A mesenchymal stem cell culture and expansion medium and its preparation method

Abstract

The present invention relates to the technical field of cell culture, and specifically relates to a mesenchymal stem cell culture and amplification medium and a preparation method thereof. The mesenchymal stem cell culture and amplification medium uses DMEM / F12 medium as a basic component, and the DMEM / F12 medium further includes the following components: oxygen-releasing material 8 g / L, natural extract 2 g / L, growth factor 30 ng / mL, nicotinamide 5 mM, cholesterol 8 μg / mL, recombinant human transferrin 20 mg / L, recombinant human insulin 10 mg / L, vitamin C 4 mg / L, L-glutamine 2 mM, reduced glutathione 60 μg / L, sodium selenite 20 mg / L. The prepared mesenchymal stem cell culture and amplification medium is beneficial to the growth and proliferation of mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a culture medium for culturing and amplifying mesenchymal stem cells and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells with certain differentiation potential and can differentiate into various cell types such as skeletal muscle, cardiac muscle, adipose tissue, cartilage, and bone. When applied clinically, they can be used for the replacement of target cell populations and the repair of damaged and diseased tissues and organs. They have an immunomodulatory function and can play a role in immune reconstruction. The allogeneic transplantation rejection reaction is relatively mild, and the requirement for tissue matching is not strict, so they are widely used. Mesenchymal stem cells mainly exist in bone marrow, and can also be isolated and prepared from tissues such as adipose tissue, synovium, bone, muscle, lung, liver, pancreas, as well as amniotic fluid and umbilical cord blood. After isolating human mesenchymal stem cells, it is necessary to culture human mesenchymal stem cells to a sufficient amount for differentiation into the target tissue. It is also necessary to maintain the differentiation potential during the culture process. When culturing mesenchymal stem cells with a traditional cell culture medium (DMEM + 10% FBS), obvious senescence characteristics will appear in the cells at the third passage. In addition, the culture system of stem cells mainly uses a culture medium containing animal serum (such as fetal bovine serum). The heterologous contamination brought by animal serum and the contamination by known or unknown pathogens such as viruses and mycoplasmas. It is not known what changes will occur in the internal structure of the stem cells growing in such an environment, which brings potential risks to the clinical research of MSCs. The commercially available serum-free medium for MSCs on the market is expensive, and the cell growth cycle is short, the cell proliferation effect is not ideal, the cells age quickly, and the cell proliferation rate and multi-directional differentiation ability decline. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides a culture medium for culturing and amplifying mesenchymal stem cells and a preparation method thereof.

[0004] The present invention is achieved by the following technical solutions:

[0005] A culture medium for culturing and amplifying mesenchymal stem cells, with DMEM / F12 medium as the basic component, and the DMEM / F12 medium further includes the following components: oxygen-releasing material 8 g / L, natural extract 2 g / L, growth factor 30 ng / mL, nicotinamide 5 mM, cholesterol 8 μg / mL, recombinant human transferrin 20 mg / L, recombinant human insulin 10 mg / L, vitamin C 4 mg / L, L-glutamine 2 mM, reduced glutathione 60 μg / L, sodium selenite 20 mg / L.

[0006] Further, the growth factors are 10 ng / mL of basic fibroblast growth factor, 10 ng / mL of transforming growth factor-β, and 10 ng / mL of platelet-derived growth factor-BB.

[0007] Further, the preparation method of the oxygen slow-release material comprises the following steps:

[0008] (1) Prepare 0.1 mol / L aqueous sodium carbonate solution, 0.1 mol / L aqueous calcium chloride solution, 10 mmol / L aqueous sodium dodecyl sulfate solution, and 2 g / L aqueous tartaric acid solution; divide the aqueous tartaric acid solution into two equal volumes and pour them into the sodium carbonate solution and the calcium chloride solution respectively, stir at 60 °C and 200 - 300 rpm for 18 - 25 min to obtain mixture A and mixture B respectively;

[0009] (2) Pour the aqueous sodium dodecyl sulfate solution described in step (1) into mixture A obtained in step (1), react in a water bath at 80 °C for 20 - 30 min, add mixture B obtained in step (1), continue the water bath reaction for 0.8 - 1.2 h, keep the stirring speed at 200 r / min, centrifuge at 5000 rpm for 10 min to collect the precipitate, wash with deionized water and absolute ethanol, and dry at 60 °C to obtain porous nanoparticles;

