Mesenchymal stem cell exosome separation and purification method and application of mesenchymal stem cell exosome in anti-inflammatory treatment

Abstract

The invention belongs to the technical field of biological medicines, and discloses a separation and purification method of mesenchymal stem cell exosomes and application of the mesenchymal stem cell exosomes in anti-inflammatory treatment. The mesenchymal stem cell exosome separation and purification method comprises the following steps: S1, preparing a sample; and S2, differential centrifugation. And S3, ultrafiltration. And S4, carrying out immunomagnetic bead separation. And S5, collecting and purifying the exosome. The method is simple to operate and low in cost, can obtain the exosome with high purity and high activity, and is suitable for large-scale production; the mesenchymal stem cell exosome is rich in various anti-inflammatory factors and immunomodulatory molecules and can inhibit activation of inflammatory cells and relieve tissue damage, the mesenchymal stem cell exosome serves as a novel medicine for anti-inflammatory treatment, the exosome is conveyed to an inflammatory part through intravenous injection and the like, the anti-inflammatory effect is achieved, and the mesenchymal stem cell exosome is used for treating inflammatory diseases. Important breakthrough and contribution are expected to be brought to the field of anti-inflammatory treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for separating and purifying mesenchymal stem cell exosomes and an application thereof in anti-inflammatory treatment. Background Art

[0002] Mesenchymal stem cells (MSCs) have shown great potential in the treatment of various diseases due to their immunomodulatory and anti-inflammatory properties. In recent years, studies have found that exosomes released by MSCs also have anti-inflammatory effects, and because of their smaller size and lower immunogenicity, they are more suitable as therapeutic carriers. However, the effective separation and purification of exosomes is a key challenge in their clinical application. The present invention aims to provide an innovative separation and purification technology for mesenchymal stem cell exosomes, and explore its new application in anti-inflammatory treatment. Summary of the invention

[0003] In order to overcome the above technical problems, the present invention provides a method for separating and purifying mesenchymal stem cell exosomes and its application in anti-inflammatory treatment.

[0004] The present invention adopts the following technical solutions:

[0005] A method for separating and purifying mesenchymal stem cell exosomes, comprising:

[0006] S1. Sample preparation:

[0007] Collect biological samples containing exosomes and pre-treat the samples to initially remove impurities such as cell debris and protein aggregates;

[0008] S2. Differential centrifugation:

[0009] The pretreated samples were centrifuged at low speed to remove larger cell debris and particulate matter;

[0010] This is followed by high-speed centrifugation to separate the exosomes from other smaller biomolecules;

[0011] Repeat the centrifugation step and adjust the centrifugation speed and time as needed to further optimize the isolation of exosomes;

[0012] S3.Ultrafiltration:

[0013] Use ultrafiltration membrane to filter the centrifuged sample to further remove residual proteins and other small molecules;

[0014] S4. Immunomagnetic separation:

[0015] Prepare antibodies that specifically bind to exosome surface markers and couple them to magnetic beads;

[0016] The antibody-magnetic bead complex is added to the ultrafiltered sample to allow the magnetic beads to bind to the exosomes;

[0017] The magnetic beads bound to exosomes are separated from the sample using a magnetic field;

[0018] Wash the magnetic beads to remove unbound nonspecific substances;

[0019] S5. Exosome collection and purification:

[0020] Exosomes were released from the magnetic beads and the released exosomes were further purified.

[0021] In S1, the biological sample can be cell culture supernatant, blood, or urine.

[0022] In S5, the release of exosomes is achieved by changing the pH, ionic strength or using a specific eluent.

[0023] In S5, further purification is achieved by additional centrifugation, ultrafiltration or chromatography.

[0024] The present invention also explores the application of mesenchymal stem cell exosomes in anti-inflammatory treatment.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The method for separating and purifying exosomes from mesenchymal stem cells of the present invention is simple to operate, low in cost, and can obtain high-purity and high-activity exosomes, and is suitable for large-scale production;

[0027] 2. Mesenchymal stem cell exosomes are rich in a variety of anti-inflammatory factors and immunomodulatory molecules, such as TGF-β, IL-10, etc. These molecules can inhibit the activation of inflammatory cells and reduce tissue damage. The present invention proposes to use mesenchymal stem cell exosomes as a new type of anti-inflammatory drug, and deliver the exosomes to the inflammatory site by intravenous injection or other means to exert anti-inflammatory effects, which is expected to bring important breakthroughs and contributions to the field of anti-inflammatory treatment. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. Unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are conventional methods in the art. The following embodiments are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0029] Based on the physical properties and surface markers of exosomes, differential centrifugation, ultrafiltration and immunomagnetic bead separation are used to achieve efficient and high-purity exosome isolation.

