Method for preparing a cell-derived exosome subpopulation, product and use thereof

Abstract

This invention discloses a method for preparing cell-derived exosome subsets, as well as products and applications, belonging to the field of biomedical technology. The preparation method includes: filtering cell culture medium to obtain a cell culture supernatant; concentrating the supernatant 5-30 times using a tangential flow system with a 300-500 kDa hollow fiber filter, followed by 10-20 times washing to obtain a concentrated solution; and performing Sephacryl S-400 HR gel filtration column chromatography, collecting the eluent from two 280 nm UV absorption peaks to obtain exosome subsets S1 and S2; the protein concentration of the concentrated solution is 20-50 mg / mL. This invention achieves, for the first time, precise separation of functional exosome subsets. Subset S1 exhibits strong immunomodulatory activity, and subset S2 demonstrates excellent cell regeneration promotion effects. Furthermore, the process is highly versatile and suitable for large-scale production. These subsets can be used to prepare immunomodulatory or cell regeneration-promoting drugs, providing precise technical solutions for the treatment of related diseases.

Description

Preparation methods, products and applications of cell-derived exosome subsets Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation method, products, and applications of cell-derived exosome subsets. Background Technology

[0002] Exosomes are small lipid bilayer membrane vesicles with a diameter of 30-150 nm secreted by cells. They are widely distributed in biological fluids such as cell culture media, blood, and urine, and encapsulate various bioactive molecules, including proteins, lipids, mRNA, and miRNA. As important mediators of intercellular signaling, exosomes can target and deliver their bioactive substances to recipient cells, regulating various physiological and pathological processes such as cell proliferation, differentiation, apoptosis, and immune responses. They show great potential for applications in immune regulation, tissue repair, disease diagnosis, and treatment.

[0003] In recent years, research and translation of exosomes in the biomedical field have progressed rapidly, especially in the treatment of diseases related to immune regulation and cell regeneration, where many breakthroughs have been achieved. For example, exosomes derived from mesenchymal stem cells have been shown to exert significant immunomodulatory effects by inhibiting the expression of inflammatory factors (such as IL-6 and TNFα) and promoting the secretion of anti-inflammatory factors (such as IL-10), providing new therapeutic directions for autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. At the same time, the active molecules carried by exosomes, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF), can promote cell proliferation and repair in damaged tissues, showing excellent application prospects in scenarios such as skin wound healing, myocardial injury repair, and osteoarthritis treatment.

[0004] However, the heterogeneity of exosomes is a core technological bottleneck restricting their clinical translation. Exosomes secreted by the same cell type vary significantly in particle size distribution, molecular composition, and biological activity, forming a diverse mixed system. Existing exosome separation technologies (such as differential centrifugation, density gradient centrifugation, ultrafiltration, and Capto Core 700 chromatography) mainly focus on the overall purification of exosomes, failing to achieve precise separation of functional subpopulations. Such mixed exosomes present several drawbacks in clinical applications: on the one hand, the effects of different functional subpopulations may antagonize each other, significantly reducing therapeutic efficacy; on the other hand, the presence of non-targeted subpopulations may increase the risk of potential side effects, reducing the safety and targeting of treatment. For example, in inflammation treatment, some pro-inflammatory subpopulations of mixed exosomes may weaken the therapeutic effect of anti-inflammatory subpopulations; in tissue repair, the presence of non-proliferative regulatory subpopulations reduces the repair efficiency per unit dose, increasing clinical usage costs.

[0005] In addition, existing exosome separation technologies have many shortcomings: differential centrifugation is cumbersome and time-consuming, has a low exosome recovery rate, and is prone to exosome aggregation, which destroys its structural integrity; ultrafiltration can achieve concentration, but it is difficult to effectively remove small molecule impurities and protein contamination, resulting in insufficient product purity; although Capto Core 700 chromatography can improve exosome purity, it can only separate exosomes from impurities and cannot distinguish the functional subgroups within exosomes.

[0006] Therefore, developing a preparation method that can achieve precise separation of functional exosome subpopulations while taking into account efficiency, purity, and bioactivity is of great significance for elucidating the mechanism of action of exosomes, improving clinical treatment effects, and promoting the industrialization of exosome drugs. Summary of the Invention

[0007] Based on the deficiencies of the existing technology, this invention aims to provide a method for preparing cell-derived exosome subsets, as well as products and applications. By optimizing the process route and key parameters, it achieves efficient separation of functional exosome subsets (S1 and S2), providing a new technical solution and product for the precision treatment of diseases related to immune regulation and cell regeneration.

[0008] the term:

[0009] In this invention, the term "exosome subset" refers to a subset of exosomes with specific functional targeting, specifically two types of exosomes (S1 and S2) that are isolated by the preparation method of this invention and have clear differences in molecular composition (proteins, nucleic acids, etc.) and biological activity. The S1 subset is enriched with immune-regulating active molecules, while the S2 subset is rich in cell regeneration-related functional factors. Both maintain the integrity of the lipid bilayer membrane structure.

[0010] In this invention, the term "double-layer composite structure filter membrane" refers to a composite filter material used for clarifying and filtering cell culture medium. It is composed of an upper layer with a pore size of 1.5 μm (retaining cell clumps and large impurities) and a lower layer with a pore size of 0.8 μm (retaining cell debris and small impurities). The material is selected from biocompatible materials such as polyethersulfone and polyvinylidene fluoride, which can achieve stepwise impurity retention and ensure the purity of raw materials for subsequent exosome separation.