[0010] (3) Place the porous nanoparticles prepared in step (2) in a reaction kettle and evacuate it. When the pressure in the reaction kettle is constant, stop evacuating, fill it with oxygen. When the pressure in the reaction kettle reaches 0.35 MPa, stop filling with oxygen to obtain oxygen-carrying porous nanoparticles;

[0011] (4) Add γ-cyclodextrin, malic acid, and sodium hypophosphite to deionized water, ultrasonicate at 300 - 400 W for 30 min, react at 100 °C for 3 h, react at 140 °C for 10 - 15 min, add distilled water and stir evenly, add absolute ethanol, let it stand at 4 °C for 3 - 4 h, centrifuge at 8000 rpm for 5 min, wash the precipitate with absolute ethanol, and dry it under vacuum at 50 °C to obtain malic acid-cyclodextrin;

[0012] (5) Add the malic acid-cyclodextrin, resveratrol, and sulfamic acid obtained in step (4) to benzene, stir at 100 rpm, heat and reflux at 100 - 110 °C for 5 - 6 h, filter while it is hot under vacuum, let the filtrate stand at 4 - 6 °C for 3 - 4 h, centrifuge at 5000 rpm for 15 - 20 min, recrystallize the precipitate with ethyl acetate, wash with absolute ethanol, and dry it under vacuum at 60 °C to obtain modified cyclodextrin;

[0013] (6) Add the modified cyclodextrin obtained in step (5) to deionized water, stir at 30 °C and 180 rpm for 30 min to obtain a modified cyclodextrin solution; add the modified cyclodextrin solution to the reaction kettle described in step (3), stir at 40 - 60 °C and 100 - 150 rpm for 2 - 3 h, let it stand at 4 °C for 2 h, centrifuge at 8000 - 10000 rpm for 5 - 8 min, wash the precipitate with absolute ethanol and deionized water, and dry it under vacuum to obtain the oxygen slow-release material.

[0014] Further, in step (1), the volume ratio of the prepared aqueous sodium carbonate solution, aqueous calcium chloride solution, aqueous sodium dodecyl sulfate solution and aqueous tartaric acid solution is 2:2:1:1.

[0015] Further, in step (4), the dosage ratio of γ-cyclodextrin, malic acid, sodium hypophosphite to deionized water is 1 g:1 g:0.6 - 0.8 g:20 mL.

[0016] Further, in step (4), the volume ratio of distilled water to deionized water is 1:4.

[0017] Further, in step (4), the volume ratio of the added absolute ethanol to distilled water is 10 - 12:1.

[0018] Further, in step (5), the dosage ratio of malic acid-cyclodextrin, resveratrol, sulfamic acid and benzene is 1 g:1.2 - 1.5 g:0.05 g:10 - 12 mL.

[0019] Further, in step (6), the dosage ratio of the modified cyclodextrin to deionized water is 1 g:5 - 7 mL.

[0020] Further, in step (6), the mass ratio of the modified cyclodextrin solution to the oxygen-carrying porous nanoparticles in the reaction kettle is 5:1.

[0021] Further, the preparation method of the natural extract includes the following steps: Dry astragalus membranaceus and dandelion at 100 °C, grind them through a 100-mesh sieve to obtain a mixed powder; take the mixed powder, add 10 times the weight of deionized water, heat and extract at 100 °C for 1 h, filter to obtain a filtrate, extract 3 times, combine the filtrates, concentrate at 60 °C and 10 -3 MPa to 20% of the original volume, add a 95 vol% ethanol solution until the final ethanol concentration reaches 60 wt%, let it stand at 4 °C for 6 - 8 h, centrifuge at 5000 rpm for 30 min, filter the supernatant through a 0.22 μm filter membrane, and freeze-dry at -20 °C for 20 - 24 h to obtain the natural extract.

[0022] Further, the mass ratio of dandelion to astragalus membranaceus is 1:1.