[0030] Cell source and preparation:

[0031] Mesenchymal stem cells (MSCs) are usually derived from bone marrow, adipose tissue, umbilical cord blood, etc. MSCs exosomes are separated and purified from MSCs culture supernatant by ultracentrifugation, tangential flow ultrafiltration and other methods.

[0032] Sample preparation:

[0033] Collect biological samples containing exosomes - cell culture supernatant, and pre-treat the samples to initially remove impurities such as cell debris and protein aggregates.

[0034] Differential Centrifugation:

[0035] The pretreated samples were centrifuged at low speed to remove larger cell debris and particulate matter;

[0036] Subsequently, high-speed centrifugation is performed to separate the exosomes from other smaller biomolecules (such as proteins, RNA, etc.);

[0037] Repeat the centrifugation step and adjust the centrifugation speed and time as needed to further optimize the isolation of exosomes.

[0038] Ultrafiltration:

[0039] Use an ultrafiltration membrane to filter the centrifuged sample to further remove residual proteins and other small molecules. Select an ultrafiltration membrane with an appropriate pore size to ensure that exosomes can pass through while impurities are retained.

[0040] Immunomagnetic separation:

[0041] Prepare antibodies that specifically bind to exosome surface markers and couple them to magnetic beads;

[0042] The antibody-magnetic bead complex is added to the ultrafiltered sample to allow the magnetic beads to bind to the exosomes;

[0043] The magnetic beads bound to exosomes are separated from the sample using a magnetic field;

[0044] Wash the beads to remove unbound nonspecific substances.

[0045] Exosome collection and purification:

[0046] Release of exosomes from magnetic beads is usually achieved by changing the pH, ionic strength or using a specific elution buffer.

[0047] The released exosomes are further purified, such as by re-centrifugation, ultrafiltration or chromatography.

[0048] Quality Control and Assessment:

[0049] Perform quality control on the isolated exosomes, including morphological observation, particle size analysis, protein marker detection, etc.; evaluate the purity and concentration of exosomes to ensure that they meet the needs of subsequent experiments or treatments.

[0050] Animal Model:

[0051] Use appropriate inflammatory animal models, such as arthritis models, acute lung injury models, etc.

[0052] Dosage:

[0053] MSCs exosomes are delivered into animals through intravenous injection, local injection, etc.

[0054] Observation indicators:

[0055] Inflammation-related indicators: such as the levels of inflammatory factors (TNF-α, IL-6, etc.) and inflammatory cell infiltration; tissue damage and repair: such as histopathological scores and functional recovery.

[0056] Typical experimental results:

[0057] Arthritis Model:

[0058] Levels of inflammatory factors: In the arthritis model, after intravenous injection of MSCs exosomes, the levels of inflammatory factors such as TNF-α and IL-6 in the animals were significantly reduced, indicating that exosomes have anti-inflammatory effects.

[0059] Histopathological score: The infiltration of inflammatory cells in the synovial joints of animals in the exosome treatment group was reduced, cartilage destruction was alleviated, and the histopathological score was significantly lower than that in the control group.

[0060] Acute lung injury model:

[0061] Pulmonary function indicators: In the acute lung injury model, the lung function indicators of animals (such as oxygenation index) were improved after intravenous injection of MSCs exosomes, indicating that exosomes have a repairing effect on lung damage.

[0062] Inflammatory cell infiltration: The infiltration of inflammatory cells in the lung tissue of animals in the exosome treatment group was reduced, and pulmonary edema was alleviated.

[0063] In many studies, MSCs exosomes have shown good safety, and no obvious toxic side effects have been observed.

[0064] Experiment to evaluate the therapeutic effect of MSCs exosomes on arthritis model:

[0065] To evaluate the effects of intravenous injection of mesenchymal stem cell (MSCs) exosomes on inflammatory response and tissue damage repair in arthritis model mice.

[0066] Experimental procedures

[0067] Establishment of animal model: Healthy adult mice were selected and an arthritis model was established by intra-articular injection of a specific dose of collagen inducer.

