Preparation method of mesenchymal stem cell exosome

Abstract

The invention relates to a preparation method of mesenchymal stem cell exosomes, which comprises the following steps: S10, culturing mesenchymal stem cells by using a cell culture medium containing human umbilical cord matrigel and a platelet lysis buffer to obtain a cell culture solution; s20, performing differential centrifugation on the cell culture fluid, and removing cells, cell debris and apoptotic bodies in the cell culture fluid to obtain supernate; s30, carrying out tangential flow ultrafiltration on the supernatant to obtain an ultrafiltrate; and S40, centrifuging and filtering the ultrafiltrate to obtain a high-content and high-purity exosome solution. Compared with the prior art, the mesenchymal stem cells are cultured by adopting the cell culture medium added with the human umbilical cord matrigel, so that the proliferation of the mesenchymal stem cells is remarkably promoted, and the concentration of exosomes in the cell culture solution is improved; meanwhile, the exosome in the cell culture fluid is separated and purified by adopting a method of combining differential centrifugation with tangential flow ultrafiltration, so that the purity and the extraction rate of the exosome are effectively improved.

Description

Technical Field

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

[0002] Exosomes, as nanoscale extracellular vesicles secreted by cells, have unique structural characteristics and rich biological functions. The diameter of these vesicles is usually between 40nm and 100nm, and they contain nucleic acids (including DNA fragments, mRNA, and various non-coding RNAs such as microRNA), proteins, lipids, and other biologically active substances. Exosomes play a vital role in information transmission between cells, material exchange, and maintaining body homeostasis. In 1983, exosomes were first discovered in sheep reticulocytes. Subsequently, in 1987, Johnstone officially named them "exosomes", a term that is still widely used today. It is worth noting that the secretion of exosomes is not exclusive to a specific cell type, but is widely present in a variety of cell types, and can be produced in both normal physiological and pathological states. They are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal particles, and are finally released into the extracellular matrix after a series of complex biological processes, including the fusion of the multivesicular outer membrane with the cell membrane. In fact, almost all cultured cell types can secrete exosomes, and these vesicles also exist naturally in various body fluids, such as blood, saliva, urine, cerebrospinal fluid, and milk, providing a rich source for the research and application of exosomes.

[0003] In recent years, exosomes have attracted much attention from the academic and medical communities due to their potential as efficient carriers of microRNA (miRNA), disease biomarkers, and value as potential therapeutic targets. In particular, in the field of plastic surgery, exosomes derived from mesenchymal stem cells have been found to have a significant effect in promoting wound healing, providing new possibilities for cell-free therapy of skin regeneration. However, although exosomes have shown great application prospects in the biomedical field, the limitations of their extraction efficiency and purification technology have become bottlenecks restricting their clinical applications.

[0004] At present, exosomes are mainly extracted by ultracentrifugation, that is, the solution containing exosomes is filtered with a 0.22μm sterile filter membrane, the filtrate is centrifuged at 0-20℃, 50000-200000g for 30-120min, the supernatant is discarded, and the exosome precipitate is obtained. However, the ultracentrifugation method has the disadvantages of low extraction efficiency, low purity of exosomes, and high equipment requirements. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing mesenchymal stem cell exosomes.

[0006] The present invention is achieved through the following technical solution: a method for preparing mesenchymal stem cell exosomes, comprising the following steps:

[0007] S10, culturing mesenchymal stem cells using a cell culture medium containing human umbilical cord matrix gel and platelet lysate to obtain a cell culture medium;

[0008] S20, performing differential centrifugation on the cell culture fluid to remove cells, cell debris and apoptotic bodies in the cell culture fluid to obtain a supernatant;

[0009] S30, performing tangential flow ultrafiltration on the supernatant to obtain an ultrafiltrate;

[0010] S40, centrifuging and filtering the ultrafiltrate to obtain a high-content and high-purity exosome solution.

