Method for efficiently separating and culturing mesenchymal stem cells

Abstract

The invention provides a method for efficiently separating and culturing mesenchymal stem cells. The method comprises the steps of separating adipose-derived mesenchymal stem cells, culturing the cells and the like. In the steps of adipose tissue treatment and cell separation, a specific digestive enzyme mixed solution is used for digestion treatment, so that the adipose tissue is fully digested, the cell damage is reduced to the greatest extent, the separation of adipose cells is promoted to the greatest extent, and the yield of the cells is remarkably improved. For cell culture, a serum-free culture medium is innovatively adopted. The formula of the culture medium is elaborately blended, a DMEM-F12 culture medium is used as a matrix, and platelet lysate, human serum albumin, recombinant insulin, EGF, bFGF, special oligopeptide and other components in a specific proportion are added. The components have a synergistic effect, so that the potential risk caused by serum is avoided, a suitable growth environment is provided for cells, the growth and proliferation of the cells are promoted, and the undifferentiated state of the cells is maintained.

Description

Technical Field

[0001] The invention relates to the technical field of stem cells and provides a method for efficiently separating and culturing mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) have the potential for self-renewal and multidirectional differentiation, and have broad application prospects in the fields of regenerative medicine and treatment of autoimmune diseases. At present, the sources of mesenchymal stem cells mainly include bone marrow, fat, umbilical cord and other tissues.

[0003] Traditional methods for isolating and culturing mesenchymal stem cells often have problems such as low cell yield, greater cell damage, and low cell purity. For example, some enzymatic digestion methods may excessively damage cell surface markers, affecting the subsequent functions and applications of the cells. During the culture process, it is common practice to use culture media containing serum, but serum may contain pathogens, immunogenic substances, and factors of unclear composition, which not only increases the risk of cell culture, but may also lead to unstable cell characteristics. In addition, existing culture methods also have shortcomings in terms of cell expansion efficiency, cell viability, and cell stability. Low cell expansion efficiency may not be able to meet the needs of clinical and scientific research for a large number of cells, and low cell viability affects the function and therapeutic effect of the cells; and poor cell stability may cause cells to lose their original characteristics and differentiation potential during long-term culture.

[0004] In summary, the existing mesenchymal stem cell isolation and culture technology has many shortcomings, which limits its wide application in clinical and scientific research. Therefore, it is urgent to develop an efficient isolation and culture method to improve the yield and quality of mesenchymal stem cells to meet the needs of clinical and scientific research. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for efficiently isolating and culturing mesenchymal stem cells.

[0006] The scheme of the present invention includes the following core contents:

[0007] A method for efficiently isolating and culturing mesenchymal stem cells comprises the following steps:

[0008] (1) Isolation of adipose-derived mesenchymal stem cells

[0009] The fat sample is centrifuged to remove fat and retain the fat tissue and cell pellet; a digestive enzyme mixture is added to digest and the digestion is terminated; the sample is centrifuged, the supernatant is discarded, the cell pellet is resuspended in the culture medium, and filtered to obtain primary adipose-derived mesenchymal stem cells;

[0010] The digestive enzyme mixture comprises type I collagenase, type II collagenase, neutral protease and polyphenol polymer;

[0011] The preparation method of the polyphenol polymer comprises: 1) adding polyvinyl pyrrolidone to an ethanol solution, adjusting the pH to 8.0-8.5 with sodium hydroxide to obtain a solution A; 2) taking a tannic acid aqueous solution, adding it to the solution A, stirring, then adding a glyoxal solution, and continuing to stir;

[0012] (2) Cell culture

[0013] The primary adipose-derived mesenchymal stem cells were inoculated into a culture bottle, primary cell culture medium was added, and culture was performed to harvest the P1 adipose-derived mesenchymal stem cells;

[0014] The primary cell culture medium is based on DMEM-F12 medium, supplemented with platelet lysate, human serum albumin, glutamine, epidermal growth factor EGF, basic fibroblast growth factor bFGF and short peptide YLWIMR.

