Exosome for promoting cartilage repair as well as preparation method and application of exosome

Abstract

The invention discloses an exosome for promoting cartilage repair and a preparation method and application thereof, the preparation method comprises the following steps: step 1, carrying out separation and subculture on cells to obtain a cell culture; the cells are mesenchymal stem cells, nerve cells and cartilage cells; step 2, centrifuging and filtering the cell culture to obtain a concentrated solution; and step 3, carrying out differential centrifugation on the concentrated solution to obtain the purified exosome. The particle size distribution of the exosome is concentrated while the purity of the exosome is improved, the yield and purity of the obtained exosome are high, and the effect is good when the exosome is used for cartilage repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to exosomes that promote cartilage repair, and a preparation method and application thereof. Background Art

[0002] The main function of articular cartilage is to transmit and distribute motion loads, maintain and withstand contact stress, and thus smoothly complete joint functional activities. Since there are no blood vessels, lymph nodes, and nerves in articular cartilage, its own repair ability is weak and the repair process is long. Non-drug treatment, pharmacological treatment, and surgical treatment are needed to improve articular cartilage damage, and the treatment effects are mostly unsatisfactory.

[0003] Current research shows that mesenchymal stem cells (MSC) have unique proliferation and directional differentiation capabilities, and can improve joint function by producing matrices such as type II collagen and proteoglycans through paracrine cytokines, making them an ideal material for repairing articular cartilage damage. However, due to the difficulty in preserving and transporting mesenchymal stem cells and the risks of transplantation, mesenchymal stem cell-derived exosomes (MSC-Exos) are difficult to apply clinically to repair articular cartilage. Mesenchymal stem cell exosomes (MSC-Exos) are drug delivery vehicles with similar biological functions to mesenchymal stem cells (MSC). They are relatively easy to store and transport, and have no transplantation risks. They play a vital role and have good development prospects in promoting cartilage repair and regeneration. However, the MSC-Exos obtained by existing methods have low purity and poor cartilage repair effect. Summary of the Invention

[0004] The purpose of the present invention is to provide an exosome that promotes cartilage repair, and a preparation method and application thereof. The exosomes prepared by the present invention have high yield and purity, and have a good cartilage repair effect.

[0005] In order to achieve the above object, the technical solution of the present invention is implemented as follows: a method for preparing exosomes that promote cartilage repair, comprising the following steps:

[0006] Step 1, isolating and subculturing cells to obtain cell culture; the cells are mesenchymal stem cells, neural cells and chondrocytes;

[0007] Step 2, centrifuging and filtering the cell culture to obtain a concentrate;

[0008] Step 3: performing differential centrifugation on the concentrated solution to obtain purified exosomes.

[0009] Furthermore, when the cells are mesenchymal stem cells or neural cells, the cells are separated and subcultured, specifically comprising:

[0010] The synovial fluid is filtered and centrifuged, and then resuspended in culture medium to obtain a resuspended liquid;

[0011] The resuspension was inoculated into a culture flask and cultured at 5% CO2 and 37°C until the cell coverage rate reached 70%-80%. The culture medium was replaced every 3 days during the culture process.

[0012] The cells were digested with 0.25% trypsin-EDTA and resuspended in a culture flask; then subcultured to obtain a cell culture.

[0013] Furthermore, the synovial fluid is filtered and centrifuged, and then resuspended in culture medium to obtain a resuspension, specifically comprising:

[0014] The synovial fluid was filtered using a 40 μm filter, centrifuged at 4° C. and 1500 rpm for 7 min, and then resuspended in DMEM-F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to obtain a resuspension.

[0015] Furthermore, when the cells are chondrocytes, the cells are isolated and subcultured, specifically comprising:

[0016] The cartilage was digested and centrifuged, then washed with physiological saline and resuspended to obtain resuspended cell fluid;

[0017] The resuspended cell solution was inoculated into a culture flask, and chondrocytes were inoculated every 4 hours until the cartilage fragments were completely digested. The culture medium was replaced every 3 days during the culture process, and cell culture was obtained after 5 days of culture.

