A preparation method and application of mesenchymal stem cell exosomes

Abstract

The present invention provides a method for preparing mesenchymal stem cell exosomes and its application. N-acetylcysteine and quercetin are added to the culture medium used for culturing MSC cells, so as to stimulate MSC to produce exosomes with anti-aging and anti-apoptosis effects. Moreover, by gradually reducing the oxygen partial pressure for culturing, the exosomes produced by stimulating MSC further have anti-aging effects. And after the cells are induced and cultured with N-acetylcysteine and quercetin, it can inhibit the adverse effects such as acute oxidative stress, decreased proliferation ability and unstable quality produced under hypoxic conditions without damaging the activity of cell and exosome-related components. This method shows good anti-aging and anti-apoptosis effects in vitro, providing a new idea for the future application of exosomes in the treatment of senile diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing mesenchymal stem cell exosomes and its application. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with self-renewal ability and multi-directional differentiation potential. They have the advantages of rich sources, which can be obtained from tissues such as bone marrow, umbilical cord, umbilical cord blood, and adipose tissue, and low immunogenicity. Cell therapy based on MSCs has been successfully applied to the treatment of diseases such as cardiovascular system diseases, bone and cartilage defects, and diabetes. MSCs release a variety of cytokines and growth factors through paracrine and autocrine, and these secreted bioactive factors can inhibit fibrosis and apoptosis, enhance angiogenesis, and participate in tissue repair and regeneration.

[0003] Exosomes are nanoscale lipid-encapsulated structures with a diameter of 30-150 nm, which encapsulate substances such as proteins, mRNAs, and microRNAs inside. Exosomes naturally exist in body fluids, including blood, saliva, urine, breast milk, etc. Exosomes are membranous vesicles secreted by living cells and derived from late endosomes (also known as multivesicular bodies). Almost all cells, including tumors, can produce and release exosomes. Exosomes are secreted and released by cells, spread in body fluids such as blood, and can finally be phagocytosed by other cells. They are important mediators of cell communication. Exosomes secreted by host cells or tumor cells are involved in cell growth, proliferation, metabolism, and regulation.

[0004] N-acetylcysteine (NAC) is produced by the acetylation of L-cysteine, and its precursor cysteine (Cys) is a common polar α-amino acid containing a sulfhydryl group in the body. It is a non-essential amino acid that can be metabolically converted into glucose, can be produced by methionine metabolism in the human body, and can also be converted into cystine. The biological activity of N-acetylcysteine is mainly attributed to the sulfhydryl group (-SH) in its structure, and its acetyl group protects it from oxidation and metabolism, thus maintaining the persistence of its biological activity. And its intracellular metabolite glutathione (GSH) plays a great role in protecting cells.

[0005] Studies have shown that reactive oxygen species (ROS) are important factors in cell senescence and apoptosis, and can induce the transduction of cell senescence and apoptosis signals. Seon-Hee Oh et al. found that in cells with impaired oxidative function, the content of glutathione in them decreased sharply, resulting in the loss of intracellular redox function, and the cells began to age and undergo programmed death. If N-acetylcysteine is added, it can scavenge intracellular reactive oxygen species, reduce damage, metabolize to produce a large amount of glutathione, restore the intracellular redox function, and inhibit cell apoptosis. In addition, in the apoptosis experiment of endothelial cells, it was found that sodium arsenite (Ars) might induce the expression of intercellular adhesion molecule-1 (ICAM-1), causing cell apoptosis. After adding N-acetylcysteine (NAC), the content of sodium arsenite (Ars) in cells can be significantly reduced, the expression of intercellular adhesion molecules can be decreased, the intracellular oxidative environment can be changed, and the adhesion between cells can be reduced, thereby inhibiting cell apoptosis.

[0006] Quercetin, also known as quercetagetin and quercetin, is a natural flavonoid substance discovered by Hungarian physiologist Szent Gyorgyi Albert in 1936. Quercetin has rich biological activities, and the most remarkable one is its powerful antioxidant ability: quercetin can effectively scavenge free radicals, protect cells from oxidative damage; quercetin can protect cell DNA from oxidative damage, thus delaying cell senescence; at the same time, quercetin has an anti-inflammatory effect and can reduce the inflammatory response.

[0007] Oxygen concentration plays an important role in the processes of mesenchymal stem cell proliferation, differentiation, and self-renewal. However, under in vitro culture conditions, MSCs are usually exposed to normoxia (21% O2), which is very different from the oxygen concentration in the body under natural physiological conditions. In fact, a large part of MSCs exist in a hypoxic (2% - 8% O2 or even lower) environment in the body. Some studies have shown that exosomes are isolated from MSCs cultured under hypoxic conditions, and these exosomes are grown in a medium similar to peripheral artery disease (0% fetal bovine serum, 1% oxygen), and it is found that these exosomes contain many angiogenic factors, which may be beneficial to ischemic tissues. Another study found that after hypoxia induction, exosomes derived from bone marrow mesenchymal stem cells showed increased vascularization, reduced cardiomyocyte apoptosis rate, and increased recruitment of cardiac progenitor cells. Therefore, on the premise of hypoxic induction of MSCs, their biological functions and activities can be significantly enhanced, thereby improving the transplantation treatment effect of MSCs and their secreted exosomes in disease models. However, in current studies, the extraction of exosomes under hypoxic conditions usually may adopt the method of directly reducing the oxygen concentration, that is, directly reducing the partial pressure of oxygen from 21% to less than 5%. This will not only have an adverse impact on cell growth, such as: ① Suddenly reducing the partial pressure of oxygen will trigger an acute oxidative stress response in MSCs, resulting in an instant increase in the level of reactive oxygen species (ROS); ② Suddenly reducing the partial pressure of oxygen may trigger hypoxic apoptosis or necrosis of MSCs; ③ Rapidly reducing the partial pressure of oxygen may lead to changes in the pH value of the culture medium or accumulation of metabolites (such as lactic acid); thus, the composition of miRNAs, proteins, and lipids in exosomes may be abnormally changed, affecting their specific functions.