[0015] Preferably, the digestive enzyme mixture comprises, by mass concentration, 0.05% to 0.1% type I collagenase, 0.05% to 0.1% type II collagenase, 0.02% to 0.05% neutral protease and 0.2% to 0.5% polyphenol polymer.

[0016] Preferably, the digestive enzyme mixture comprises, by mass concentration, 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer.

[0017] Preferably, the volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:6-7:0.3-0.5.

[0018] Preferably, the volume ratio of tannic acid solution, solution A, and glyoxal solution is 1:6:0.3.

[0019] Preferably, the preparation method of the polyphenol polymer comprises: 1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% to 50% according to a solid-liquid ratio of 5 to 6 mg: 1 mL, and adjusting the pH to 8.0 to 8.5 with sodium hydroxide to obtain solution A; (2) taking a tannic acid aqueous solution with a mass concentration of 0.025 to 0.055 g / mL, adding it to solution A, stirring, and then adding a glyoxal solution with a mass concentration of 2.5% to 3.5%, and continuing to stir for at least 12 hours.

[0020] Preferably, the primary cell culture medium uses DMEM-F12 culture medium as the basal culture medium, and is supplemented with 1% to 2% platelet lysate, 1% to 2% human serum albumin, 4 to 6 mM glutamine, 1 to 2 μg / mL epidermal growth factor EGF, 2 to 4 μg / mL basic fibroblast growth factor bFGF, and 1% to 5% short peptide YLWIMR.

[0021] Preferably, the primary cell culture medium uses DMEM-F12 culture medium as the basal culture medium, and is supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

[0022] Further explanation: The short peptide of the present invention, such as the short peptide YLWIMR, can be synthesized by conventional synthesis methods, such as conventional liquid phase synthesis or solid phase synthesis. The present invention has no particular limitation on the synthesis method of the short peptide.

[0023] The present invention also relates to cultured mesenchymal stem cells obtained by the method for efficiently separating and culturing mesenchymal stem cells.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. High cell yield

[0026] A large number of adipose-derived mesenchymal stem cells can be obtained from adipose tissue by using an optimized digestive enzyme mixture and a specific culture medium. The digestive enzyme mixture can significantly increase the number of nucleated cells harvested, and the complete culture medium can further increase the cell yield during the passage process.

[0027] 2. High cell viability

[0028] During the entire separation and culture process, the cell viability can be maintained at a high level (above 95%), ensuring the quality of the cells.

[0029] 3. Reduce serum dependence

[0030] The use of serum-free culture medium reduces the potential risks brought by serum, such as immunogenicity and viral contamination.

[0031] 4. Efficient training system

[0032] The entire process from fat sample processing to cell subculture has been optimized, the operation is relatively simple, and it can be completed in a relatively short time, which is conducive to timely clinical treatment. DETAILED DESCRIPTION

[0033] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0034] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0036] Example 1: Method for efficiently isolating and culturing mesenchymal stem cells

[0037] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0038] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 7.2×10 8 The living cells were counted by trypan blue staining and the cell viability was calculated. The cell viability was above 96%.

[0039] The digestive enzyme mixture (in terms of mass percentage concentration) contains 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer, and is prepared with D-Hanks solution.

[0040] The preparation method of the polyphenol polymer is as follows: (1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% according to a solid-liquid ratio of 5 mg:1 mL, adjusting the pH to 8.0-8.5 with sodium hydroxide, and obtaining solution A. (2) taking a tannic acid aqueous solution with a concentration of 0.025 g / mL, adding it to the solution obtained in the above step (1), stirring, and then adding a glyoxal solution with a mass concentration of 2.5%, and continuing to stir for 12 hours. The volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:6:0.3.

[0041] 2. Primary cell culture and subculture

[0042] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 6.8×10 7 cells / cm 2.