[0011] In this invention, the term "tangential flow filtration system" refers to a separation device used for exosome ultrafiltration concentration. The core component is a hollow fiber filter (with a molecular weight cutoff of 300-500 kDa). The sample flows parallel to the membrane surface through tangential flow, and exosomes are retained and small molecule impurities are separated under transmembrane pressure. Combined with 15-20 times the volume of washing filtration, culture medium residues and protein contamination can be efficiently removed.

[0012] In this invention, the term "washing" refers to the impurity replacement operation in the tangential flow filtration process. It means that while ultrafiltration concentration is being carried out, buffer solution is continuously added and an equal volume of dialysate containing impurities is discharged. The washing ratio is defined as the ratio of the total volume of buffer solution added to the volume of the initial supernatant before concentration. The purpose is to remove impurities such as salts and small molecule metabolites from the sample and improve the purity of exosomes.

[0013] In this invention, the term "gel filtration chromatography" is used as the core step in achieving the separation of exosome subpopulations. A SephacrylS-400HR gel filtration column is used. Based on the elution volume difference of exosome subpopulations in gel particles, the eluent corresponding to two independent absorption peaks is collected by detecting the 280nm ultraviolet signal, thereby achieving precise separation of functional subpopulations.

[0014] In this invention, the term "immunomodulation" refers to the core biological function of exosome subset S1, which means regulating the balance of the body's immune response by inhibiting the expression of inflammatory factors (such as IL-6, TNFα, IL-1β) and regulating the polarization of immune cells. It is applicable to the intervention of inflammation-related diseases or autoimmune diseases.

[0015] In this invention, the term "promoting cell regeneration" refers to the core biological function of exosome subset S2, which means accelerating the repair and regeneration of damaged tissues by promoting the proliferation of target cells such as fibroblasts and epithelial cells (e.g., increasing the positive rate of Ki67 and BrdU), and is applicable to scenarios such as wound healing and tissue damage repair.

[0016] In this invention, the term "mammal" refers to vertebrate groups with mammalian characteristics (viviparous, lactating), whose derived cells have the ability to stably secrete functional exosomes, including but not limited to species such as humans, mice, rats, rabbits, and monkeys. This invention preferably uses human mammalian cells (such as mesenchymal stem cells, neural stem cells, etc.), whose secreted exosomes have high compatibility with the human physiological environment in biological functions such as immune regulation and cell regeneration, and have better safety and efficacy in clinical applications.

[0017] In this invention, the term "CV" is an abbreviation for "Column Volume," which refers to the total volume of the gel packing material inside a gel filtration chromatography column (such as a SephacrylS-400HR chromatography column). It is the core unit of measurement for the amount of buffer solution used, the volume of sample loading, and the volume of washing during chromatography operations.

[0018] The technical solution of the present invention includes:

[0019] On the one hand, the present invention provides a method for preparing cell-derived exosome subsets, the preparation method comprising the following steps:

[0020] Step (1) Filter the cell culture medium to obtain the cell culture supernatant;

[0021] 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;

[0022] Step (3) The concentrate is separated by chromatography using a Sephacryl S-400 HR gel filter column. The elution buffers in the 50-500 mAU range and the 500-200 mAU range are collected based on the 280 nm UV absorption peak to obtain cell-derived exosome subsets S1 and S2. The protein concentration of the concentrate is 20-50 mg / mL.

[0023] Specifically, the cells mentioned in step (1) are mammalian cells.

[0024] Preferably, the mammalian cells are derived from any one or more of mesenchymal stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, cardiomyocytes, airway epithelial cells, and fibroblasts.

[0025] More preferably, the mesenchymal stem cells are derived from any one or more of the following: adipose tissue, bone marrow, umbilical cord, dental pulp, placenta, amnion, umbilical cord blood, synovium, muscle tissue, and periodontal ligament.

[0026] Specifically, the cell culture medium used includes: serum-free medium, serum substitute medium, and exosome-free serum medium.

[0027] Preferably, the cell culture medium used is a serum-free medium.

[0028] Specifically, the filtration capacity described in step (1) is 150-300 L / m³. 2 The filtration flow rate is 100-300 LMH.

[0029] More specifically, the filtration capacity described 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 .

[0030] Preferably, the filter loading in step (1) is 160-170, 170-180, 180-190 or 190-200 L / m³. 2 .

[0031] More preferably, the filtration capacity in step (1) is 183 L / m³. 2 .

[0032] More specifically, the filtration flow rate 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.

[0033] Preferably, the flow rate of the filter 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.

[0034] More preferably, the flow rate of the filter in step (1) is 150 LMH.

[0035] Specifically, the filtration 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.

[0036] Preferably, the material of the double-layer composite structure filter membrane is hydrophilic polyethersulfone.

[0037] Preferably, the dual-layer core structure has an upper layer of 1.5 μm and a lower layer of 0.8 μm.

[0038] Specifically, 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.

[0039] Preferably, the hollow fiber filter described in step (2) is made of polysulfone.

[0040] Specifically, the hollow fiber filter described in step (2) has a molecular weight cutoff of 300-350, 350-400, 400-450 or 450-500 kDa.

[0041] Preferably, the hollow fiber filter described in step (2) has a molecular weight cutoff of 300 kDa.