[0008] Although exosomes derived from MSCs have many advantages such as promoting angiogenesis, promoting cell proliferation, growth, migration, etc., the role of exosomes derived from MSCs cultured by conventional methods in cell anti-aging regulation is not obvious at present. At the same time, some related studies have proved that N-acetylcysteine can inhibit the senescence and apoptosis of T cells during the regulation of immune cells, but it has not been applied to the anti-aging effect of exosomes produced by MSC cells, nor is there a study on the combination of N-acetylcysteine and quercetin in cell anti-aging. Summary of the Invention

[0009] In view of this, the present invention aims to provide a method for preparing mesenchymal stem cell exosomes and its application. In the culture medium used for culturing MSC cells, N-acetylcysteine and quercetin are added to stimulate MSC to produce exosomes with anti-aging and anti-apoptotic effects. Moreover, by gradually reducing the oxygen partial pressure for culturing, the exosomes produced by MSC are further endowed with anti-aging efficacy. After the cells are induced and cultured with N-acetylcysteine and quercetin, it can inhibit the adverse effects such as acute oxidative stress, decreased proliferation ability and unstable quality under hypoxic conditions without damaging the activity of cell and exosome-related components. This method shows good anti-aging and anti-apoptotic effects in vitro, providing a new idea for the future application of exosomes in the treatment of aging diseases.

[0010] To achieve the above object, the technical solution of the present invention is realized as follows:

[0011] A method for preparing mesenchymal stem cell exosomes, the method comprising the following steps:

[0012] S1. Primary culture:

[0013] The umbilical cord mesenchymal stem cells are subjected to primary culture using a serum-free complete medium to obtain P0-generation cells;

[0014] S2. Acclimation culture:

[0015] N-acetylcysteine and quercetin are added to the serum-free complete medium to prepare an acclimation culture medium, and the P0-generation cells are passaged and cultured to P5-generation using the acclimation culture medium;

[0016] S3. Gradual hypoxic partial pressure separation and extraction of mesenchymal stem cell exosomes:

[0017] The P5-generation mesenchymal stem cells are continuously acclimation cultured until the cell confluence > 80%, then replaced with a serum-free basal medium, and the culture flask is placed in an incubator containing 5Vt% CO2 and 15Vt% O2 and cultured at 37°C for 24 hours. Thereafter, the O2 content is decreased by 4 - 9Vt% every 24h, and adjusted 2 - 3 times. The supernatant is aspirated and subjected to gradient centrifugation for extraction to obtain exosomes.

[0018] Adding N-acetylcysteine during the MSC culture process can cause changes in the MSC microenvironment, thereby increasing the expression levels of certain microRNAs (miRNAs) that regulate cellular senescence levels in MSCs, such as miR-21, miR-146a, etc. These miRNAs can be taken up by exosomes produced by the cells, generating exosome particles with anti-aging levels. Additionally, the added quercetin can regulate the gene expression of MSCs, altering the composition and function of miRNAs in the exosomes they secrete, enabling the exosomes to potentially carry more autophagy-related functional proteins or miRNAs (such as miR-100), promoting autophagic activity in recipient cells, and enhancing their ability to cope with aging damage.

[0019] In some embodiments, S1 specifically includes the following steps:

[0020] 1) Use 0.9% saline containing 1% volume ratio of penicillin and 1% volume ratio of streptomycin as the tissue cleaning solution to clean the surface of the placenta to remove surface congestion, cut the umbilical cord and remove the internal blood vessels;

[0021] 2) Peel the Wharton's jelly in the umbilical cord, clean it with the tissue cleaning solution, cut it into small pieces of 2 - 3 mm 3 and transfer them to a T75 culture flask, 1 mL per flask;

[0022] 3) Slowly add 10 mL of serum-free complete medium (serum-free basal medium containing 2% volume ratio of human platelet lysate) to the culture flask, gently shake the culture flask to make the umbilical cord tissue pieces spread flat in the culture flask, and statically culture it in an incubator at 37°C and 5% CO2. Replenish the medium once every 2 - 3 days (add 3 - 4 mL of serum-free complete medium each time), and after replenishing 2 - 3 times in total, remove the tissue pieces and completely replace the serum-free complete medium and continue to culture until the cell confluence reaches 70%, and then digest the obtained cells;

[0023] 4) Aspirate and discard the medium in the culture flask, aspirate 20 mL of PBS to rinse the culture flask to remove the residual medium, add 3 mL of recombinant trypsin solution for digestion, gently tap the culture flask to make the cells fall off to the bottom of the culture flask, add 10 mL of MSC serum-free complete medium to neutralize the trypsin to terminate the digestion process, disperse the cells with a pipette, collect the cell suspension in a centrifuge tube, centrifuge and discard the supernatant to obtain a cell pellet, which is recorded as the cells of the P0 passage.