[0043] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

[0044] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0045] 3. Cell Detection and Identification

[0046] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0047] By the 10th generation, the morphological activity, proliferation ability and flow cytometry phenotype of each generation of cells were stable. The cell viability was detected by trypan blue staining, and the cell viability of each generation was above 95%. They could be induced to differentiate into cells such as adipocytes, osteoblasts and chondrocytes.

[0048] Example 2 Method for efficiently isolating and culturing mesenchymal stem cells

[0049] The difference from Example 1 is that the content of the ingredients in the digestive enzyme mixture and the content of the ingredients in the cell culture medium are slightly changed.

[0050] The specific method is as follows:

[0051] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0052] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 6.8×10 8 The living cells were counted by trypan blue staining and the cell viability was calculated. The cell viability was above 96%.

[0053] The digestive enzyme mixture (in terms of mass percentage concentration) comprises 0.1% type I collagenase, 0.1% type II collagenase, 0.05% neutral protease and 0.5% polyphenol polymer, and is prepared with D-Hanks solution.

[0054] The preparation method of the polyphenol polymer is as follows: (1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% according to a solid-liquid ratio of 5 mg:1 mL, adjusting the pH to 8.0-8.5 with sodium hydroxide, and obtaining solution A. (2) taking a 0.025 g / mL tannic acid aqueous solution, adding it to the solution obtained in the above step (1), stirring, and then adding a glyoxal solution with a mass concentration of 2.5%, and continuing to stir for 12 hours. The volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:6:0.3.

[0055] 2. Primary cell culture and subculture

[0056] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 6.9×10 7 cells / cm 2 .

[0057] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 2% platelet lysate, 2% human serum albumin, 6 mM glutamine, 2 μg / mL epidermal growth factor EGF, 4 μg / mL basic fibroblast growth factor bFGF, and 5% short peptide YLWIMR.

[0058] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0059] 3. Cell Detection and Identification

[0060] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected. These markers were negative (expression rate less than 2%).

[0061] By the 10th generation, the morphological activity, proliferation ability and flow cytometry phenotype of each generation of cells were stable. The cell viability was detected by trypan blue staining, and the cell viability of each generation was above 95%.

[0062] Example 3 Method for efficiently isolating and culturing mesenchymal stem cells

[0063] The difference from Example 1 is that the preparation method of the polyphenol polymer is slightly changed. The specific method is as follows:

[0064] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0065] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the lower layer of cell pellet. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm for 30 minutes at 37°C to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet with DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 7.1×108 The living cells were counted by trypan blue staining and the cell viability was calculated. The cell viability was above 96%.

[0066] The digestive enzyme mixture (in terms of mass percentage concentration) contains 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer, and is prepared with D-Hanks solution.

[0067] The preparation method of the polyphenol polymer is as follows: (1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 50% according to a solid-liquid ratio of 6 mg:1 mL, adjusting the pH to 8.0-8.5 with sodium hydroxide, and obtaining solution A. (2) taking a tannic acid aqueous solution with a concentration of 0.055 g / mL, adding it to the solution obtained in the above step (1), stirring, and then adding a glyoxal solution with a mass concentration of 3.5%, and continuing to stir for 12 hours. The volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:7:0.5.

[0068] 2. Primary cell culture and subculture

[0069] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 7.0×10 7 cells / cm 2 .

[0070] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

[0071] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0072] 3. Cell Detection and Identification

[0073] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0074] By the 10th generation, the morphological activity, proliferation ability and flow cytometry phenotype of each generation of cells were stable. The cell viability was detected by trypan blue staining, and the cell viability of each generation was above 95%. They could be induced to differentiate into cells such as adipocytes, osteoblasts and chondrocytes.

[0075] Comparative Example 1: Method for isolating and culturing mesenchymal stem cells

[0076] The digestive enzyme mixture of this comparative example does not contain polyphenol polymer, and its amount is replaced by D-Hanks solution.