[0042] Specifically, the washing and filtration in step (2) is 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19 or 19-20 times.

[0043] More preferably, the washing and filtration in step (2) is 20 times the washing and filtration.

[0044] Specifically, 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.

[0045] Preferably, the buffer solution used for washing and filtering in step (2) is PBS buffer.

[0046] Specifically, the transmembrane pressure of the tangential flow filtration system described in step (2) is 0.2-2 bar.

[0047] More specifically, the transmembrane pressure of the tangential flow filtration system described 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.

[0048] Preferably, the transmembrane pressure of the tangential flow filtration system described 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.

[0049] Preferably, the transmembrane pressure of the tangential flow filtration system described in step (2) is 0.5 bar.

[0050] Specifically, the ultrafiltration concentration described in step (2) is to concentrate the cell culture supernatant by 5-30 times.

[0051] More specifically, the ultrafiltration concentration in step (2) is to concentrate the cell culture supernatant volume by 5-10, 10-15, 15-20, 20-25 or 25-30 times.

[0052] Preferably, the ultrafiltration concentration in step (2) is to concentrate the cell culture supernatant by 10-15 or 15-20 times.

[0053] More preferably, the ultrafiltration concentration in step (2) is to concentrate the cell culture supernatant by 20 times.

[0054] Specifically, the chromatographic separation process described in step (3) includes: equilibration, loading of concentrated solution, elution and in-situ washing.

[0055] More specifically, the equilibration includes: equilibrating the Sephacryl S-400 HR gel filter column using a buffer solution;

[0056] Preferably, in the equilibration step, the buffer solution includes any one or more of the following: PBS buffer, Tris-HCl buffer, HEPES buffer, physiological saline, PBST buffer, and MES buffer.

[0057] More preferably, the buffer solution comprises PBS buffer.

[0058] Preferably, in the equilibration step, the volume of the buffer solution is 1-3 CV and the flow rate is 20-40 cm / h.

[0059] More preferably, in the equilibration step, the volume of the buffer solution is 2 CV and the flow rate is 30 cm / h.

[0060] More specifically, the sample loading concentrate includes: a concentrate with a protein concentration of 20-50 mg / mL, a loading volume of 2%-5% of the Sephacryl S-400 HR gel filter column, and a flow rate of 5-15 cm / h for loading.

[0061] Preferably, during the sample loading concentration process: the concentration includes a protein concentration of 20-30, 30-40, or 40-50 mg / mL.

[0062] More preferably, during the sample loading concentration process: the concentration comprises a concentration with a protein concentration of 35 mg / mL.

[0063] Preferably, during the loading of the concentrated solution: the loading volume of the concentrated solution includes 2%-3%, 3%-4%, or 4%-5% of the volume of the SephacrylS-400 HR gel filter column.

[0064] More preferably, during the sample loading process: the sample loading volume of the concentrate includes 3% of the Sephacryl S-400 HR gel filter column volume.

[0065] Preferably, during the sample loading process, the sample loading flow rate of the concentrate includes 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 or 14-15 cm / h.

[0066] More preferably, during the sample loading process, the sample loading flow rate of the concentrate is 15 cm / h.

[0067] More specifically, the elution includes: eluting the Sephacryl S-400 HR gel filter column with buffer at a flow rate of 5-15 cm / h, starting collection when the UV signal rises at 280 nm, stopping collection when it approaches the baseline level, collecting elution buffer in the 50-500 mAU range and elution buffer in the 500-200 mAU range to obtain cell-derived exosome subsets S1 and S2.

[0068] Preferably, during the elution process, the flow rate includes 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, or 14-15 cm / h.

[0069] More preferably, during the elution process, the flow rate includes 15 cm / h.

[0070] Preferably, in the elution process: the buffer solution includes any one or more of the following: PBS buffer, Tris-HCl buffer, HEPES buffer, physiological saline, PBST buffer, and MES buffer.

[0071] More preferably, in the elution process: the buffer solution comprises PBS buffer.

[0072] More specifically, the in-situ cleaning includes rinsing with NaOH solution.

[0073] Preferably, the NaOH solution is a 0.25-1M NaOH solution.

[0074] More preferably, the NaOH solution is a 0.25-0.5 or 0.5-1M NaOH solution.

[0075] More preferably, the NaOH solution is a 0.5M NaOH solution.

[0076] Preferably, the volume of the NaOH solution is 2-5 CV.

[0077] More preferably, the volume of the NaOH solution is 2-3, 3-4, or 4-5 CV.

[0078] More preferably, the volume of the NaOH solution is 3 Cv.

[0079] Preferably, the flow rate of the NaOH solution is 10-30 cm / h.

[0080] More preferably, the flow rate of the NaOH solution includes 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.

[0081] More preferably, the flow rate of the NaOH solution is 26 cm / h.

[0082] Specifically, the concentration of the cell-derived exosome subset mentioned in step (3) is 1 × 10⁻⁶. 7 -1×10 11 particles / mL.

[0083] More specifically, the concentration of the cell-derived exosome subset mentioned in step (3) is 1 × 10⁻⁶. 7 -1×10 8 1×10 8 -1×10 9 1×10 9 -1×10 10 Or 1×10 10 -1×10 11 particles / mL.

[0084] Preferably, the concentration of the cell-derived exosome subset described in step (3) is 1 × 10⁻⁶. 10 particles / mL.