[0011] Compared with the prior art, the present invention uses a cell culture medium added with human umbilical cord matrix gel to culture mesenchymal stem cells, which significantly promotes the proliferation of mesenchymal stem cells and increases the concentration of exosomes in the cell culture fluid; at the same time, a differential centrifugation combined with tangential flow ultrafiltration method is used to separate and purify the exosomes in the cell culture fluid, effectively improving the purity and extraction rate of the exosomes.

[0012] Furthermore, in step S10, the cell culture medium contains human umbilical cord matrix gel, platelet lysate and mesenchymal stem cell serum-free culture medium in a volume ratio of 1-2:1:16-20.

[0013] Furthermore, the preparation method of the human umbilical cord matrix gel comprises the following steps: washing a fresh human umbilical cord with water, NaCl solution and distilled water in sequence, then degreasing and removing polysaccharides from the washed umbilical cord with a degreasing agent, guanidine hydrochloride solution and citric acid in sequence to obtain a degreased and depolysaccharide-free umbilical cord; then treating the degreased and depolysaccharide-free umbilical cord with a decellularized matrix with pancreatic enzyme to obtain a pure umbilical cord decellularized matrix; freeze-drying, crushing and sterilizing the umbilical cord decellularized matrix, then adding acetic acid solution and pepsin to hydrolyze, centrifuging to obtain a supernatant, and salting out the supernatant at least twice, dissolving the precipitate after salting out with a hydrochloric acid aqueous solution to obtain a human umbilical cord matrix gel.

[0014] Furthermore, the tangential flow ultrafiltration in step S30 includes: passing the supernatant into a hollow fiber column ultrafilter, adding HEPES buffer for tangential flow ultrafiltration, and stopping ultrafiltration after the HEPES buffer completely replaces the solution of the supernatant to obtain an ultrafiltrate with an equal volume to the cell culture medium.

[0015] Furthermore, the molecular weight cut-off of the tangential flow ultrafiltration in step S30 is 100 KDa.

[0016] Furthermore, the differential centrifugation in step S20 includes: centrifuging the cell culture medium at 1000g for 5-15min to remove the cells, centrifuging the supernatant after the cells are removed at 4°C and 2000g for 20-40min to remove cell debris, and then centrifuging the supernatant after the cell debris are removed at 4°C and 10000g for 20-40min to remove large vesicles such as apoptotic bodies to obtain a supernatant.

[0017] Furthermore, the centrifugation in step S40 includes centrifugation at 4°C and 10,000 g for 20-40 min to obtain an exosome solution; and the filtration includes filtering the exosome solution with a 0.1 μm filter membrane to obtain a high-content and high-purity exosome solution.

[0018] Furthermore, the method further includes S50: freeze-drying the high-content and high-purity exosome solution to obtain exosome freeze-dried powder.

[0019] Furthermore, in step S50, the high-content and high-purity exosome solution is mixed with 40% trehalose solution and 5% mannitol solution in a volume ratio of 100:5:2, incubated for 20-40 minutes, and then freeze-dried.

[0020] The present invention also provides mesenchymal stem cell exosomes prepared by the above method.

[0021] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscopy (TEM) image of the exosomes prepared in Example 1, with a scale bar of 100 nm.

[0023] Figure 2 This is a transmission electron microscopy (TEM) image of the exosomes prepared in Example 1, with a scale bar of 200 nm.

[0024] Figure 3 Schematic diagram of the finished product of the lyophilized exosome powder prepared in Example 1.

[0025] Figure 4 This is a comparison chart of the marker proteins of exosomes and cells, where CL stands for cells and EV stands for exosomes.

[0026] Figure 5 This is the gel electrophoresis picture of Test Example 1, where 1 is the Marker, 2 is the exosomes prepared in Example 1, and 3 is the purchased exosome standard.

[0027] Figure 6 This is the Western blot picture of Test Example 1.