[0024] In some embodiments, the concentration of N-acetylcysteine is 3 - 7 mmol / L, and the concentration of quercetin is 1 - 3 μmol / L.

[0025] Preferably, the concentration of N-acetylcysteine is 5 mmol / L, and the concentration of quercetin is 1 μmol / L.

[0026] In some embodiments, in step S3, after the last oxygen content adjustment, the oxygen content in the environment is controlled at 1-5 Vt%.

[0027] In some embodiments, in step S3, the O2 content is adjusted twice: after the first 24 hours of cultivation, the oxygen content is adjusted to 7-8 Vt%, and cultivation continues for 24 hours; after 24 hours, the oxygen content is adjusted to 2-3 Vt%, and cultivation continues for 24 hours; the total cultivation time is 72 hours.

[0028] In some embodiments, in step S2, the seeding density of MSC cells for each subculture is 1×10 4 cells / cm 2 , and they are cultivated in an incubator at 37°C and 5 Vt% CO2, and the culture medium is changed every 48 hours.

[0029] In some embodiments, in step S3, the seeding density of MSC cells is 1×10 4 cells / cm 2 .

[0030] The present invention also provides an application of the mesenchymal stem cell exosomes prepared by the above-mentioned preparation method in cell anti-aging.

[0031] In some embodiments, the addition amount of exosomes is 2.5×10 8 particles / mL - 2.5×10 9 particles / mL.

[0032] Compared with the prior art, the preparation method of the mesenchymal stem cell exosomes of the present invention has the following advantages:

[0033] In the preparation method of the mesenchymal stem cell exosomes of the present invention, N-acetylcysteine and quercetin are added to the culture medium used for culturing MSC cells, so as to stimulate MSC to produce exosomes with anti-aging and anti-apoptotic effects. And by gradually reducing the oxygen partial pressure for cultivation, the exosomes produced by stimulating MSC further have anti-aging effects. And after the cells are induced and cultured with N-acetylcysteine and quercetin, it can inhibit the adverse effects such as acute oxidative stress, decreased proliferation ability and unstable quality under hypoxic conditions without damaging the activity of cells and exosome-related components. This method shows good anti-aging and anti-apoptotic effects in vitro, providing a new idea for the future treatment of senile diseases with exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1 It is the electron microscopy image of exosomes;

[0036] Figure 2 It is the particle size graph of exosomes;

[0037] Figure 3 It is the detection of surface membrane proteins CD81 and CD9 of exosomes by flow cytometry. (a) is surface membrane protein CD81, and (b) is surface membrane protein CD9;

[0038] Figure 4 It is the detection of intracellular proteins of exosomes by Western Blot;

[0039] Figure 5 It is the establishment of an IMR-90 cell senescence model using D-galactose. (a) is 0 mg / mL galactose, (b) is 20 mg / mL galactose, (c) is 40 mg / mL galactose, and (d) is 80 mg / mL galactose;

[0040] Figure 6 It is the result of ROS staining in the oxidative stress experiment. (a) is the negative group, (b) is the positive group, (c) is the normal group, (d) is the Min group, (e) is the Max group, (f) is the normoxia group, (g) is the single quercetin group, (h) is the single acetyl group, (i) is the factor group, (j) is the one-step hypoxia group, and (k) is this patent;

[0041] Figure 7 It is the expression result of p16 cell cycle inhibitory protein;

[0042] Figure 8 It is the expression result of p53 cell cycle inhibitory protein. Detailed implementation manners

[0043] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] Example 1 Preparation of exosomes

[0046] The preparation method includes the following steps:

[0047] S1. Primary culture of umbilical cord mesenchymal stem cells

[0048] S11. Place items such as a stainless-steel tray, filter, stainless-steel cup, lunch box, tissue cleaning solution (0.9% normal saline containing 1% penicillin and 1% streptomycin), and pipette in appropriate positions in the biosafety cabinet. Use surgical forceps to take out the placental tissue and place it in the stainless-steel tray. Clean the surface of the placenta with the tissue cleaning solution to remove the congestion on the placenta surface. Use surgical scissors and forceps to cut the umbilical cord at the placental connection, and cut the other end behind the umbilical cord sterile clip. Take out the umbilical cord, wash it 3 times with the tissue cleaning solution to remove the surface congestion and blood clots. Hold one end of the umbilical cord with forceps, and use another pair of forceps to hold the umbilical cord and slide along the umbilical cord tissue to remove the residual umbilical blood in the blood vessels. First, cut the umbilical cord into several segments 2 - 3 cm long, then cut it along the longitudinal section of the umbilical cord with scissors, flatten it, remove the 3 blood vessels inside (2 umbilical arteries and 1 umbilical vein), and transfer it to a new petri dish.