[0085] In another aspect, the present invention provides a cell-derived exosome subset obtained by any of the preparation methods described above.

[0086] In another aspect, the present invention provides the use of the cell-derived exosome subsets described in any of the above claims in the preparation of immunomodulatory drugs or drugs that promote cell regeneration.

[0087] In another aspect, the present invention provides an immunomodulatory or cell-regenerating drug, said drug comprising any of the cell-derived exosome subsets described above.

[0088] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0089] Preferably, the excipients include, but are not limited to, any one or more of the following: fillers, binders, disintegrants, lubricants, emulsifiers, antioxidants, antibacterial agents, isotonic regulators, suspending agents, solubilizers, cosolvents, preservatives, and flavoring agents.

[0090] In another aspect, the present invention provides an immunomodulatory drug or a method for promoting cell regeneration, the method comprising using any of the cell-derived exosome subsets or drugs described above.

[0091] Specifically, the method includes administering an effective amount of cell-derived exosome subsets or drugs.

[0092] Preferably, the effective amount includes 1×10 7 -1×10 8 1×10 8 -1×10 9 1×10 9 -1×10 10 Or 1×10 10 -1×10 11 Cell-derived exosome subsets with particles / mL.

[0093] In another aspect, the present invention provides the application of the above-mentioned cell-derived exosome subsets in immune regulation, wherein the method of applying the immune regulation includes the following steps:

[0094] a. Adding LPS stimulates mouse macrophages to polarize from M0 to M1, generating an inflammatory response;

[0095] b. Add LPS along with cell-derived exosome subsets;

[0096] c. Evaluate the expression of inflammatory factors.

[0097] Specifically, the inflammatory factors include, but are not limited to, IL-6, TNFα, and IL-1β.

[0098] Specifically, the concentration of the cell-derived exosome subset mentioned in step b is 1 × 10⁻⁶. 7 -1×10 11 particles / mL.

[0099] More specifically, the concentration of the cell-derived exosome subset mentioned in step b is 1 × 10⁻⁶. 7 -1×10 8 1×10 8 -1×10 9 1×10 9 -1×10 10 Or 1×10 10 -1×10 11particles / mL.

[0100] Preferably, the concentration of the cell-derived exosome subset described in step b is 1 × 10⁻⁶. 10 particles / mL.

[0101] In another aspect, the present invention provides the application of the above-mentioned cell-derived exosome subsets in cell regeneration, and the method for applying the cell regeneration method includes the following steps:

[0102] a. After the fibroblasts have reached a certain degree of confluence, remove the complete culture medium and replace it with a serum-free basal culture medium;

[0103] b. Add cell-derived exosome subsets to the basal culture medium;

[0104] c. Evaluate cell proliferation.

[0105] Specifically, the cell proliferation evaluation indicators include, but are not limited to, CCK8, Ki67, and BrdU.

[0106] Specifically, the concentration of the cell-derived exosome subset mentioned in step b is 1 × 10⁻⁶. 7 -1×10 11 particles / mL.

[0107] More specifically, the concentration of the cell-derived exosome subset mentioned in step b is 1 × 10⁻⁶. 7 -1×10 8 1×10 8 -1×10 9 1×10 9 -1×10 10 Or 1×10 10 -1×10 11 particles / mL.

[0108] Preferably, the concentration of the cell-derived exosome subset described in step b is 1 × 10⁻⁶. 10 particles / mL.

[0109] The beneficial effects of this invention are as follows:

[0110] 1. In existing technologies, exosome isolation often involves purification using mixed systems (such as differential centrifugation and CaptoCore 700 chromatography), which cannot distinguish functionally specific subpopulations, leading to limited or even antagonistic therapeutic effects. The preparation method of this invention achieves, for the first time, the separation of functional exosome subpopulations (S1 and S2): the S1 subpopulation is enriched with immunomodulatory active molecules, and its IL-6 inhibition rate is increased by more than 40% compared to traditional mixed exosomes; the S2 subpopulation is rich in cell regeneration-related factors, and its fibroblast proliferation rate is increased by more than 35% compared to traditional methods, fundamentally solving the clinical translation bottleneck caused by exosome heterogeneity.

[0111] 2. The preparation method of this invention is applicable to various mammalian cells, including mesenchymal stem cells (derived from adipose tissue, bone marrow, umbilical cord, etc.), neural stem cells, and cardiomyocytes. Experimental verification shows that exosomes from different cell sources can be separated into subpopulations using this process, and the immunomodulatory activity of the S1 subpopulation and the proliferative activity of the S2 subpopulation remain consistent, demonstrating significantly better universality than existing targeted separation techniques. Furthermore, the tangential flow filtration system in the process supports large-scale continuous operation, and gel filtration chromatography can be mass-produced through column volume scaling.

[0112] 3. The exosome subsets isolated in this invention exhibit strong functional specificity: Subset S1 can significantly inhibit LPS-induced expression of macrophage inflammatory factors (IL-6, TNFα, IL-1β), making it suitable for the treatment of autoimmune diseases and inflammation-related diseases; Subset S2 can efficiently promote the proliferation of fibroblasts, epithelial cells, etc., and can be used in wound repair, tissue regeneration, and other scenarios. Compared with traditional mixed exosomes, the subset products have stronger therapeutic targeting and fewer side effects, providing a novel precision treatment strategy for the fields of immune regulation and cell regeneration, with broad prospects for clinical translation. Attached Figure Description

[0113] Figure 1 shows the effect of optimizing the tangential flow filtration system conditions; A in the figure shows the effect of different hollow fiber filters in terms of molecular weight cutoff; B shows the effect of different washing ratios.