[0028] Figure 7 This is the particle size detection picture of exosomes in test example 2. DETAILED DESCRIPTION

[0029] The present invention further studies the problems of low extraction efficiency and low purity of exosomes in the prior art exosome preparation method, aiming to propose an efficient and high-purity method for preparing mesenchymal stem cell exosomes. The present invention finds that in the prior art, the concentration of exosomes in the cell culture medium used to prepare exosomes is usually low, and there are complex interactions between exosomes and other cell components (such as proteins, lipids, etc.), which makes separation difficult and directly affects the extraction efficiency of exosomes. In addition, when separating exosomes, the traditional ultracentrifugation method is prone to cause other nanoscale particles (such as protein aggregates, liposomes, etc.) to be precipitated together in addition to the target exosomes, thereby seriously reducing the purity of the exosomes.

[0030] Based on this, the present invention intends to optimize the preparation method of exosomes from two aspects:

[0031] First, the present invention intends to optimize the culture method of mesenchymal stem cells, and promote the proliferation and / or exosome secretion of mesenchymal stem cells by optimizing the culture conditions (such as culture medium components, culture time, etc.), thereby increasing the concentration of exosomes in the cell culture fluid. To this end, the present invention creatively introduces a special cell culture medium containing human umbilical cord matrix gel and platelet lysate, which significantly promotes the secretion of exosomes by mesenchymal stem cells and greatly increases the natural concentration of exosomes in the cell culture fluid.

[0032] Next, the present invention analyzes the traditional ultracentrifugation method and proposes a more sophisticated purification method based on its limitations: First, large impurities such as cells, debris and apoptotic bodies in the cell culture medium are effectively removed by differential centrifugation with a centrifugal force of less than or equal to 10,000 g to obtain a relatively clear supernatant. Subsequently, tangential flow ultrafiltration technology is introduced to separate and purify exosomes. This technology can gently and efficiently separate exosomes from other cellular components based on differences in molecular size and shape, avoiding the loss of exosomes and the decrease in purity that may be caused by traditional ultracentrifugation.

[0033] Based on the above analysis, the method for preparing mesenchymal stem cell exosomes provided by the present invention comprises the following steps:

[0034] S10, culturing mesenchymal stem cells using a cell culture medium containing human umbilical cord matrix gel and platelet lysate;

[0035] S20, performing differential centrifugation on the cell culture fluid to remove cells, cell debris, apoptotic bodies and other large vesicles in the cell culture fluid to obtain a supernatant;

[0036] S30, performing tangential flow ultrafiltration on the supernatant to obtain an ultrafiltrate;

[0037] S40, centrifuging and filtering the ultrafiltrate to obtain a high-content and high-purity exosome solution.

[0038] The cell culture medium is a mesenchymal stem cell serum-free culture medium purchased from Xirui Unlimited Biotechnology Co., Ltd. The preparation method of the human umbilical cord matrix gel is as follows:

[0039] The fresh human umbilical cord is washed thoroughly with water, NaCl solution and distilled water in sequence to obtain the washed umbilical cord; the washed umbilical cord is degreased and depolysaccharide-free with a degreasing agent, a guanidine hydrochloride solution and citric acid in sequence to obtain the degreased and depolysaccharide-free umbilical cord; the degreased and depolysaccharide-free umbilical cord is decellularized with pancreatic enzyme to obtain the umbilical cord decellularized matrix; the umbilical cord decellularized matrix is ​​freeze-dried, crushed and sterilized, and then sterile HAc solution (acetic acid solution) and pepsin are added to hydrolyze it, centrifuged to obtain a hydrolysis supernatant, and the hydrolysis supernatant is salted out at least twice, and the precipitate after salting out is dissolved with a hydrochloric acid aqueous solution with a pH of 2-3 to obtain human umbilical cord matrix glue.

[0040] In step S30, a hollow fiber column ultrafilter is used to perform tangential flow ultrafiltration.

[0041] The present invention is further described in detail below in conjunction with specific examples. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0042] Example 1

[0043] This embodiment provides a method for preparing mesenchymal stem cell exosomes, which specifically includes the following steps:

[0044] S10. Culturing mesenchymal stem cells using a cell culture medium containing human umbilical cord matrix gel and platelet lysate to obtain a cell culture medium, wherein the cell culture medium contains human umbilical cord matrix gel, platelet lysate and mesenchymal stem cell serum-free culture medium in a volume ratio of 1-2:1:16-20.