[0049] S12. Use forceps to peel off the Wharton's jelly and place it in a petri dish containing the tissue cleaning solution. Wash the Wharton's jelly with the tissue cleaning solution and tear it into long strips. After processing, transfer the Wharton's jelly to a 50 mL centrifuge tube with forceps and cut it into small pieces 2 - 3 mm 3 in size with scissors. Transfer 1 mL to each T75 culture flask with a 3 mL sterile dropper. The tip of the dropper needs to be cut off with sterile scissors before use.

[0050] S13. Slowly add 10 mL of serum-free complete medium (serum-free basal medium containing 2% human platelet lysate, the serum-free basal medium is MSC NutriStem® XF Basal Medium from Biological industries, product number 05 - 200 - 1A) to the culture flask, and gently shake the culture flask to make the umbilical cord tissue pieces spread flat in the culture flask. Place the T75 culture flask in an incubator at 37°C and 5% CO2 for static culture. Take out the culture flask from the incubator on the third day of culture, and supplement 3.5 mL of serum-free complete medium. Then, replenish the medium once every 3 days. After replenishing the medium 3 times, cells will crawl out from the bottom of the culture flask. Remove the tissue pieces and replace all the serum-free complete medium and continue the culture. When the cell confluence reaches 70%, the obtained cells will be digested next.

[0051] S14. Before subculture of cells, equilibrate recombinant trypsin, serum-free complete medium, and PBS at room temperature. Take the culture flask out of the incubator, aspirate the medium in the culture flask with a pipette, and pipette 20 mL of PBS to wash the culture flask to remove the residual medium. Add 3 mL of recombinant trypsin solution to the culture flask for digestion, gently shake the culture flask to make the trypsin fully cover the cell surface, digest for about 1 min until the cells shrink into round shapes under the microscope, gently tap the culture flask to make the cells fall off like quicksand to the bottom of the culture flask, add 10 mL of serum-free complete medium to neutralize the trypsin and terminate the digestion process, disperse the cells with a pipette, and collect the cell suspension in a 15 mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 1400 rpm for 5 min, then remove the supernatant. The precipitated cells are designated as P0 generation cells.

[0052] S2. Domestication of umbilical cord mesenchymal stem cells

[0053] S21. In serum-free complete medium, add 5 mmol / L of N-acetylcysteine and 1 μmol / L of quercetin to prepare the domestication medium.

[0054] S22. Resuspend the P0 generation cells with 10 mL of the domestication medium, perform cell counting, and then inoculate them into a T75 culture flask at an inoculation density of 1×10 4 MSC cells / cm 2 Add the above domestication medium to 30 mL, and statically culture in an incubator at 37 °C and 5% CO2. Replace the fresh domestication medium every 48 hours until the cell confluence reaches 80%. Aspirate the medium in the culture flask, pipette 20 mL of PBS to wash the culture flask to remove the residual medium, add 3 mL of recombinant trypsin solution for digestion, gently tap the culture flask to make the cells fall off to the bottom of the culture flask, add 10 mL of the domestication medium to neutralize the trypsin and terminate the digestion process, disperse the cells with a pipette, collect the cell suspension in a centrifuge tube, centrifuge and discard the supernatant to obtain the cell precipitate designated as P1 generation cells.

[0055] S23. To ensure that the mesenchymal stem cells can be fully and continuously nourished by the domestication medium, amplify, digest, and subculture the P1 generation cells in the above manner until the cell passage number reaches P5 generation.

[0056] S3. Gradual separation and extraction of mesenchymal stem cell exosomes under low oxygen partial pressure

[0057] S31. Resuspend the P5 generation cells with 10 mL of the domestication medium, perform cell counting, and then inoculate them into a T75 culture flask at an inoculation density of 1×10 4 MSC cells / cm 2Inoculate into a T75 culture flask, supplement the above-mentioned acclimation medium to 30 mL, and statically culture in a 37 °C, 5% CO₂ incubator. Replace the fresh acclimation medium every 48 hours until the cell confluence > 80%.

[0058] S32. Take out the culture flask, aspirate and discard the medium, wash the culture flask 3 times with PBS, then add 30 mL of serum-free basal medium (MSC NutriStem® XF Basal Medium from Biological industries, catalog number 05-200-1A, without 2% human platelet lysate) to the flask, and then place the culture flask in a 37 °C, 15 Vt % O₂, 5 Vt % CO₂ incubator for 24 hours. Then adjust the oxygen concentration to 7.5 Vt % and continue culturing for 24 hours. After 24 hours, adjust the oxygen concentration to 2 Vt % and continue culturing for 24 hours; after a total of 72 hours of culturing, collect the cell supernatant into a 50 mL centrifuge tube.

[0059] S33. Centrifuge the centrifuge tube at 300 g, 4 °C for 10 minutes to remove dead cells and large cell debris; then aspirate the supernatant into a new 50 mL centrifuge tube.

[0060] S34. Centrifuge the centrifuge tube at 2000 g, 4 °C for 20 minutes to further remove impurities such as cell debris; then aspirate the supernatant into a high-speed centrifuge tube (Beckman Coulter).

