiva, item number 28935605) with PBS buffer (0.0067M PO4, pH 7.0-7.2), 2CV, flow rate 30cm / h.

[0161] Loading: the protein concentration of the concentrated solution is 55mg / mL, the loading volume is 3% of the column volume, and the flow rate is 15cm / h.

[0162] Elution: use PBS buffer (0.0067M PO4, pH 7.0-7.2) to flush, flow rate 15cm / h, start collection when the ultraviolet signal rises at 280nm, stop collection when it approaches the baseline level, collect the eluate in the peak range of 50-500mAU ultraviolet absorption as S1, and collect the eluate in the peak range of 500-200mAU ultraviolet absorption as S2.

[0163] CIP: use 0.5M NaOH solution to flush 2CV, flow rate 10cm / h.

[0164] Experimental Example 2 Immunomodulatory experiment and cell proliferation experiment verification of Comparative Example 2 1. Immunomodulatory experiment (1) Day 1: Place mouse macrophage RAW264.7 at 2x10 5 cells / well in a 6-well plate and culture overnight to adhere; (2) Day 2: Remove the old culture medium and add fresh culture medium containing 100ng / mL lipopolysaccharide (LPS) and continue to culture for 24h, and add 1x10 10 ​1x10 (3) Day 3: Collect the cell supernatant and centrifuge at 2000xg for 5 minutes, then take the supernatant for enzyme-linked immunosorbent assay to detect IL-6 expression.

[0165] 2. Cell proliferation experiment (1) Day 1: Human dermal fibroblasts HSF were seeded at a density of 4x10 4 / mL cell density into a 96-well cell culture plate overnight to adhere; (2) Day 2: Remove the old culture medium, add fresh culture medium and continue to culture for 72h, the experimental group adds 1x10 10 / mL exosomes (S1, S2 or exosome product) or concentrate (prepared in step 2), and the blank group adds an equal amount of PBS; (3) Day 5: Remove the old culture medium, add 100 μL DMEM / F12 basic culture medium and 10 μL CCK8 solution to each well of the cell culture plate, continue to incubate for 30 minutes, and measure the absorbance value (A) at 450nm. The proliferation rate%=OD experimental group / OD blank groupx100%.

[0166] 5. Determination results During the chromatography process, the protein concentration of the sample will affect the resolution of the chromatography column, resulting in failure of subpopulation separation. The results are shown in Figure 7 Table 2. The immunomodulatory activity and cell proliferation activity of Comparative Example 2 show that S1 and S2 have no significant difference and the activity is not improved compared with TFF and TFF+core700 group, indicating that when the protein concentration of the sample is more than 50mg / ml, the subpopulation separation fails.

[0167] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing a cell-derived exosome subset, characterized in that, The preparation method includes the following steps: Step (1) Filter the cell culture medium to obtain the cell culture supernatant; Step (2) The cell culture supernatant was concentrated 5-30 times using a tangential flow filtration system with a 300-500kDa hollow fiber filter, and washed 10-20 times to obtain the concentrate; Step (3) The concentrate is separated by chromatography using a Sephacryl S-400 HR gel filter column. Based on the 280 nm UV absorption peak, the elution buffer in the 50-500 mAU range and the elution buffer in the 500-200 mAU range are collected to obtain cell-derived exosome subset S1 and cell-derived exosome subset S2. The protein concentration of the concentrate is 20-50 mg / mL.

2. The preparation method according to claim 1, characterized in that, The cells mentioned in step (1) are mammalian cells.

3. The preparation method according to claim 2, characterized in that, The mammalian cells mentioned are derived from any one or more of the following: mesenchymal stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, cardiomyocytes, airway epithelial cells, and fibroblasts.

4. The preparation method according to claim 3, characterized in that, The mesenchymal stem cells mentioned are derived from any one or more of the following: bone marrow, umbilical cord, dental pulp, placenta, amnion, umbilical cord blood, synovium, muscle tissue, and periodontal ligament.

5. The preparation method according to claim 1, characterized in that, The filtration capacity mentioned in step (1) is 150-300 L / m³. 2 The filtration flow rate is 100-300 LMH.

6. The preparation method according to claim 1, characterized in that, The filtration described in step (1) is performed using a double-layer composite structure filter membrane. The material of the double-layer composite structure filter membrane includes any one or more of the following: polyethersulfone, polyvinylidene fluoride, polytetrafluoroethylene, mixed cellulose ester, cellulose acetate, and glass fiber.

7. The preparation method according to claim 1, characterized in that, The hollow fiber filter material mentioned in step (2) includes any one or more of the following: polysulfone, polyethersulfone, polyvinylidene fluoride, sulfonated polysulfone, and polyether ether ketone.

8. The preparation method according to claim 1, characterized in that, The buffer solution used for washing and filtration in step (2) includes any one or more of the following: PBS buffer, Tris-HCl buffer, HEPES buffer, physiological saline, PBST buffer, and MES buffer.

9. The preparation method according to claim 1, characterized in that, The hollow fiber filter described in step (2) has a molecular weight cutoff of 300 kDa; the washing filter is 20 times the washing filter; and the transmembrane pressure of the tangential flow filtration system is 0.2-2 bar.

10. The preparation method according to claim 1, characterized in that, The concentration mentioned in step (2) is to concentrate the volume of cell culture supernatant by 5-30 times.

11. The preparation method according to claim 1, characterized in that, The chromatographic separation process described in step (3) includes: equilibration, loading of concentrated solution, elution, and in-situ washing.

12. The preparation method according to claim 11, characterized in that, The volume of the concentrated solution loaded is 2%-5% of the volume of the Sephacryl S-400 HR gel filter column, and the elution flow rate is 5-15 cm / h.

13. Cell-derived exosome subsets obtained by the preparation method according to any one of claims 1-12.

14. The use of the cell-derived exosome subsets as described in claim 13 in the preparation of immunomodulatory drugs or drugs that promote cell regeneration.

15. A drug for immunomodulation or promoting cell regeneration, characterized in that, The drug comprises the cell-derived exosome subset as described in claim 13.

16. The medicament according to claim 15, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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