[0045] Specifically, under sterile conditions, 10 mL of human umbilical cord matrix gel was added to a 250 mL shake flask and 5×10 5hUMSC cells from the third to fifth generations were placed in a shake flask at 37°C and 5% CO2 for 30 min to form a gel. Then 100 mL of mesenchymal stem cell serum-free medium and 5% platelet lysate were added to the shake flask and blown evenly to disperse the gel into tiny droplets. The shake flask was then placed in a shake flask at 37°C and 5% CO2 for 4-5 days. The cell culture medium was obtained and counted. The number of cells was 2x10 7 Pieces / mL.

[0046] Wherein, the preparation method of the human umbilical cord matrix gel comprises the following steps:

[0047] S11, washing pretreatment: Take 50g of fresh human umbilical cord and wash it with water to remove blood and impurities on the surface of the umbilical cord to obtain a clean umbilical cord. Cut the clean umbilical cord into segments, place it in a 10% by mass NaCl solution, stir it at room temperature for 24 hours to remove impurities. Then use distilled water to fully wash the umbilical cord to remove impurities and remove the NaCl solution.

[0048] S12, degreasing and polysaccharide removal: The washed umbilical cord is mixed with a degreasing solution (the degreasing solution is an acetone-water solution, wherein acetone: water = 1:10 (V / V)) at a ratio of 1 g: 20 mL, and stirred at 4°C for 48 hours, and then rinsed with distilled water 3 to 5 times to remove the degreasing solution. The rinsed umbilical cord is mixed with a 4 mol / L guanidine hydrochloride solution at a ratio of 1 g: 10 mL, and stirred at 4°C for 48 hours, and then centrifuged at 12000×g for 30 minutes, and the precipitate is thoroughly washed 3 to 5 times with 0.1 mol / L citric acid to obtain the umbilical cord after degreasing and polysaccharide removal.

[0049] S13. Decellularization: The umbilical cord after defatting and polysaccharide removal was decellularized for 1 hour using 0.25% pancreatic enzyme EDTA solution, and then fully washed with distilled water to obtain a pure umbilical cord decellularized matrix.

[0050] S14, sterilization: freeze-drying and crushing the umbilical cord decellularized matrix, and sterilizing it with 10 kGy Co-60 to obtain a sterile umbilical cord decellularized matrix.

[0051] S15, extraction and purification: add 100mL of sterile 0.5M HAc solution to the sterile umbilical cord decellularized matrix, then add 100mg of sterile pepsin, hydrolyze at 4°C for 48h, centrifuge and remove the precipitate to obtain a supernatant solution, and salt out the supernatant with 0.7M sodium chloride, including adding 0.7M sodium chloride to the supernatant for salting out, then centrifuge and remove the supernatant to obtain a salting out precipitate. Redissolve the salting out precipitate with 0.1M HAc, and salt out the reconstituted solution twice with 0.7M sodium chloride to obtain a final salting out precipitate, and dissolve the final salting out precipitate with a hydrochloric acid aqueous solution of pH 2-3 to obtain human umbilical cord matrix glue.

[0052] S20, performing differential centrifugation on the cell culture fluid to remove cells, cell debris, apoptotic bodies and other large vesicles in the cell culture fluid to obtain a supernatant.

[0053] Specifically, 100 mL of cell culture fluid was centrifuged at 1000 g for 10 min to remove cells, the supernatant after the cells were removed was centrifuged at 4° C. and 2000 g for 30 min to remove cell debris, and then the supernatant after the cell debris was removed was centrifuged at 4° C. and 10000 g for 30 min to remove apoptotic bodies and other large vesicles to obtain a supernatant. The collection of cell culture fluid, precipitation and separation of supernatant were all performed under sterile conditions.