[0018] Furthermore, the digestion of the cartilage followed by centrifugation specifically includes:

[0019] Cut the cartilage into 1mm pieces 3 Size, digested in DMEM medium containing 1 mg / mL type II collagenase, in a thermostatic shaker at 37°C for 10 h to obtain the digestion solution;

[0020] The digestion solution was centrifuged at 4°C and 1500 rpm for 7 min.

[0021] Furthermore, the centrifugal acceleration in step S2 is 2000g-5000g, the filtration is tangential flow filtration, and the retention capacity is 200KDa-500KDa.

[0022] Furthermore, the process of differential centrifugation in step S3 is as follows:

[0023] Centrifuge at an acceleration of 1500 g to 2500 g for 15 min to 20 min to obtain supernatant I;

[0024] Centrifuging the supernatant I at an acceleration of 8000 g to 10000 g for 30 min to 40 min to obtain supernatant II;

[0025] The supernatant II is centrifuged at an acceleration of 120,000 g to 150,000 g for 70 min to 90 min.

[0026] The second technical solution of the present invention is achieved as follows: exosomes are prepared according to the above-mentioned method for preparing exosomes that promote cartilage repair.

[0027] The third technical solution of the present invention is achieved as follows: the above-mentioned exosomes that promote cartilage repair are used in cartilage repair.

[0028] Furthermore, it is used to prepare cartilage repair scaffolds, cartilage repair sustained-release microspheres and cartilage injury treatment preparations.

[0029] Compared with the existing technology, the present invention has the following beneficial effects: the present invention removes cell debris and impurities by centrifuging and filtering the mesenchymal stem cell culture, and purifies the concentrate by differential centrifugation, thereby improving the purity of the exosomes while concentrating their particle size distribution. The obtained exosomes have a high yield and purity, and are more effective when used for cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Flowchart for the preparation of exosomes to promote cartilage repair;

[0031] Figure 2 This is a base composition distribution diagram of the raw data after sequencing of exosomes prepared in Example 1;

[0032] Figure 3 This is a base composition distribution diagram in the clean data after exosome sequencing prepared in Example 1;

[0033] Figure 4 This is the expression diagram of intracellular inflammatory factor IL-6 detected by ELISA;

[0034] Figure 5 This is the expression diagram of intracellular inflammatory factor TNF-α detected by ELISA;

[0035] Figure 6 This is an ELISA test for the expression of the intracellular anti-inflammatory factor IL-10. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] In the description of the present invention, it should be clarified that the terms "vertical", "transverse", "longitudinal", "front", "rear", "left", "right", "up", "down", "horizontal", etc. indicating orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, which are only for the convenience of describing the present invention, and do not mean that the device or element referred to must have a specific orientation or position, and therefore cannot be understood as a limitation on the present invention. In the description of the present invention, it should be noted that, unless otherwise clearly stipulated and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0038] Bone marrow mesenchymal stem cells have the potential to be easy to separate, proliferate and highly differentiated, and can alleviate articular cartilage damage by promoting cartilage repair and reducing inflammation levels; embryonic mesenchymal stem cells are a type of omnipotent stem cell that repairs cartilage damage by enhancing cell proliferation and migration, inhibiting apoptosis and regulating immune responses; adipose-derived mesenchymal stem cells have abundant tissue sources and are easy to obtain, and can withstand cell apoptosis caused by the ischemic and hypoxic cartilage microenvironment, thereby promoting articular cartilage repair; synovial mesenchymal stem cells are differentiated from the same cells as cartilage tissue, and have a relatively similar tissue structure, which is conducive to cartilage repair; therefore, although the mechanisms of action of mesenchymal stem cells derived from bone marrow, embryos, fat and synovium are different, they can all serve as the basis of exosomes for cartilage repair.

[0039] Exosomes produced by neural cells play an indispensable role in the development of neurons and neural circuits. They can not only repair brain cells damaged by developmental diseases, but also increase the number of developing neural cells and promote the proliferation of the dentate gyrus in mice.