[0061] S35. Place the centrifuge tube in a high-speed centrifuge and centrifuge at 10000 g, 4 °C for 30 minutes to further remove smaller cell debris and impurities; then aspirate the supernatant into a 50 mL centrifuge tube.

[0062] S36. Filter the supernatant with a 0.22 μm PES filter membrane (millipore), place the filtered supernatant in an ultracentrifuge tube (Beckman Coulter), balance it, and then put it into an ultracentrifuge.

[0063] S37. Centrifuge the filtered supernatant at 100000 g, 4 °C for 90 minutes. After centrifugation, aspirate and discard the supernatant, resuspend it with 20 mL of sterile PBS, and repeatedly pipette the bottom of the ultracentrifuge tube to resuspend the exosome precipitate at the bottom; balance it and put it into the ultracentrifuge again for 100000 g, 4 °C, 90 minutes of centrifugation. After centrifugation, discard the supernatant, resuspend it with 1 mL of sterile PBS, repeatedly pipette the bottom of the centrifuge tube, and then store the extracted exosome suspension at -80 °C.

[0064] Experiment 1 Transmission electron microscopy observation and particle size detection of exosome particles

[0065] The exosomes obtained in Example 1 were observed as follows:

[0066] 1. Take 50 μl of the purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and fix it in a refrigerator at 4°C for 1 h;

[0067] 2. Drop 20 μl of the fixed exosome suspension onto the front of the copper grid and let it stand for 20 min;

[0068] 3. Carefully suck off the excess solution with absorbent filter paper; then wash the copper grid 5 times with ultrapure water for 30 seconds each time, and dry it with filter paper;

[0069] 4. Drop 1 drop of 2% uranyl acetate staining solution onto the front of the copper grid, stain for 1 min, and then suck off the excess staining solution along the edge of the copper grid with filter paper;

[0070] 5. Place the copper grid in the air at room temperature to dry naturally, and wait until it is dry before observing it on the machine.

[0071] 6. Dilute 50 μl of the purified exosome precipitate 100 - 500 times with PBS, and take 50 μl of the sample for detecting the particle size and peak value of the exosomes with a nano particle size analyzer.

[0072] As Figure 1 the morphology of the exosomes could be observed. The exosomes had a complete envelope under the electron microscope and had a double - layer lipid membrane structure. The morphology of the exosomes presented a typical "tea - saucer - like" structure. At the same time, nano particle size analysis was performed on the exosomes, and the results were as Figure 2 shown. It could be seen from the figure that the exosomes had peak shapes at 81 nm and 110 nm in particle size, and the number of exosome particles with a particle size less than 200 nm accounted for about 99.9% of the total number of particles.

[0073] Experiment 2 Detection of the surface protein expression of exosome particles by flow cytometry

[0074] The surface protein expression of the exosomes obtained in Example 1 was detected as follows:

[0075] 1. Invert and mix the components of the Thermofisher CD63 magnetic beads (product number 10606D) for 10 min; aspirate 20 μl of the magnetic bead suspension into a 1.5 mL round - bottom EP tube;

[0076] 2. Add 200 μl of magnetic bead washing solution to the EP tube and mix well with a pipette tip;

[0077] 3. Place the EP tube on the magnetic rack for 1 min; then aspirate and discard the supernatant;

[0078] 4. Take 50 μl of the extracted exosome suspension, add 50 μl of washing solution to a final volume of 100 μl, and mix well.

[0079] 5. Place the exosome-washing solution mixture on a rotary mixer, set the rotation speed to 10 rpm, and incubate at 2 - 8°C overnight with rotation.

[0080] 6. The next day, centrifuge the sample rapidly for 3 - 5 seconds to collect the precipitate.

[0081] 7. Add 300 μl of washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0082] 8. Place the sample on a magnetic stand for about 1 minute, and aspirate the supernatant.

[0083] 9. Add 400 μl of washing solution to the sample and mix well with a pipette tip for 30 seconds.

[0084] 10. Place the sample on a magnetic stand for about 1 minute, aspirate the supernatant, and resuspend with 300 μl of washing solution.

[0085] 11. Take 100 μl of each sample, add CD9-PE and CD81-FITC flow antibodies to the samples respectively, and incubate at 4°C in the dark for 30 minutes.

[0086] 12. Place the incubated sample on a magnetic stand for about 1 minute, aspirate the supernatant, and add 300 μl of washing solution for washing.

[0087] 13. After repeating step 12 once, resuspend the sample with 300 μl of PB and then detect it on the machine.

[0088] The test results are as Figure 3 shown. It can be seen from the figure that when labeling exosome particles with CD81-PE antibody, the proportion of exosome particles with positive CD81 expression detected is 97.53%; when labeling exosome particles with CD9-PE antibody, the proportion of exosome particles with positive CD9 expression detected is 95.51%.

[0089] Experiment 3 Detection of Intracellular Proteins in Exosomes

[0090] Perform detection of intracellular proteins in the exosomes obtained in Example 1. The steps are as follows:

[0091] 1. Protein quantification: Quantitatively detect the exosome protein according to the QubitTM Protein Assay Kit instructions.