[0054] S30, performing tangential flow ultrafiltration on the supernatant to completely replace the supernatant with HEPES buffer to obtain an ultrafiltrate, wherein the molecular weight cutoff of the tangential flow ultrafiltration is 100 KDa.

[0055] Specifically, under sterile conditions, the supernatant is passed through a hollow fiber column ultrafilter with a molecular weight cutoff of 100 KDa, and HEPES buffer is added for tangential flow ultrafiltration. After the HEPES buffer completely replaces the solution of the supernatant, the ultrafiltration is stopped, and an ultrafiltrate with an equal volume to the cell culture medium is obtained. In this embodiment, the volume of the ultrafiltrate is 100 mL, wherein the flow rate of the tangential flow ultrafiltration is 5-6 mL / min, the volume of the HEPES buffer used for ultrafiltration is 6 V, and the time is 2-3 h.

[0056] The HEPES buffer solution is composed of 0.8 g / L NaCl and 20 mmol / L HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and the pH of the HEPES buffer solution is adjusted to 6.9-7.2 by 1 M NaOH, and then sterile filtered using a 0.22 um filter membrane to meet the use requirements.

[0057] In addition, before performing tangential flow ultrafiltration, the hollow fiber column ultrafilter was thoroughly washed with water to remove impurities, and then cleaned with 0.2-0.5M sodium hydroxide solution to remove organic matter and adjust the pH value. Finally, the hollow fiber column ultrafilter was rinsed with sterile water under a clean bench to ensure the sterility of the hollow fiber column ultrafilter.

[0058] S40, centrifuging and filtering the ultrafiltrate to obtain a high-content and high-purity exosome solution.

[0059] Specifically, the ultrafiltrate was centrifuged at 4°C and 10,000 g for 30 min to remove a small amount of residual apoptotic bodies and other large vesicles to obtain an exosome solution. The exosome solution was then filtered using a 0.1 μm filter membrane to remove a small amount of cell debris and bacteria to obtain about 100 mL of a high-content, high-purity exosome solution. The concentration of exosomes in the high-content, high-purity exosome solution was 7x10 9 Pieces / mL.

[0060] The structure of the obtained exosomes was observed by transmission electron microscopy, such as Figure 1-2 As shown, the obtained exosomes present a bowl-shaped structure under the microscope, and thus can be qualitatively identified as exosomes.

[0061] S50, freeze-drying the high-content and high-purity exosome solution to obtain exosome freeze-dried powder.

[0062] Specifically, the exosome solution was packaged under sterile conditions. In this embodiment, 6 mL of exosome solution was packaged in each molded bottle, 0.42 mL of lyophilization protective agent was added to each membrane bottle, mixed, incubated at 37°C for 30 min, placed in a -80°C refrigerator for pre-freezing for 12 h, and transferred to a vacuum freeze dryer for freeze-drying to obtain exosome lyophilized powder. Figure 3 As shown, the content of the lyophilized exosome powder in each molded bottle is greater than or equal to 40 billion. In other embodiments, the packaging amount of the exosome solution can be determined according to the specific volume of the membrane bottle.

[0063] The freeze-drying protective agent includes 40% trehalose solution sterilized by 0.22 μm filter membrane and 5% mannitol solution sterilized by 0.22 μm filter membrane in a volume ratio of 5:2.

[0064] Test Example 1

[0065] refer to Figure 4 According to the relevant literature, the marker proteins of exosomes include CD63, CD81, CD9 and Tsg101, while the marker proteins in cells do not contain CD63 and Tsg101. Therefore, the exosomes in Example 1 were identified by SDS-PAGE and Western blot, and the exosomes obtained in this example were run on gel, and Western blot was used to determine whether the marker proteins CD63, CD81, CD9 and Tsg101 of the exosomes could be run out, and further identification of whether the exosomes were obtained in Example 1 specifically included the following steps:

[0066] (1) Cleaning the glass plate: Use detergent to scrub the glass plate, then rinse with tap water and distilled water and dry it.