[0040] When the cell sources are mesenchymal stem cells and neural cells, the method for preparing exosomes that promote cartilage repair comprises the following steps:

[0041] S1.1 After obtaining the patient's consent, bone marrow samples were obtained from the medullary cavity of the femoral shaft of patients undergoing hemiarthroplasty and total hip replacement surgery. Heparinized saline was added to the collected specimens, mixed thoroughly, and bone marrow mesenchymal stem cells were isolated and cultured within 2 hours.

[0042] Measure 10 mL of sample containing heparinized saline, dilute the sample with PBS buffer at a volume ratio of 1:1, repeatedly aspirate and mix, and centrifuge at 360g for 8-12 minutes at room temperature to remove cell debris and impurities in the upper layer to obtain the centrifuge fluid.

[0043] Add PBS solution to the centrifuge to 10 mL, mix well, add an equal volume of 1.067 g / mL Percoll centrifuge, centrifuge at 800g-900g for 15min-25min to obtain liquid stratification, and aspirate the mononuclear cell layer to obtain synovial fluid.

[0044] The synovial fluid was filtered using a 40 μm filter, centrifuged at 4° C. and 1500 rpm for 7 min, and resuspended in DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to obtain a resuspension.

[0045] The resuspension was inoculated into a culture flask and cultured at 5% CO2 and 37°C until the cell coverage rate reached 70%-80%. The culture medium was replaced every 3 days during the culture process.

[0046] The cells were digested with 0.25% trypsin-EDTA and resuspended in a culture flask; then subcultured to obtain a cell culture.

[0047] The culture medium used for the culture is prepared from a basic culture medium and additives, wherein the additives are selected from one or more of human serum albumin, vitamin C, EGF, PDGF-BB and norepinephrine.

[0048] Among them, human serum albumin can promote the stable growth of mesenchymal stem cells, and can also synergize with other growth factors to promote cell generation; vitamin C can reduce reactive oxygen species in cells, help maintain cell activity, and can also increase the activity of certain key enzymes to enable stable cell growth; EGF can promote the division of mesenchymal stem cells and increase the production of mesenchymal stem cells. PDGF-BB, as an important mitogen for mesenchymal stem cells, can promote the proliferation of mesenchymal stem cells; norepinephrine, as a neurotransmitter, can promote the secretion of exosomes.

[0049] The content of human serum albumin is 2g / L-6g / L, the content of vitamin C is 40mg / L-120mg / L, the content of EGF is 20μg / L-35μg / L, the content of PDGF-BB is 20μg / L-35μg / L, and the content of norepinephrine is 3μM-8μM.

[0050] S1.2 Centrifuge the cell culture at a centrifugal acceleration of 2000g-5000g for 5min-20min to remove cell debris, and then perform tangential flow filtration to obtain a concentrate.

[0051] The tangential flow filtration medium uses a hollow fiber column with a retention capacity of 200KDa-500KDa, which is not easy to clog during use and has high efficiency.

[0052] S1.3 The concentrated solution obtained in 1.2 is subjected to differential centrifugation to obtain purified exosomes, specifically as follows: the concentrated solution is centrifuged at 1500g-2500g for 15-20 min to obtain supernatant I; supernatant I is centrifuged at a centrifugal acceleration of 8000g-10000g for 30-40 min to obtain supernatant II; supernatant II is centrifuged at a centrifugal acceleration of 120000g-150000g for 70-90 min; differential centrifugation is used to separate exosomes of the same size grade to improve the purity of the exosomes.

[0053] When the cell source is chondrocytes, cell culture is obtained through the following process:

[0054] Cut the cartilage into 1mm pieces 3 The cells were digested in DMEM medium containing 1 mg / mL type II collagenase for 10 h in a thermostatic shaker at 37°C to obtain the digestion solution.