[0092] 2. Sample preparation: Mix the exosome sample with Loading buffer, then place it in a 100°C water bath for 10 minutes to denature the protein. After cooling, centrifuge.

[0093] 3. Prepare the precast gel: Place the ExoressPAGE Gels precast gel in the electrophoresis tank, add pre-cooled Running Buffer to the electrophoresis tank, and gently pull out the comb with both hands.

[0094] 4. Loading: Load 20 - 70 μL into each well and record the loading order.

[0095] 5. Electrophoresis: 80 v for the upper gel for 30 min; 110 v for the lower gel for 120 min. After electrophoresis, rinse the gel with 1X PBS for 2 min, and then soak it in Transfer buffer for equilibration.

[0096] 6. Soak the PVDF membrane in 100% ethanol for 10 min, then soak it in ddH2O for 5 min, and finally soak it in Transfer buffer for equilibration for 20 min.

[0097] 7. Blotting: Perform semi-dry blotting for 30 min on the Trans-Blot® Turbo™ Transfer System, with the blotting condition of 25 V / 1.3 mA / cm 2 , and after blotting, soak the membrane in PBS for 3 - 5 min.

[0098] 8. Blocking: Place the blotted membrane in 20 ml of blocking solution and incubate it on a shaker at room temperature for 0.5 - 1 h.

[0099] 9. Washing the membrane: Wash the membrane with 1X PBST (1000 ml 1×PBS + 1 ml Tween-20) for 5 - 10 min, and wash it four times.

[0100] 10. Incubating with primary antibody: Dilute the primary antibody (1:1000) according to the antibody instruction manual, incubate it overnight at 4°C on a shaker. Recover the primary antibody solution and rinse the membrane with 1X PBST 5 times, 5 - 10 min each time. The primary antibody is rabbit anti-human antibody, and the antibodies are: CD63, CD81, CD9, Alix, Calnexin, Flot1 in sequence.

[0101] 11. Incubating with secondary antibody: Add the secondary antibody (1:3000), incubate it at 4°C on a shaker for 0.5 - 1 h. Recover the secondary antibody solution, and use 1×PBST. The secondary antibody is mouse anti-rabbit antibody.

[0102] 12. Rinse the membrane 5 times, 5 - 10 min each time.

[0103] 13. Chemiluminescent gel imaging: Add ECL luminescent solution for luminescence reaction for 2 min, and image it in a chemiluminescent imaging system.

[0104] Figure 4 The results of Western Blot detection showed that bands of intracellular proteins Alix and Flot1 of the extracted exosome particles appeared, and the protein sizes were in line with the expected levels. Exosomes expressed transmembrane proteins CD63, CD9, and CD81, so bands of these several proteins all appeared, and the sizes were in line with the expected expression. Exosomes did not express the Calnexin protein, so there was no band for this protein.

[0105] Experiment 4 Comparison of different preparation methods

[0106] In order to verify the influence of the culture method on the anti-aging function of exosomes, the following experiment was designed:

[0107] 1. Exosomes of the control group (hereinafter referred to as the ordinary group):

[0108] On the basis of Example 1, the acclimation medium used in step S2 was completely changed to a serum-free complete medium, and the others were the same as in Example 1, and finally exosomes were prepared.

[0109] 2. Exosomes of the low-dose group (hereinafter referred to as the Min group):

[0110] On the basis of Example 1, the N-acetylcysteine added in the acclimation medium used in step S2 was 1 mmol / L, and the added quercetin was 0.5 μmol / L, and the others were the same as in Example 1, and finally exosomes were prepared.

[0111] 3. Exosomes of the high-dose group (hereinafter referred to as the Max group):

[0112] On the basis of Example 1, the N-acetylcysteine added in the acclimation medium used in step S2 was 10 mmol / L, and the added quercetin was 5 μmol / L, and the others were the same as in Example 1, and finally exosomes were prepared.

[0113] 4. N-acetylcysteine exosomes (hereinafter referred to as the mono-acetyl group):

[0114] On the basis of Example 1, quercetin was not added to the acclimation medium used in step S2, and the others were the same as in Example 1, and finally exosomes were prepared.

[0115] 5. Quercetin exosomes (hereinafter referred to as the mono-quercetin group):

[0116] On the basis of Example 1, N-acetylcysteine was not added to the acclimation medium used in step S2, and the others were the same as in Example 1, and finally exosomes were prepared.

[0117] 6. Normoxia group:

[0118] Based on Example 1, in step S3, mesenchymal stem cell exosomes are isolated and extracted under normal oxygen partial pressure:

[0119] Domesticate and culture the P5 generation of mesenchymal stem cells according to step S31 of Example 1 until the cell confluence > 80%, then replace it with a serum-free basal medium. Place the culture flask in an incubator at 37°C, 5% CO2, and 21% O2 for 72 hours, and obtain exosomes after 72 hours.

[0120] 7. One-step hypoxic group:

[0121] Based on Example 1, in step S3, mesenchymal stem cell exosomes are isolated and extracted using one-step hypoxic partial pressure:

[0122] Domesticate and culture the P5 generation of mesenchymal stem cells according to step S31 of Example 1 until the cell confluence > 80%, then replace it with a serum-free basal medium. Place the culture flask in an incubator at 37°C, 5% CO2, and 2% O2 for 72 hours, and obtain exosomes after 72 hours.