[0114] Figure 2 shows the purification spectrum of the chromatography column.

[0115] Figure 3 shows the effect of optimized chromatography system conditions; A) IL-6 inhibition rate of component F1-component F10; B) IL-6 proliferation rate of component F1-component F10; C) IL-6 inhibition rate of different chromatography purification methods; D) IL-6 proliferation rate of different chromatography purification methods; ns in the figure represents no significant difference between groups; **** represents a significant difference p<0.0001.

[0116] Figure 4 shows the effect of different elution flow rates on subpopulation separation.

[0117] Figure 5 shows the application effects of the preparation method of cell-derived exosome subsets in different cells; A in the figure is the IL-6 inhibition rate; B is the proliferation rate.

[0118] Figure 6 is a comparison of the exosome purity between Comparative Example 1 and Example 3; * in the figure represents a significant difference p<0.05.

[0119] Figure 7 shows a comparison of the immunomodulation and cell proliferation experiments of Comparative Example 2 and Example 3; A in the figure represents the IL-6 inhibition rate; B represents the cell proliferation rate; ns represents no significant difference between groups; **** represents a significant difference (p<0.0001). Detailed Implementation

[0120] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0121] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0122] Basic Example 1: Preparation of Cell Culture Medium

[0123] 1. Preparation of culture medium for human umbilical cord mesenchymal stem cells:

[0124] Human umbilical cord mesenchymal stem cells were purchased from Pronosei (CAT: CP-CL11), at a concentration of 10,000 cells / cm². 2 The cells were densely seeded and cultured in complete medium (CAT: CM-CL11). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0125] 2. Preparation of human bone marrow mesenchymal stem cell culture medium:

[0126] Human bone marrow mesenchymal stem cells were purchased from Pronossa (CAT: CP-H166), at a rate of 10,000 cells / cm³. 2 The cells were densely seeded and cultured in complete medium (CAT: CM-H166). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0127] 3. Preparation of culture medium for human dental pulp mesenchymal stem cells:

[0128] Human dental pulp mesenchymal stem cells were purchased from Cyagen Biosciences (CAT: HUXDP-01001) at a concentration of 10,000 cells / cm³. 2 The cells were densely seeded and cultured in complete medium (CAT: HUXDP-90011). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0129] 4. Preparation of culture medium for human adipose-derived mesenchymal stem cells:

[0130] Human adipose-derived mesenchymal stem cells were purchased from Pronosei (CAT: CP-H202), at a concentration of 10,000 cells / cm². 2 The cells were densely seeded and cultured in complete medium (CAT: CM-H202). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0131] 5. Preparation of human neural stem cell culture medium:

[0132] Human neural stem cells were purchased from Shangen Biotechnology (CAT: SNP-H235), at a rate of 20,000 cells / cm³. 2 The cells were densely seeded and cultured in complete medium (CAT: SNPM-H235). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours and then the cell culture solution was collected for exosome purification.

[0133] 6. Preparation of human cardiomyocyte culture medium:

[0134] Human cardiomyocytes were purchased from Pronossa (CAT: CP-H076) at a concentration of 20,000 cells / cm². 2 The cells were densely seeded and cultured in complete culture medium (CAT: CM-H076). After the cells were fully adhered, the complete culture medium was removed and replaced with serum-free basal culture medium. The cells were cultured for another 48 hours and then the cell culture medium was collected for exosome purification.

[0135] 7. Preparation of culture medium for human small airway epithelial cells:

[0136] Human small airway epithelial cells were purchased from Pronosel (CAT: CP-H010), at a concentration of 10,000 cells / cm². 2 The cells were densely seeded and cultured in complete medium (CAT: CM-H010). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0137] 8. Preparation of human embryonic stem cell culture medium:

[0138] Human embryonic stem cells were purchased from Pronosei (CAT: CL-0890), at a concentration of 100,000 cells / cm³. 2 The cells were densely seeded and cultured in complete culture medium (CAT: CM-0890). After the cells reached 80% confluence, the culture medium was replaced with fresh medium and cultured for another 48 hours. The cell culture medium was then collected for exosome purification.

[0139] 9. Preparation of human induced pluripotent stem cell culture medium:

[0140] Human induced pluripotent stem cells were purchased from Pronosei (CAT: CL-1059), at a rate of 100,000 cells / cm². 2 The cells were densely seeded and cultured in complete culture medium (CAT: CM-1059). After the cells reached 80% confluence, the culture medium was replaced with fresh medium and cultured for another 48 hours. The cell culture medium was then collected for exosome purification.

[0141] 10. Preparation of culture medium for human dermal fibroblasts:

[0142] Human dermal fibroblasts were purchased from Pronossa (CAT: CP-H103), at a concentration of 10,000 cells / cm³. 2 The cells were densely seeded and cultured in complete medium (CAT: CM-H103). After the cells reached 80% confluence, the complete medium was removed and replaced with serum-free basal medium. The cells were cultured for another 48 hours, and then the cell culture medium was collected for exosome purification.

[0143] Example 1: Condition Optimization of Tangential Flow Filtration System

[0144] 1. Preparation of cell culture supernatant

[0145] 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.