[0067] (2) Glue pouring: Align the glass plates and put them into the clamps to clamp them tightly, then clamp them vertically on the rack to prepare for glue pouring; prepare 12% separation gel, add TEMED and shake well immediately before pouring; after the separation gel solidifies, prepare 5% concentrated gel, add TEMED and shake well immediately before pouring; after the concentrated gel is filled, insert the comb into the concentrated gel, and after the concentrated gel solidifies, pinch the two sides of the comb with both hands and gently pull it out vertically upwards. Place the gel in the electrophoresis tank.

[0068] (3) Loading: Measure the protein content and calculate the volume of solution containing 50 ng of protein as the loading volume. Add 5× SDS loading buffer to the sample to a final concentration of 1×. Boil the sample in boiling water for 5 minutes before loading to denature the protein. After adding enough electrophoresis buffer, prepare for loading.

[0069] (4) Electrophoresis: The electrophoresis time is generally 1.5 hours, the voltage is 80V, 30 minutes, and then 120V, 50 minutes. The electrophoresis can be terminated when bromophenol blue just runs out, and the membrane can be transferred.

[0070] (5) After transfer, stain the membrane with bromophenol blue staining solution on a shaker for 40 minutes. Pour away the staining solution and add destaining solution on a shaker to destain until the background color is completely removed and the protein bands are visible.

[0071] Electrophoresis results refer to Figure 5 .

[0072] (6) The primary antibody for Western blot was monoclonal antibodies against exosome characteristic proteins CD63, CD81, and TSG101, as well as monoclonal antibodies against calregulin, a common impurity protein in exosomes. The secondary antibody was HRP-labeled IgG antibody.

[0073] like Figure 6 As shown, the exosomes prepared in this example contain the above-mentioned proteins.

[0074] Test Example 2

[0075] The particle size of the obtained exosomes was analyzed by nanoparticle tracking analyzer (NTA). Specifically, the high-content and high-purity exosome solution in step S40 was diluted with HEPES buffer to a particle concentration of 1×10 7 / mL and 1×10 9 / mL range. Then the nanoparticle tracking analyzer (NTA) was used for detection, and the analysis results were as follows Figure 7 As shown, the particle size of the exosomes prepared in this example is mainly distributed below 200 nm.

[0076] Comparative Example 1

[0077] Comparative Example 1 provides a method for preparing mesenchymal stem cell exosomes, comprising: S1, culturing mesenchymal stem cells; S2, preparing exosomes; wherein step S1 is similar to step S10 of Example 1. The method for preparing mesenchymal stem cell exosomes specifically comprises the following steps:

[0078] S1. Cultivation of mesenchymal stem cells: Under sterile conditions, add 10 mL of human umbilical cord matrix gel to a 250 mL shake flask and inoculate 5x10 5 hUMSC cells from the third to fifth generations were placed in a shake flask at 37°C and 5% CO2 for 30 min to form a gel. Then 100 mL of mesenchymal stem cell serum-free medium and 5% platelet lysate were added to the shake flask and blown evenly to disperse the gel into tiny droplets. The shake flask was then placed in a shake flask at 37°C and 5% CO2 for 4-5 days. The cell culture medium was obtained and counted. The number of cells was 2x10 7 Pieces / mL.

[0079] S2. Preparation of exosomes: 100 mL of cell culture medium was centrifuged at 1000 g for 10 min to remove cells, and the supernatant after removing cells was centrifuged at 4 ° C and 2000 g for 30 min to remove cell debris, and then the supernatant after removing cell debris was centrifuged at 4 ° C and 10000 g for 30 min to remove apoptotic bodies and other large vesicles to obtain supernatant, and then the supernatant was centrifuged at 4 ° C and 100000 g for 2 h, the supernatant was discarded, and the precipitate was re-dissolved with 100 mL PBS to obtain exosome solution. Among them, the collection of cell culture medium and the separation of precipitate and supernatant were all carried out under sterile conditions.