[0123] Experiment 5. Research on the anti-aging effect of exosomes at the in vitro cell level

[0124] 1. Establish an IMR-90 cell senescence model using D-galactose

[0125] 1) Take 2.5 g of D-galactose powder, fully dissolve it with 34.5 mL of sterile PBS, and aliquot it into 1 mL EP tubes, so that the D-galactose concentration in each tube is 80 mg / mL.

[0126] 2) Resuscitate the IMR-90 cell line in a 37°C water bath, and inoculate it into several 6-well plates according to the seeding density of 2×10 4 / cm 2 , add 2 mL of DMEM medium containing 10% FBS, and change the fresh medium every 2 - 3 days until the IMR-90 cell confluence ≥ 80%. Add 0 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL of D-galactose to the medium respectively and culture for 24 hours.

[0127] 3) Use the SA-β-galactosidase labeling method to detect the phenotypic changes of cell senescence under different concentrations of D-galactose to screen for a suitable D-galactose concentration.

[0128] SA-β-gal is a typical marker in senescent cells, and the senescence state of cells can be detected by changes in β-galactosidase activity.

[0129] The steps are as follows:

[0130] I. After aspirating the culture medium supernatant in each well after modeling, wash the cells 2 - 3 times with PBS, and then fix the cells with 4% paraformaldehyde for 15 - 30 minutes.

[0131] II. Stain the cells with the staining solution (usually containing X-gal substrate) in the SA-β-galactosidase kit. The staining process needs to be carried out at 37 °C for 12 hours. During the staining process, β-galactosidase will convert X-gal into a blue product, and the blue cells are senescent cells. After staining, wash away the unbound dye with PBS. Observe the cells under a microscope and record the number and morphology of the blue-stained cells.

[0132] III. Calculate the number of senescent cells induced by different concentrations of D-galactose in IMR-90 cells. The well with the largest number of senescent cells is the optimal D-galactose induction concentration.

[0133] As Figure 5 shown, the senescence of cells at different concentrations. It can be seen from the figure that the best cell senescence model is obtained with 80 mg / mL galactose.

[0134] 2. Detection of the inhibitory effect of exosomes on the senescence of IMR-90 cells by oxidative stress experiment

[0135] Oxidative Stress refers to a state in which excessive reactive oxygen species (ROS) are produced inside cells, exceeding the antioxidant defense ability of the cells, thereby triggering a series of cell damage and senescence.

[0136] Establish a cell senescence model using D-galactose, add the exosomes obtained by the method of this patent and the exosomes obtained in each group of Experiment 4 to the cells during modeling, and set up a control group at the same time to observe the antioxidant stress effect of each group of exosomes on the process of cell senescence.

[0137] The method is as follows:

[0138] 1) Select 80 mg / mL D-galactose to induce senescence of IMR-90 cells for 24 hours, and design the experimental group and the control group as follows:

[0139] The positive control group (hereinafter referred to as the positive group) is IMR-90 cells after 24 hours of D-galactose induction;

[0140] The negative control group (hereinafter referred to as the negative group) is IMR-90 cells without senescence induction;

[0141] The experimental group is IMR-90 cells after 24 hours of D-galactose induction and adding 1×10 9Exosomes in each group of Experiment 4 (including normal group, Min group, Max group, monoacetyl group, monoquercetin group, normoxia group and one-step hypoxia group) per mL of particles;

[0142] The positive control only-addition factor group (hereinafter referred to as the factor group) was that only N-acetylcysteine and quercetin at the concentration described in Example 1 were added to IMR-90 cells after being induced by D-galactose for 24 hours, and no exosomes were added.

[0143] And 2 mL of DEME medium containing 10% FBS was added to each well. The well plate was placed in an incubator at 37°C with 5% CO2 for 24 hours.

[0144] 2) After 24 hours, the well plate was taken out, the supernatant medium was aspirated and discarded, and then the cells in each group were stained using a ROS detection kit (Shanghai Zhuocai Biotechnology Co., Ltd., product number: ZC-A4108), and the change degree of intracellular reactive oxygen species was observed. When the cells showed green fluorescence, it indicated that the cells had a higher content of reactive oxygen species components and the cells were in a senescent state.

[0145] The results were as Figure 6 shown. It could be seen from the figure that in the ROS oxidative stress experiment, by observing IMR-90 cells in each group under a fluorescence microscope and evaluating the degree of cell senescence by comparing the fluorescence intensity, it was found that the exosomes prepared by the method of the present patent group had the strongest antioxidant and anti-aging effects on cells, almost the same as the negative group, that is, no fluorescence was visible to the naked eye, and different intensities of fluorescence were generated in other groups.

[0146] 3. Detection of the inhibitory effect of exosomes on IMR-90 cell senescence by cyclin experiments

[0147] Senescence can cause an increase in cyclin inhibitors P16 and P21, leading to cell cycle arrest. The expression levels of cyclin inhibitors were detected by the Elisa method to illustrate the anti-aging effect of exosomes.