[0055] The digestion solution was centrifuged at 4°C and 1500 rpm for 7 minutes, and then washed with physiological saline and resuspended to obtain a resuspended cell solution;

[0056] The resuspended cell solution was inoculated into a culture flask, and chondrocytes were inoculated every 4 hours until the cartilage fragments were completely digested. The culture medium was replaced every 3 days during the culture process, and cell culture was obtained after 5 days of culture.

[0057] Then repeat S1.2 and S1.3 to obtain chondrocyte-derived exosomes.

[0058] Example 1

[0059] Exosomes were prepared as follows:

[0060] S1.1 After obtaining the patient's consent, bone marrow material was obtained from the medullary cavity of the femoral shaft of patients undergoing hemiarthroplasty and total hip replacement surgery. Heparinized saline was added to the collected specimen, mixed thoroughly, and bone marrow mesenchymal stem cells were isolated and cultured within 2 hours.

[0061] Measure 10 mL of sample containing heparinized saline, dilute the sample with PBS buffer at a volume ratio of 1:1, repeatedly aspirate and mix, and centrifuge at 360g for 8-12 minutes at room temperature to remove cell debris and impurities in the upper layer to obtain the centrifuge fluid.

[0062] Add PBS solution to the centrifuge to 10 mL, mix well, add an equal volume of 1.067 g / mL Percoll centrifuge, centrifuge at 800g-900g for 15min-25min to obtain liquid layer, and aspirate the mononuclear cell layer into a sterile centrifuge tube to obtain synovial fluid.

[0063] The synovial fluid was filtered using a 40 μm filter, centrifuged at 1500 rpm for 7 min at 4°C, and resuspended in DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to obtain a resuspension;

[0064] The resuspension was inoculated into a culture flask and cultured at 5% CO2 and 37°C until the cell coverage rate reached 70%-80%. The culture medium was replaced every 3 days during the culture process.

[0065] The cells were digested with 0.25% trypsin-EDTA and resuspended in a culture flask; then subcultured to obtain a cell culture.

[0066] The culture medium consists of a basic culture medium and additives, wherein the content of human serum albumin is 5 g / L, the content of vitamin C is 100 mg / L, the content of EGF is 31 μg / L, the content of PDGF-BB is 31 μg / L, and the content of norepinephrine is 6 μM.

[0067] S1.2 The mesenchymal stem cell culture was centrifuged at 3000 g for 10 min, and then filtered using a hollow fiber column with a cutoff of 300 kDa.

[0068] S1.3 Centrifuge the concentrate at 2000g for 18 min to obtain supernatant I; centrifuge supernatant I at 9000g for 35 min to obtain supernatant II; centrifuge supernatant II at 135000g for 80 min to obtain purified exosomes.

[0069] The exosomes prepared by the above method were sequenced, and the base composition distribution in the sequencing raw data and clean data was as follows: Figure 2 、 Figure 3 shown.

[0070] Example 2

[0071] While keeping other preparation conditions unchanged in Example 1, the amounts of additives in the culture medium were adjusted to: 2 g / L human serum albumin, 40 mg / L vitamin C, 20 μg / L EGF, 20 μg / L PDGF-BB, and 3 μM norepinephrine.

[0072] Example 3

[0073] While keeping other preparation conditions unchanged in Example 1, the amounts of additives in the culture medium were adjusted to: 3 g / L human serum albumin, 60 mg / L vitamin C, 24 μg / L EGF, 24 μg / L PDGF-BB, and 4 μM norepinephrine.

[0074] Example 4

[0075] While keeping other preparation conditions unchanged in Example 1, the amounts of additives in the culture medium were adjusted to: 4 g / L human serum albumin, 80 mg / L vitamin C, 28 μg / L EGF, 28 μg / L PDGF-BB, and 5 μM norepinephrine.

[0076] Example 5

[0077] While keeping other preparation conditions unchanged in Example 1, the amounts of additives in the culture medium were adjusted to: 6 g / L human serum albumin, 120 mg / L vitamin C, 35 μg / L EGF, 35 μg / L PDGF-BB, and 8 μM norepinephrine.