[0077] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0078] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 4.3×10 7 The living cells were counted by trypan blue staining and the cell viability was calculated, which was 92%.

[0079] The digestive enzyme mixture (by mass concentration) contains 0.05% type I collagenase, 0.05% type II collagenase, and 0.02% neutral protease, and is prepared with D-Hanks solution.

[0080] 2. Primary cell culture and subculture

[0081] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 6.7×10 7 cells / cm 2 .

[0082] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

[0083] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0084] 3. Cell Detection and Identification

[0085] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0086] By the 10th generation, the cell viability of each generation was above 95%.

[0087] Comparative Example 2: Method for isolating and culturing mesenchymal stem cells

[0088] The digestive enzyme mixture of this comparative example does not contain polyphenol polymers, but is replaced by tannic acid.

[0089] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0090] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 5.7×10 7 The living cells were counted by trypan blue staining and the cell viability was calculated, which was 91%.

[0091] The digestive enzyme mixture (in terms of mass percentage concentration) contains 0.1% type I collagenase, 0.1% type II collagenase, 0.05% neutral protease and 0.5% tannic acid, and is prepared with D-Hanks solution.

[0092] 2. Primary cell culture and subculture

[0093] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 6.7×10 7 cells / cm 2 .

[0094] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

[0095] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0096] 3. Cell Detection and Identification

[0097] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0098] By the 10th generation, the cell viability of each generation was 92% to 95%.

[0099] Comparative Example 3: Method for isolating and culturing mesenchymal stem cells

[0100] The short peptide added to the culture medium of this comparative example is ERWDCW

[0101] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0102] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 7.2×10 8 The living cells were counted by trypan blue staining and the cell viability was calculated. The cell viability was above 96%.

[0103] The digestive enzyme mixture (by mass concentration) comprises 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer, and is prepared with D-Hanks solution.

[0104] The preparation method of the polyphenol polymer is as follows: (1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% according to a solid-liquid ratio of 5 mg:1 mL, adjusting the pH to 8.0-8.5 with sodium hydroxide, and obtaining solution A. (2) taking a tannic acid aqueous solution with a concentration of 0.025 g / mL, adding it to the solution obtained in the above step (1), stirring, and then adding a glyoxal solution with a mass concentration of 2.5%, and continuing to stir for 12 hours. The volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:6:0.3.

[0105] 2. Primary cell culture and subculture

[0106] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 4.8×10 7 cells / cm 2 .

[0107] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide ERWDCW.

[0108] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0109] 3. Cell Detection and Identification

[0110] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0111] At the 10th generation, the cell viability was detected by trypan blue staining, and the cell viability of each generation was 85% to 92%.

[0112] Comparative Example 4: Method for efficiently isolating and culturing mesenchymal stem cells

[0113] The culture medium of this comparative example does not contain any short peptide, and the amount of the short peptide is replaced by DMEM-F12 culture medium.

[0114] 1. Isolation of Adipose-derived Mesenchymal Stem Cells

[0115] 150 mL of fat sample was obtained from the abdomen of the volunteer through liposuction surgery, placed in a sterile container, and transported to the laboratory under cold chain conditions at 4°C. The fat sample was gently rinsed with D-Hanks solution to remove impurities such as blood. The rinsed fat sample was transferred to a centrifuge tube and centrifuged at 100×g for 3 to 5 minutes to promote obvious stratification of the adipose tissue. Carefully remove the upper layer of fat, retaining the middle adipose tissue and the cell pellet in the lower layer. Add an equal volume of digestive enzyme mixture and oscillate and digest at 100rpm at 37°C for 30 minutes to fully digest the adipose tissue into single cells. After digestion, add an equal volume of DMEM-F12 medium to terminate digestion. Subsequently, centrifuge at 1500rpm for 5 minutes, discard the supernatant, and resuspend the cell pellet in DMEM-F12 medium. Filter with a 100μm filter to separate the primary adipose mesenchymal stem cells. Sampling was performed and cells were counted using a cell counting plate, and a total of 7.2×10 8 The living cells were counted by trypan blue staining and the cell viability was calculated. The cell viability was above 96%.