[0080] After testing, the content of exosomes in the exosome solution was 4x10 7 / mL. Compared with Example 1, the content of exosomes prepared by ultrahigh-speed centrifugation in Comparative Example 1 was significantly reduced. The reason may be that the shear force of ultrahigh-speed centrifugation is too high, which causes some exosome vesicles to rupture and agglomerate, resulting in exosome damage and a reduction in the content of effective exosomes. Secondly, compared with Example 1, the content of microvesicles larger than 400nm and apoptotic bodies in the exosome solution is higher, which reduces the purity of the exosomes.

[0081] Furthermore, the exosome solution was freeze-dried to obtain exosome freeze-dried powder. Specifically, under sterile conditions, 6 mL of exosome solution and 0.42 mL of freeze-dried protective agent were added to each molded bottle, mixed, incubated at 37°C for 30 min, placed in a -80°C refrigerator for pre-freezing for 12 h, and transferred to a vacuum freeze dryer for freeze-drying to obtain exosome freeze-dried powder. The content of exosome freeze-dried powder in each molded bottle was 2.4x10 8 Pieces / mL.

[0082] Comparative Example 2

[0083] Comparative Example 2 provides a method for culturing mesenchymal stem cells, which is similar to step S10 of Example 1, except that in this example, the amount of human umbilical cord matrix gel added is 2 mL:

[0084] Under sterile conditions, add 2 mL of human umbilical cord matrix gel to a 250 mL shake flask and inoculate 5x10 5 hUMSC cells from the third to fifth generations were placed in a shake flask at 37°C and 5% CO2 for 30 min to form a gel. Then 100 mL of mesenchymal stem cell serum-free medium and 5% platelet lysate were added to the shake flask and blown evenly to disperse the gel into tiny droplets. The shake flask was then placed in a shake flask at 37°C and 5% CO2 for 4-5 days. The cell culture medium was obtained and counted. The number of cells was 5x10 6 Pieces / mL.

[0085] Compared with step S10 of Example 1, it can be seen that the increase in the concentration of human umbilical cord matrix gel helps to promote the proliferation of mesenchymal stem cells, thereby helping to increase the content of exosomes in the cell culture medium.

[0086] Comparative Example 3

[0087] Comparative Example 3 provides a method for culturing mesenchymal stem cells, comprising the following steps:

[0088] Under sterile conditions, add 3 mL of mesenchymal stem cell complete medium to a 6-well plate and seed 2x10 5 The third to fifth generation hUMSC cells were evenly blown and placed in a 37°C carbon dioxide incubator for 36-72 hours. The cell culture medium was obtained and counted. The number of cells was 8x10 5 Pieces / mL.

[0089] Compared with step S10 of Example 1, it can be seen that the addition of human umbilical cord matrix gel and the use of shake flask suspension culture are helpful to promote the proliferation of mesenchymal stem cells, thereby helping to increase the content of exosomes in the cell culture medium.

[0090] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0091] (1) The present invention optimizes cell culture conditions and uses a cell culture medium containing human umbilical cord matrix gel and platelet lysate to culture mesenchymal stem cells. The umbilical cord matrix gel enables the mesenchymal stem cells to attach and grow better, thereby increasing the number of mesenchymal stem cells, thereby promoting the secretion of exosomes, increasing the concentration of exosomes in the cell culture medium, and promoting the exosomes to transmit information between cells, stimulate collagen production, and other cosmetic effects.

[0092] (2) The present invention adopts a purification method combining differential centrifugation and tangential flow ultrafiltration to extract exosomes from cell culture fluid: first, large cells, cell debris and apoptotic bodies are removed by differential centrifugation, and then tangential flow ultrafiltration is used to further separate exosomes and other cellular components, thereby effectively improving the purity and extraction rate of exosomes.

[0093] (3) The present invention avoids the use of excessively high centrifugal forces for the purification of exosomes, reduces the rupture and agglomeration of exosome vesicles, and thus protects the integrity of the exosomes. At the same time, the use of tangential flow ultrafiltration also provides a gentle and efficient separation method, further ensuring the quality and activity of the exosomes.