[0148] The operation method was as follows:

[0149] IMR-90 cells were induced to senesce by 80 mg / mL D-galactose for 24 hours to establish a model, and the experimental groups and control groups were designed as follows:

[0150] The positive group was IMR-90 cells after being induced by D-galactose for 24 hours;

[0151] The negative group was IMR-90 cells without senescence induction;

[0152] The experimental group was IMR-90 cells after being induced by D-galactose for 24 hours and adding 1×10 9Exosomes in each group of Experiment 4 (including the normal group, Min group, Max group, mono-acetyl group, mono-quercetin group, normoxia group, and stepwise hypoxia group) per mL of particles;

[0153] In the factor group, only N-acetylcysteine and quercetin at the concentrations described in Example 1 were added to IMR-90 cells after 24 hours of D-galactose induction, and no exosomes were added.

[0154] Then, 2 mL of DEME medium containing 10% FBS was added to each well, and 2 parallel wells were set for each group. The well plates were placed in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, the cell supernatant was collected, and an Elisa kit (Shanghai Zhuocai Biotechnology Co., Ltd., product numbers: ZC-32275, ZC-32019) was used to detect the changes in the expression levels of the inflammation factors p21 and p16 proteins associated with senescence.

[0155] The results are as Figure 7-8 shown. The group in this patent is Example 1. Compared with other methods, the method described in the present invention (i.e., Example 1) has the least content of the cyclin inhibitors p16 and p21 released by cells, indicating that the method described in the present invention has the best anti-aging effect on cells.

[0156] At the same time, adding only the two factors without adding exosomes (factor group) and adding unmodified exosomes (normal group) have a worse anti-aging effect on cells than the group in this patent, indicating that it is not simply the two factors N-acetylcysteine and quercetin that play a role in the anti-aging level of cells, nor is it the exosomes of unmodified MSCs, but the exosomes after being modified by the two factors. Adding only one factor, that is, adding only N-acetylcysteine (mono-acetyl group) or only quercetin (mono-quercetin group), also does not have a better anti-aging level on cells than the present invention, indicating that the two factors described in this patent must exist simultaneously and the exosomes produced after modifying MSCs can achieve the best anti-aging effect on cells. After reducing the addition concentration of the two factors (such as the Min group), the anti-aging effect of exosomes on cells is worse than the method described in this patent. After increasing the addition concentration of the two factors (Max group), there is no significant improvement in the anti-aging effect on cells. Therefore, it is considered that the concentration of the two factors used in the present invention is the optimal concentration range for modifying MSCs.

[0157] In addition, it can be seen from the attached drawings that when the two factors coexist and the use concentrations are the same, the exosomes obtained by the stepwise hypoxia method have more advantages in the cell anti-aging level than the exosomes obtained by the normoxia group and the stepwise hypoxia group, indicating that the exosomes obtained by the stepwise hypoxia method are also more beneficial to the anti-aging effect of cells.

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The method includes the following steps: S1. Primary culture: Primary culture of umbilical cord mesenchymal stem cells is carried out using serum-free complete medium to obtain P0 generation cells; S2. Acclimation culture: N-acetylcysteine and quercetin are added to the serum-free complete medium to prepare an acclimation medium, and the P0 generation cells are passaged and cultured to P5 generation using the acclimation medium; S3. Gradual separation and extraction of mesenchymal stem cell exosomes under low oxygen partial pressure: The P5 generation of mesenchymal stem cells is continuously acclimation cultured until the cell confluence > 80%, then replaced with serum-free basal medium. The culture flask is placed in an incubator containing 5Vt% CO2 and 15Vt% O2 and cultured at 37°C for 24 hours. After that, the O2 content is decreased by 4 - 9Vt% every 24h, adjusted 2 - 3 times, and the supernatant is aspirated for gradient centrifugation to extract exosomes.

2. The preparation method of the mesenchymal stem cell exosomes according to claim 1, wherein: The concentration of N-acetylcysteine is 3 - 7 mmol / L, and the concentration of quercetin is 1 - 3 μmol / L.

3. The preparation method of the mesenchymal stem cell exosomes according to claim 1, characterized in that: In step S3, after the last oxygen content adjustment, the oxygen content in the environment is controlled at 1 - 5Vt%.

4. The preparation method of the mesenchymal stem cell exosomes according to claim 3, characterized in that: In step S3, the O2 content is adjusted twice: after the first 24-hour culture, the oxygen content is adjusted to 7 - 8 Vt%, and the culture is continued for 24 hours; after 24 hours, the oxygen content is adjusted to 2 - 3 Vt %, and the culture is continued for 24 hours; the total culture time is 72 hours.

5. The preparation method of the mesenchymal stem cell exosomes according to claim 1, characterized in that: In step S2, the seeding density of MSC cells for each subculture is 1×10 4 cells / cm 2 , and they are cultured in an incubator at 37 °C and 5% CO2. The culture medium is changed every 48 hours.

6. The preparation method of the mesenchymal stem cell exosomes according to claim 1, characterized in that: In step S3, the MSC cell seeding density is 1×10 4 cells / cm 2 .

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