[0068] Grouping and treatment: The mice with successful modeling were randomly divided into two groups: a treatment group (n=10) and a control group (n=10). The treatment group was intravenously injected with MSCs exosomes, and the control group was injected with an equal volume of normal saline.

[0069] Sample collection: Blood samples were collected from mice on the 7th, 14th, and 21st days after treatment for detection of inflammatory factors, and joint tissues were taken for histopathological analysis.

[0070] Index detection: ELISA method was used to determine the levels of TNF-α and IL-6 in serum; histological staining and scoring criteria were used to evaluate the degree of inflammatory cell infiltration in the synovial membrane and cartilage destruction.

[0071] Experimental equipment

[0072] Microplate reader (for ELISA detection), microscope and imaging system (for histopathological observation), centrifuge, pipette and other conventional laboratory equipment.

[0073] Experimental conditions

[0074] Experimental environment temperature: 22±2℃.

[0075] Relative humidity: 50±10%

[0076] Photoperiod: 12 h light / 12 h dark.

[0077] Experimental data

[0078] Table 1: Changes in serum inflammatory factor levels (pg / mL)

[0079] Group Time point (day) TNF-α IL-6 Treatment Group 7 25.4±3.1 30.8±4.2 Treatment Group 14 18.6±2.5 22.1±3.0 Treatment Group 21 12.0±1.8 15.5±2.1 Control group 7 45.7±5.6 50.3±6.1 Control group 14 40.1±4.9 43.8±5.4 Control group 21 35.2±4.2 38.6±4.7

[0080] Table 2: Histopathological scores (0-4, higher scores indicate more severe damage)

[0081] Group Time point (day) Inflammatory cell infiltration Cartilage destruction Overall rating Treatment Group 21 1.2±0.4 0.8±0.3 2.0±0.5 Control group 21 2.7±0.6 2.3±0.5 5.0±0.8

[0082] Data sources and collection methods:

[0083] Serum samples were collected by cardiac puncture and immediately placed on ice, after which serum was separated by centrifugation and stored at −80°C until analysis.

[0084] After the tissue samples were fixed, sectioned, and stained, they were scored by two independent observers according to the preset criteria, and the average value was taken as the final result.

[0085] Inflammatory factors were detected using commercial ELISA kits in strict accordance with the instructions.

[0086] in conclusion

[0087] Experimental data showed that intravenous injection of MSCs exosomes can effectively reduce the levels of inflammatory factors such as TNF-α and IL-6 in arthritis model mice, reduce inflammatory cell infiltration in the synovial membrane and cartilage destruction, indicating that MSCs exosomes have significant anti-inflammatory and tissue repair effects.

[0088] Although the embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the above embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for separating and purifying exosomes of mesenchymal stem cells, characterized in that: include: S1. Sample preparation: Collect biological samples containing exosomes and pre-treat the samples to initially remove impurities such as cell debris and protein aggregates; S2. Differential centrifugation: The pretreated samples were centrifuged at low speed to remove larger cell debris and particulate matter; This is followed by high-speed centrifugation to separate the exosomes from other smaller biomolecules; Repeat the centrifugation step and adjust the centrifugation speed and time as needed to further optimize the isolation of exosomes; S3.Ultrafiltration: Use ultrafiltration membrane to filter the centrifuged sample to further remove residual proteins and other small molecules; S4. Immunomagnetic separation: Prepare antibodies that specifically bind to exosome surface markers and couple them to magnetic beads; The antibody-magnetic bead complex is added to the ultrafiltered sample to allow the magnetic beads to bind to the exosomes; The magnetic beads bound to exosomes are separated from the sample using a magnetic field; Wash the magnetic beads to remove unbound nonspecific substances; S5. Exosome collection and purification: Exosomes were released from the magnetic beads and the released exosomes were further purified.

2. The method for separating and purifying mesenchymal stem cell exosomes according to claim 1, characterized in that: In S1, the biological sample can be cell culture supernatant, blood, or urine.

3. The method for separating and purifying mesenchymal stem cell exosomes according to claim 1, characterized in that: In S5, the release of exosomes is achieved by changing the pH, ionic strength or using a specific eluent.

4. The method for separating and purifying mesenchymal stem cell exosomes according to claim 1, characterized in that: In S5, further purification is achieved by additional centrifugation, ultrafiltration or chromatography.

5. Application of mesenchymal stem cell exosomes in anti-inflammatory treatment.