[0146] 2. Optimization of tangential flow filtration system conditions

[0147] 2.1 Hollow Fiber Filter Condition Optimization

[0148] The tangential flow concentration system used 100kDa, 300kDa, 500kDa, or 750kDa hollow fiber filters (polysulfone) to concentrate the cell culture supernatant by 20-fold, followed by 20-fold washing with PBS buffer (0.0067M PO4, pH 7.0-7.2), transmembrane pressure (TMP): 0.5 bar, flow rate 385 LMH, to obtain the concentrated solution.

[0149] 2.2 Optimization of Filtration Ratio

[0150] The tangential flow concentration system uses a 300 kDa hollow fiber filter (polysulfone material) to concentrate the cell culture supernatant by 20 times. The supernatant is then washed with PBS buffer (0.0067 M PO4, pH 7.0-7.2) for 5, 10, 15, 20 or 25 times the supernatant, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH to obtain the concentrate.

[0151] 3. Comparison of differential centrifugation systems

[0152] The human umbilical cord mesenchymal stem cell culture medium collected in Basic Example 1 was centrifuged at 300×g for 10 minutes and the supernatant was collected; centrifuged at 2000×g for 10 minutes and the supernatant was collected; centrifuged at 10000×g for 30 minutes and the supernatant was collected; centrifuged at 100000×g at 4℃ for 90 minutes and the supernatant was removed. The remaining precipitate was resuspended in PBS and centrifuged again at 100000×g for 90 minutes. Finally, 1 mL of PBS was added to resuspend the precipitate to obtain exosomes.

[0153] 4. Determination of exosome recovery rate and exosome purity

[0154] Different concentrated solutions obtained from tangential flow filtration systems under different conditions, and cell suspensions obtained from differential centrifugation systems as a control, were analyzed using nanoparticle tracking (NTA) technology to determine the particle size distribution and exosome concentration (particales / μL) in exosome subpopulations. The protein concentration (μg / μL) of exosome subpopulations was analyzed using BCA technology to calculate the purity of exosome subpopulations.

[0155] Exosome recovery rate (%) = Total number of exosome particles harvested / Total number of exosome particles in human umbilical cord mesenchymal stem cell culture medium × 100%.

[0156] Exosome purity (particales / μg) = Exosome concentration (particales / μL) / Protein content (μg / μL).

[0157] 5. Measurement Results

[0158] In the tangential flow ultrafiltration concentration process, this embodiment considered hollow fiber filters with different intercept molecular weights (100kDa-750kDa) and different washing folds. Differential centrifugation (UC), a classic method for exosome purification, was used as a control. The recovery rate and purity of exosome particles were used as evaluation indicators. The results are shown in Figure 1.

[0159] Regarding exosome recovery rate, differential centrifugation yielded the lowest recovery rate, below 20%; 100 kDa and 300 kDa filters yielded the highest recovery rates, both above 95%; followed by 500 kDa and 750 kDa. As for exosome purity, 100 kDa showed the lowest purity, while the purity of exosomes from 300 kDa to 750 kDa was all above 3 × 10⁻⁶. 8 The particales / μg concentration is close to the purity of exosomes obtained by differential centrifugation. Therefore, hollow fiber filters with molecular weight interception of 300kDa and 500kDa were selected, with 300kDa hollow fiber filters being preferred.

[0160] 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.

[0161] Example 2: Optimization of Chromatography System Conditions

[0162] 1. Preparation of cell culture supernatant

[0163] 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.

[0164] 2. Concentration of cell culture supernatant

[0165] 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.

[0166] 3. Optimization of chromatography system conditions

[0167] 3.1 Purification using Sephacryl S-400 HR chromatography column

[0168] 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.

[0169] 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.

[0170] 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.

[0171] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution at a flow rate of 10cm / h.

[0172] 3.2 Capto Core 700 chromatography column purification

[0173] Equilibration: Equilibrate the Capto Core 700 gel filter column (purchased from Cytiva, catalog number 17548115) with PBS buffer (0.0067M PO4, pH 7.0-7.2) at 10 CV and a flow rate of 155 cm / h.

[0174] Sample loading: The protein concentration of the concentrated solution was 35 mg / mL, and the loading volume was 5 CV. During the loading process, collection began when the 280 nm UV signal rose and stopped when it approached the baseline level to obtain exosome products.

[0175] Regeneration: Rinse 3CV with PBS buffer at a flow rate of 155 cm / h.

[0176] Clean in place (CIP): Rinse 3CV with 0.5M NaOH solution at a flow rate of 26 cm / h.

[0177] 4. Effect Verification

[0178] 4.1 Immunomodulatory Experiments

[0179] (1) Day 1: Mouse macrophages RAW264.7 were injected at a dose of 2×10⁻⁶. 5 Cells / wells were placed in 6-well plates and cultured overnight to allow them to adhere.

[0180] (2) Day 2: Remove the old culture medium and add fresh culture medium containing 100 ng / mL lipopolysaccharide (LPS) and continue culturing for 24 h. The experimental group was given 1×10 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2), and an equal amount of PBS was added to the blank group;

[0181] (3) Day 3: Collect cell supernatant, centrifuge at 2000×g for 5 minutes, and use the supernatant for enzyme-linked immunosorbent assay to detect IL-6 expression.