[0116] The digestive enzyme mixture (by mass concentration) comprises 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer, and is prepared with D-Hanks solution.

[0117] The preparation method of the polyphenol polymer is as follows: (1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% according to a solid-liquid ratio of 5 mg:1 mL, adjusting the pH to 8.0-8.5 with sodium hydroxide, and obtaining solution A. (2) taking a tannic acid aqueous solution with a concentration of 0.025 g / mL, adding it to the solution obtained in the above step (1), stirring, and then adding a glyoxal solution with a mass concentration of 2.5%, and continuing to stir for 12 hours. The volume ratio of the tannic acid solution, solution A, and glyoxal solution is 1:6:0.3.

[0118] 2. Primary cell culture and subculture

[0119] The isolated primary adipose-derived mesenchymal stem cells were cultured at 3000 cells / cm 2 The cells were inoculated into T75 culture flasks, primary cell culture medium was added, and culture was carried out in a CO incubator containing 5% CO, 37°C, and saturated humidity. After 3 days of culture, the medium was completely replaced once, and culture was continued for 5 days. Adipose-derived mesenchymal stem cells of generation P1 were harvested to obtain 5.0×10 7 cells / cm2 .

[0120] The primary cell culture medium is based on DMEM-F12 medium, supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, and 2 μg / mL basic fibroblast growth factor bFGF.

[0121] The P1 generation adipose-derived mesenchymal stem cells are taken for subculture. When the cell confluence reaches 80%-90% again, the next subculture can be performed to obtain P2, P3, and other generations of adipose-derived mesenchymal stem cells.

[0122] 3. Cell Detection and Identification

[0123] During the culture process, the cell morphology was observed regularly under a microscope. The mesenchymal stem cells were spindle-shaped or polygonal and grew adherently. Flow cytometry was used to identify the immunophenotype of the cells, and the expression of cell surface markers CD73, CD90, and CD105 were detected. All three were positive (expression rate greater than 98%); at the same time, the expression of CD19, CD11b, CD31, CD45, HLADR, and CD34 were detected, and the results were negative.

[0124] At the 10th generation, the cell viability was detected by trypan blue staining, and the cell viability of each generation was 89% to 85%.

[0125] The separation and culture effects of the above embodiments and comparative examples are summarized as follows:

[0126] Table 1

[0127] The number of primary cells isolated Number of cells in P1 generation Cell viability from P1 to P10 Example 1 <![CDATA[7.2×10 8 cells]]> <![CDATA[6.8×10 7 cells / cm 2 ]]> More than 95% Example 2 <![CDATA[6.8×10 8 cells]]> <![CDATA[6.9×10 7 cells / cm 2 ]]> More than 95% Example 3 <![CDATA[7.1×10 8 cells]]> <![CDATA[7.0×10 7 cells / cm 2 ]]> More than 95% Comparative Example 1 <![CDATA[4.3×10 7 cells]]> <![CDATA[6.7×10 7 cells / cm 2 ]]> More than 95% Comparative Example 2 <![CDATA[5.7×10 7 cells]]> <![CDATA[6.7×10 7 cells / cm 2 ]]> 92％～95％ Comparative Example 3 <![CDATA[7.2×10 8 cells]]> <![CDATA[4.8×10 7 cells / cm 2 ]]> 85％～92％ Comparative Example 4 <![CDATA[7.2×10 8 cells]]> <![CDATA[5.0×10 7 cells / cm 2 ]]> 89％～85％

[0128] By comparing the above examples and comparative examples, it can be found that the addition of polyphenol polymers effectively promotes the separation of adipose mesenchymal stem cells, and the addition of specific short peptides effectively promotes the growth and cell viability of passaged cells. Comparative Examples 3 and 4 also show that unreasonable use of short peptides, such as replacing YLWIMR with ERWDCW, is not conducive to cell growth.