[0094] (4) By optimizing the culture conditions and purification methods, the present invention reduces the production cost of exosomes, making them more competitive in the market.

[0095] (5) Since the exosomes prepared by the present invention have high extraction efficiency and purity, they have broad application prospects in the fields of biomedicine, regenerative medicine, disease diagnosis and treatment. In particular, the exosomes prepared by the present invention are expected to play an important role in cancer treatment, tissue repair and regeneration, and drug delivery.

[0096] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A method for preparing mesenchymal stem cell exosomes, characterized in that: The steps include: S10, culturing mesenchymal stem cells using a cell culture medium containing human umbilical cord matrix gel and platelet lysate to obtain a cell culture medium; S20, performing differential centrifugation on the cell culture fluid to remove cells, cell debris and apoptotic bodies in the cell culture fluid to obtain a supernatant; S30, performing tangential flow ultrafiltration on the supernatant to obtain an ultrafiltrate; S40, centrifuging and filtering the ultrafiltrate to obtain a high-content and high-purity exosome solution.

2. The method according to claim 1, characterized in that: In step S10, the cell culture medium contains human umbilical cord matrix gel, platelet lysate and mesenchymal stem cell serum-free culture medium in a volume ratio of 1-2:1:16-20.

3. The method according to claim 2, characterized in that: The preparation method of the human umbilical cord matrix glue comprises: washing a fresh human umbilical cord with water, NaCl solution and distilled water in sequence to obtain a washed umbilical cord; degreasing and depolysaccharide-removing the washed umbilical cord with a degreasing agent, a guanidine hydrochloride solution and citric acid in sequence to obtain a degreasing and depolysaccharide-removing umbilical cord; performing a decellularized matrix treatment on the degreasing and depolysaccharide-removing umbilical cord with pancreatic enzyme to obtain an umbilical cord decellularized matrix; freeze-drying, crushing and sterilizing the umbilical cord decellularized matrix, then adding acetic acid solution and pepsin to hydrolyze it, centrifuging to obtain a hydrolysis supernatant, salting out the hydrolysis supernatant at least twice, dissolving the precipitate after salting out with a hydrochloric acid aqueous solution to obtain the human umbilical cord matrix glue.

4. The method according to claim 1, characterized in that: The tangential flow ultrafiltration in step S30 includes: passing the supernatant into a hollow fiber column ultrafilter, adding HEPES buffer for tangential flow ultrafiltration, and stopping ultrafiltration after the HEPES buffer completely replaces the solution of the supernatant to obtain an ultrafiltrate with an equal volume to the cell culture medium.

5. The method according to claim 4, characterized in that: The molecular weight cut-off of the tangential flow ultrafiltration in step S30 is 100 KDa.

6. The method according to any one of claims 1 to 5, characterized in that: The differential centrifugation in step S20 includes: centrifuging the cell culture medium at 1000g for 5-15 minutes to remove cells, centrifuging the supernatant after the cells are removed at 4°C and 2000g for 20-40 minutes to remove cell debris, and then centrifuging the supernatant after the cell debris are removed at 4°C and 10000g for 20-40 minutes to remove large vesicles such as apoptotic bodies to obtain a supernatant.

7. The method according to claim 6, characterized in that: The centrifugation in step S40 includes centrifugation at 4°C and 10,000 g for 20-40 min to obtain an exosome solution; the filtration includes filtering the exosome solution with a 0.1 μm filter membrane to obtain a high-content and high-purity exosome solution.

8. The method according to claim 7, characterized in that: The method further comprises S50: freeze-drying the high-content and high-purity exosome solution to obtain exosome freeze-dried powder.

9. The method according to claim 8, characterized in that: In step S50, the high-content and high-purity exosome solution is mixed with 40% trehalose solution and 5% mannitol solution in a volume ratio of 100:5:2, incubated for 20-40 minutes, and then freeze-dried.

10. Mesenchymal stem cell exosomes produced by the method according to any one of claims 1 to 9.

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