[0078] Example 6

[0079] While keeping other preparation conditions unchanged in Example 1, the centrifugal acceleration in S1.2 was adjusted to 2000 g, the centrifugal time to 5 min, and the cutoff flow rate to 200 kDa.

[0080] Example 7

[0081] While keeping other preparation conditions unchanged in Example 1, the centrifugal acceleration in S1.2 was adjusted to 4000 g, the centrifugal time to 15 min, and the cutoff flow rate to 400 kDa.

[0082] Example 8

[0083] While keeping other preparation conditions unchanged in Example 1, the centrifugal acceleration in S1.2 was adjusted to 5000 g, the centrifugal time to 20 min, and the cutoff flow rate to 500 kDa.

[0084] Example 9

[0085] While other preparation conditions in Example 1 remain unchanged, the treatment process of S1.3 differential centrifugation is adjusted as follows:

[0086] The concentrate was centrifuged at 1500 g for 20 min to obtain supernatant I; supernatant I was centrifuged at 8000 g for 40 min to obtain supernatant II; supernatant II was centrifuged at 120000 g for 90 min to obtain purified exosomes.

[0087] Example 10

[0088] While other preparation conditions in Example 1 remain unchanged, the treatment process of S1.3 differential centrifugation is adjusted as follows:

[0089] The concentrate was centrifuged at 2500 g for 15 min to obtain supernatant I; supernatant I was centrifuged at 10000 g for 30 min to obtain supernatant II; supernatant II was centrifuged at 150000 g for 70 min to obtain purified exosomes.

[0090] Test Example 1

[0091] The yields of purified exosomes in Examples 1-10 were detected respectively, and the results are shown in Table 1:

[0092] Table 1 Yield and impurity content of purified exosomes in Examples 1-10

[0093] Yield (%) Impurity content (%) Example 1 66.76 0.80 Example 2 58.53 1.00 Example 3 59.64 0.96 Example 4 62.36 0.84 Example 5 62.67 0.98 Example 6 73.26 3.48 Example 7 55.26 0.56 Example 8 53.28 0.24 Example 9 65.29 0.89 Example 10 66.73 0.95

[0094] Test Example 2

[0095] Fifty-five healthy mice were divided into a blank group and ten experimental groups. After incising the skin of the mouse knee joints, a physical injury modeling method was used to induce articular cartilage damage with a diameter of 4 mm and a depth of 3 mm on the knee joints of the mice. Six weeks after the knee joint injury, surgical repair was performed on the blank group mice. After surgical repair was performed on the ten experimental group mice, the exosomes prepared in Examples 1-10 were injected into the joint cavity of the mice in each experimental group once a week for a total of 12 times.

[0096] The joints of the above mice were cut into sections and stained with safranin fast green. The repair of knee joint injuries in the mice was observed under a microscope. Only fibrous tissue was repaired in the injured area of ​​the blank group mice, while hyaline cartilage rich in type II collagen appeared in the injured area of ​​the mice in the experimental groups injected with exosomes, and the articular cartilage damage was basically repaired.

[0097] ELISA detection of inflammatory factor expression levels in chondrocytes: chondrocytes from the blank group and each experimental group were collected, centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was collected. The test was performed according to the ELISA kit. The test results were as follows: Figure 4-Figure 6 shown.

[0098] Depend on Figure 4-Figure 6 It can be seen that after the joint cavity of postoperative mice was treated with the exosomes prepared in Examples 1-10, the expression levels of intracellular inflammatory factors IL-6 and TNF-α decreased, and the expression level of intracellular anti-inflammatory factor IL-10 was increased, indicating that the exosomes prepared in the examples of the present invention have an inhibitory effect on the expression of intracellular inflammatory factors and can promote cartilage repair.

[0099] Test Example 3

[0100] The particle size and particle number concentration of the exosomes prepared in Example 1 and Examples 6-8 were detected by nanoparticle tracking technology, as shown in Table 2:

[0101] As shown in Tables 1 and 2, by adjusting the components of the culture medium, the ability of mesenchymal stem cells to proliferate and secrete exosomes can be improved, and the yield of exosomes can be increased. At the same time, by combining tangential flow filtration and differential centrifugation methods, the particle size distribution of exosomes can be concentrated, and the purity of exosomes can be improved. The exosomes prepared by this method are used for cartilage repair, and the therapeutic effect is better.