[0129] On the whole, the present invention can not only increase the number of primary cells obtained by separation, but also has a fast passaging speed, rapid cell growth and high cell viability.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficiently isolating and culturing mesenchymal stem cells, characterized in that: The following steps are involved: (1) Isolation of adipose-derived mesenchymal stem cells The fat sample is centrifuged to remove the fat and retain the fat tissue and cell pellet; a digestive enzyme mixture is added to digest and the digestion is terminated; Centrifuge, discard the supernatant, resuspend the cell pellet in culture medium, filter, and obtain primary adipose-derived mesenchymal stem cells; The digestive enzyme mixture comprises type I collagenase, type II collagenase, neutral protease and polyphenol polymer; The preparation method of the polyphenol polymer comprises: 1) adding polyvinyl pyrrolidone to an ethanol solution and adjusting the pH to 8.0-8.5 to obtain a solution A; 2) Take tannic acid aqueous solution, add it to solution A, stir, then add glyoxal solution, and continue stirring; (2) Cell culture The primary adipose-derived mesenchymal stem cells were inoculated into a culture bottle, primary cell culture medium was added, and culture was performed to harvest the P1 adipose-derived mesenchymal stem cells; The primary cell culture medium is based on DMEM-F12 medium, supplemented with platelet lysate, human serum albumin, glutamine, epidermal growth factor EGF, basic fibroblast growth factor bFGF and short peptide YLWIMR.

2. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: Measured by mass concentration, the digestive enzyme mixture contains 0.05% to 0.1% type I collagenase, 0.05% to 0.1% type II collagenase, 0.02% to 0.05% neutral protease and 0.2% to 0.5% polyphenol polymer.

3. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: In terms of mass concentration, the digestive enzyme mixture contains 0.05% type I collagenase, 0.05% type II collagenase, 0.02% neutral protease and 0.2% polyphenol polymer.

4. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: The volume ratio of tannic acid solution, solution A and glyoxal solution is 1:6-7:0.3-0.

5.

5. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: The volume ratio of tannic acid solution, solution A, and glyoxal solution is 1:6:0.

3.

6. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: The preparation method of the polyphenol polymer comprises: 1) adding polyvinyl pyrrolidone to an ethanol solution with a volume concentration of 25% to 50% according to a solid-liquid ratio of 5 to 6 mg: 1 mL, adjusting the pH to 8.0 to 8.5, and obtaining a solution A; 2) taking a tannic acid aqueous solution with a concentration of 0.025 to 0.055 g / mL, adding it to the solution A, stirring, and then adding a glyoxal solution with a mass concentration of 2.5% to 3.5%, and continuing to stir for at least 12 hours.

7. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: The primary cell culture medium is based on DMEM-F12 medium, and is supplemented with 1% to 2% platelet lysate, 1% to 2% human serum albumin, 4 to 6 mM glutamine, 1 to 2 μg / mL epidermal growth factor EGF, 2 to 4 μg / mL basic fibroblast growth factor bFGF, and 1% to 5% short peptide YLWIMR.

8. The method for efficiently isolating and culturing mesenchymal stem cells according to claim 1, characterized in that: The primary cell culture medium is based on DMEM-F12 medium, and is supplemented with 1% platelet lysate, 1% human serum albumin, 4 mM glutamine, 1 μg / mL epidermal growth factor EGF, 2 μg / mL basic fibroblast growth factor bFGF, and 1% short peptide YLWIMR.

9. Cultured mesenchymal stem cells obtained by the method for efficiently isolating and culturing mesenchymal stem cells according to any one of claims 1 to 8.