[0182] 4.2 Cell proliferation experiment

[0183] (1) Day 1: Human dermal fibroblasts HSF were injected at a dose of 4 × 10⁻⁶. 4 Seeds were planted at a density of / mL into 96-well cell culture plates and incubated overnight to allow the cells to adhere.

[0184] (2) Day 2: Remove the old culture medium, add fresh culture medium and continue culturing for 72 hours. The experimental group was given 1×10⁻⁶ medium. 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2), and an equal amount of PBS was added to the blank group;

[0185] (3) Day 5: Remove the old culture medium, add 100 μL of DMEM / F12 basal culture medium and 10 μL of CCK8 solution to each well of the cell culture plate and continue incubation for 30 minutes. Measure the absorbance value (A) at 450 nm. The proliferation rate % = OD experimental group / OD blank group × 100%.

[0186] 5. Measurement Results

[0187] In the chromatographic purification process, Capto Core 700 chromatography columns are increasingly used for exosome purification. However, this chromatographic method cannot achieve the purpose of subpopulation separation. Therefore, this invention uses Sephacryl S-400 HR chromatography columns for purification. The chromatogram is shown in Figure 2. Two absorption peaks appeared during the elution process. The components were collected during the elution process, and their activity was detected by immunomodulation and fibroblast proliferation experiments. The results are shown in AB of Figure 3. Components 4-6 showed strong immunomodulatory activity, while components 7-10 were more inclined to promote proliferation. Components 4-6 were combined and named S1, and components 7-10 were combined and named S2. The activity was compared with concentrated buffer (TFF) and Capto Core 700 purified sample (TFF+core700). The results are shown in CD of Figure 2. In terms of immunomodulatory activity, subpopulation S1 was significantly better than TFF, TFF+core700 and subpopulation S2, while subpopulation S2 was significantly better than other samples in terms of pro-proliferative activity.

[0188] Example 3: Method for preparing cell-derived exosome subsets of the present invention

[0189] 1. Preparation of cell culture supernatant

[0190] 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.

[0191] 2. Concentration of cell culture supernatant

[0192] The tangential flow concentration system uses a 300kDa hollow fiber filter (polysulfone material) to concentrate the cell culture supernatant by 20 times, wash it 20 times with PBS buffer (0.0067M PO4, pH 7.0-7.2), transmembrane pressure (TMP): 0.5 bar, flow rate 385 LMH to obtain the concentrate.

[0193] 3. Purification using Sephacryl S-400 HR chromatography column

[0194] 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.

[0195] 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.

[0196] 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.

[0197] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution at a flow rate of 10cm / h.

[0198] Example 4: Validation of the method for preparing cell-derived exosome subsets

[0199] 1. Scale-up verification

[0200] (1) Preparation of cell culture supernatant

[0201] Refer to Example 3

[0202] (2) Concentration of cell culture supernatant

[0203] Refer to Example 3

[0204] (3) Purification using Sephacryl S-400 HR chromatography column

[0205] 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.

[0206] 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.

[0207] 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.

[0208] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution at a flow rate of 10cm / h.

[0209] (4) Effect verification

[0210] The effects of different elution rates on subpopulation separation were verified using the immunomodulation and cell proliferation experiments described in Example 2. The results are shown in Figure 4. No significant changes were observed between 5 cm / h and 15 cm / h, indicating good stability in the separation of surface exosome subpopulations and suggesting potential for scale-up.

[0211] 2. Universality verification

[0212] (1) Preparation of cell culture supernatant

[0213] In Basic Example 1, mesenchymal stem cells from different tissue sources and cell types were collected and their culture media were clarified and filtered to obtain cell culture supernatant. Clarification and filtration involved filtering the cell culture medium using a Quapper clarification filter membrane pack (catalog number U33CPPLELA1P) with a double-layer composite structure (1.5μm upper layer + 0.8μm lower layer) and a flow rate of 183 L / m³. 2 The filtration flow rate is 150 LMH.

[0214] (2) Concentration of cell culture supernatant

[0215] Refer to Example 3

[0216] (3) Purification using Sephacryl S-400 HR chromatography column

[0217] Refer to Example 3

[0218] (4) Effect verification

[0219] To verify the universality of the subpopulation isolation method, exosomes prepared using the cell-derived exosome subpopulation preparation method of this invention were used to verify the results of the immunomodulation experiment and cell proliferation experiment described in Example 2 for various mesenchymal stem cells from different tissue sources and different cell types.

[0220] As shown in Figure 5, although the activities of exosome subsets from different cell sources varied, the subset separation method of the present invention could successfully separate all subsets. Consistent with exosomes derived from umbilical cord mesenchymal stem cells, the S1 subset showed significant immunomodulatory activity, while the S2 subset showed significant cell proliferation-promoting activity, indicating that the subset separation method has good universality.

[0221] Comparative Example 1

[0222] The only difference between Comparative Example 1 and Example 3 is step "2. Concentration of cell culture supernatant". The specific steps are as follows:

[0223] The tangential flow concentration system uses a 100 kDa hollow fiber filter to concentrate the cell culture supernatant 20 times, wash it 25 times with PBS buffer, and obtain the concentrate at a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385 LMH.

[0224] Experimental Example 1: Comparative Example 1 - Verification of Exosome Purity

[0225] The concentrated solution prepared in Comparative Example 1 was analyzed by nanoparticle tracking (NTA) to determine the particle size distribution and exosome concentration (particales / μL) in the exosome subpopulations. The protein concentration (μg / μL) of the exosome subpopulations was analyzed by BCA, and the purity of the exosome subpopulations was calculated.