[0102] Table 2 Particle size and particle number concentration of exosomes prepared in Example 1 and Examples 6-8

[0103] Particle size distribution range (nm) Particle number concentration (particles / mL) Example 1 40-176 1.18E+12 Example 6 30-193 1.03E+12 Example 7 32-184 1.13E+12 Example 8 30-200 1.09E+12

[0104] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing exosomes that promote cartilage repair, characterized in that: The following steps are involved: Step 1, isolating and subculturing cells to obtain cell culture; the cells are mesenchymal stem cells, neural cells and chondrocytes; Step 2, centrifuging and filtering the cell culture to obtain a concentrate; Step 3: performing differential centrifugation on the concentrated solution to obtain purified exosomes.

2. The method for preparing exosomes that promote cartilage repair according to claim 1, characterized in that: When the cells are mesenchymal stem cells or neural cells, the cells are separated and subcultured, specifically comprising: The synovial fluid is filtered and centrifuged, and then resuspended in culture medium to obtain a resuspended liquid; The resuspension was inoculated into a culture flask and cultured at 5% CO2 and 37°C until the cell coverage rate reached 70%-80%. The culture medium was replaced every 3 days during the culture process. The cells were digested with 0.25% trypsin-EDTA and resuspended in a culture flask; then subcultured to obtain a cell culture.

3. The method for preparing exosomes that promote cartilage repair according to claim 2, characterized in that: The synovial fluid is filtered and centrifuged, and then resuspended in culture medium to obtain a resuspension, specifically comprising: The synovial fluid was filtered using a 40 μm filter, centrifuged at 4° C. and 1500 rpm for 7 min, and then resuspended in DMEM-F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to obtain a resuspension.

4. The method for preparing exosomes that promote cartilage repair according to claim 1, characterized in that: When the cells are chondrocytes, the cells are separated and subcultured, specifically comprising: The cartilage was digested and centrifuged, then washed with physiological saline and resuspended to obtain resuspended cell fluid; The resuspended cell solution was inoculated into a culture flask, and chondrocytes were inoculated every 4 hours until the cartilage fragments were completely digested. The culture medium was replaced every 3 days during the culture process, and cell culture was obtained after 5 days of culture.

5. The method for preparing exosomes that promote cartilage repair according to claim 4, characterized in that: The digestion and centrifugation of the cartilage specifically comprises: Cut the cartilage into 1mm pieces 3 Size, digested in DMEM medium containing 1 mg / mL type II collagenase, in a thermostatic shaker at 37°C for 10 h to obtain the digestion solution; The digestion solution was centrifuged at 4°C and 1500 rpm for 7 min.

6. The method for preparing exosomes that promote cartilage repair according to claim 1, characterized in that: The centrifugal acceleration in step S2 is 2000g-5000g, the filtration is tangential flow filtration, and the retention capacity is 200KDa-500KDa.

7. The method for preparing exosomes for promoting cartilage repair according to claim 1, characterized in that: The process of differential centrifugation in step S3 is as follows: Centrifuge at an acceleration of 1500 g to 2500 g for 15 min to 20 min to obtain supernatant I; Centrifuging the supernatant I at an acceleration of 8000 g to 10000 g for 30 min to 40 min to obtain supernatant II; The supernatant II is centrifuged at an acceleration of 120,000 g to 150,000 g for 70 min to 90 min.

8. Exosomes prepared according to the method for preparing exosomes promoting cartilage repair according to any one of claims 1 to 7.

9. Use of the exosomes promoting cartilage repair as claimed in claim 8 in cartilage repair.

10. The use of exosomes promoting cartilage repair in cartilage repair according to claim 9, characterized in that: Used to prepare cartilage repair scaffolds, cartilage repair sustained-release microspheres and cartilage injury treatment preparations.

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