[0226] Exosome recovery rate (%) = Total number of exosome particles harvested / Total number of exosome particles in human umbilical cord mesenchymal stem cell culture medium × 100%.

[0227] Exosome purity (particales / μg) = Exosome concentration (particales / μL) / Protein content (μg / μL).

[0228] As shown in Figure 6, in the tangential flow concentration stage, the average purity of exosomes in the concentrate obtained in Comparative Example 1 was 5.47E+07 particles / μg, while the average purity of exosomes in the concentrate obtained in Example 3 was 3.53E+08 particles / μg. The difference between the two is one order of magnitude, indicating that higher purity means better quality.

[0229] Comparative Example 2

[0230] The only difference between Comparative Example 2 and Example 3 is step "3. Sephacryl S-400 HR chromatography column purification". The specific steps are as follows:

[0231] 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.

[0232] Sample loading: The concentrated protein concentration is 55 mg / mL, the loading volume is 3% of the column volume, and the flow rate is 15 cm / h.

[0233] 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.

[0234] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution at a flow rate of 10cm / h.

[0235] Experimental Example 2: Comparative Example 2 - Immunomodulation and Cell Proliferation Experiments to Verify

[0236] 1. Immunomodulatory experiments

[0237] (1) Day 1: Mouse macrophages RAW264.7 were injected at a dose of 2×10⁻⁶. 5 Cells / wells were placed in 6-well plates and cultured overnight to allow them to adhere.

[0238] (2) Day 2: Remove the old culture medium and add fresh culture medium containing 100 ng / mL lipopolysaccharide (LPS) and continue culturing for 24 h. The experimental group was given 1×10 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2), and an equal amount of PBS was added to the blank group;

[0239] (3) Day 3: Collect cell supernatant, centrifuge at 2000×g for 5 minutes, and use the supernatant for enzyme-linked immunosorbent assay to detect IL-6 expression.

[0240] 2. Cell proliferation experiment

[0241] (1) Day 1: Human dermal fibroblasts HSF were injected at a dose of 4 × 10⁻⁶. 4 Seeds were planted at a density of / mL into 96-well cell culture plates and incubated overnight to allow the cells to adhere.

[0242] (2) Day 2: Remove the old culture medium, add fresh culture medium and continue culturing for 72 hours. The experimental group was given 1×10⁻⁶ medium. 10 / mL exosomes (S1, S2 or exosome product) or concentrated solution (prepared in step 2), and an equal amount of PBS was added to the blank group;

[0243] (3) Day 5: Remove the old culture medium, add 100 μL of DMEM / F12 basal culture medium and 10 μL of CCK8 solution to each well of the cell culture plate and continue incubation for 30 minutes. Measure the absorbance value (A) at 450 nm. The proliferation rate % = OD experimental group / OD blank group × 100%.

[0244] 5. Measurement Results

[0245] During chromatography, the protein concentration of the sample affects the resolution of the chromatography column, leading to the failure of subpopulation separation. As shown in Figure 7, the immunomodulatory activity and cell proliferation activity of Comparative Example 2 showed no significant difference between S1 and S2, and the activity was not improved compared with the TFF and TFF+core700 groups, indicating that subpopulation separation failed when the sample protein concentration exceeded 50 mg / ml.

[0246] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a cell-derived exosome subset, characterized in that, The preparation method includes the following steps: Step (1) filtration of cell culture medium to obtain cell culture supernatant; the filtration loading is 150-300 L / m 2 The filtration flow rate is 100-300 LMH; the filtration is performed using a double-layer composite membrane, the double-layer composite structure being an upper layer of 1.5 μm + a lower layer of 0.8 μm; in step (2), the cell culture supernatant is concentrated 5-30 times using a tangential flow filtration system with a 300-500 kDa hollow fiber filter, and washed 15-20 times to obtain a concentrated solution; in step (3), the concentrated solution is separated by chromatography using a Sephacryl S-400 HR gel filter column, the chromatography separation process including: equilibration, loading of concentrated solution, elution, and in-situ washing; the equilibration includes: equilibrating the Sephacryl S-400 HR gel filter column with buffer; in the equilibration step, the volume of the buffer is 1-3 CV, and the flow rate is 20-40 cm / h; the loading concentrated solution includes: a concentrated solution with a protein concentration of 20-50 mg / mL, and the loading volume is Sephacryl S-400 HR gel filter column. The sample is loaded at a flow rate of 5-15 cm / h onto a 2%-5% volume of the S-400HR gel filter column. The elution includes: eluting the Sephacryl S-400 HR gel filter column with buffer at a flow rate of 5-15 cm / h, starting collection when the UV signal rises at 280 nm and stopping collection when it approaches the baseline level, collecting elution buffer in the 50-500 mAU range and elution buffer in the 500-200 mAU range to obtain cell-derived exosome subsets S1 and S2. The in-situ washing includes rinsing with NaOH solution. The cells mentioned in step (1) are mammalian cells. The hollow fiber filter material mentioned in step (2) includes any one or more of polysulfone, polyethersulfone, and sulfonated polysulfone.

2. The preparation method according to claim 1, 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.

3. The preparation method according to claim 2, 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.

4. 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.

5. 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 PBS buffer, Tris-HCl buffer, and HEPES buffer.

6. 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.

7. 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.

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