[0023] Furthermore, the present invention also provides a method for preparing the mesenchymal stem cell culture and expansion medium, comprising the following steps: filtering the DMEM / F12 medium through a 0.22 μm filter membrane, adding an oxygen slow-release material, a natural extract, a growth factor, niacinamide, cholesterol, recombinant human transferrin, recombinant human insulin, vitamin C, L-glutamine, reduced glutathione, and sodium selenite, and stirring at 180 rpm for 30 - 40 min to obtain the mesenchymal stem cell culture and expansion medium.

[0024] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, cord blood mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells or placental mesenchymal stem cells.

[0025] Furthermore, the mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention prepares an oxygen slow-release material with an oxygen slow-release function. Using the pores of porous nanoparticles as the oxygen carrier and pure oxygen as the oxygen source, no strongly oxidizing and alkaline substances will be generated during use, which is beneficial to the growth and reproduction of cells. At the same time, a coating layer is used to slowly release oxygen, and the dissolved oxygen added to the medium can maintain a certain level to continuously provide oxygen for the cells. After the oxygen slow-release material releases all the oxygen, the pores of the porous medium can effectively adsorb impurities in the culture medium body, reducing the risk of cell contamination. The oxygen slow-release material of the present invention uses modified cyclodextrin as the raw material for the coating layer. Cyclodextrin can be biodegradable, provide nutrients, and promote cell proliferation. The oxygen slow-release material can provide continuous oxygen support for mesenchymal stem cells, maintain the metabolic requirements of the cells, maintain the normal metabolic activities of mesenchymal stem cells by providing continuous oxygen, avoid stress reactions caused by hypoxia, can be used for long-term slow release of oxygen, avoid oxidative stress reactions, and improve cell survival rate. By modifying cyclodextrin, connecting malic acid, and introducing resveratrol, the present invention can enhance the antioxidant activity, help the cells maintain activity, improve the dispersibility, and at the same time the modified group can appropriately reduce the size of the cavity entrance, so that the cavity size is suitable for the porous nanoparticles loaded with oxygen, improving the recognition ability of cyclodextrin, reducing the decomplexation phenomenon caused by the slightly larger molecular cavity size of γ-cyclodextrin, effectively improving the stability, enhancing the oxygen slow-release performance, and being beneficial to the amplification and growth of mesenchymal stem cells. The present invention adds extracts of Astragalus membranaceus and Taraxacum mongolicum, which contain compounds such as polysaccharides and flavonoids and have antioxidant properties. They can protect cells from ROS damage, maintain the metabolic stability of cells, ensure the efficiency of energy utilization, and the extracts of Astragalus membranaceus and Taraxacum mongolicum can have a synergistic effect, improve the cell growth environment, and promote the growth and proliferation of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 This is the microscope image of the umbilical cord mesenchymal stem cells of Passage 10 of Example 1 and the control group P1 described in Experimental Example 1 of the present invention;

[0030] Figure 2 This is the growth curve of mesenchymal stem cells cultured in the culture media described in Example 2 and Comparative Examples 1-3 of the present invention;

[0031] Figure 3 This is the oxygen concentration change graph of mesenchymal stem cells cultured in the culture media described in Example 3 and Comparative Example 2 of the present invention. Detailed implementation manners

[0032] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the following further details the present invention in conjunction with specific embodiments. However, the present invention is not limited to the following embodiments. It should be noted that unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0033] Example 1: This example provides a culture and expansion medium for mesenchymal stem cells, using DMEM / F12 medium as the basic component. The DMEM / F12 medium further includes the following components: oxygen-releasing material 8 g / L, natural extract 2 g / L, growth factors 30 ng / mL, nicotinamide 5 mM, cholesterol 8 μg / mL, recombinant human transferrin 20 mg / L, recombinant human insulin 10 mg / L, vitamin C 4 mg / L, L-glutamine 2 mM, reduced glutathione 60 μg / L, sodium selenite 20 mg / L.

[0034] The growth factors are basic fibroblast growth factor 10 ng / mL, transforming growth factor-β 10 ng / mL, and platelet-derived growth factor-BB 10 ng / mL.

[0035] The preparation method of the oxygen-releasing material includes the following steps:

[0036] (1) Prepare 100 mL of 0.1 mol / L sodium carbonate aqueous solution, 100 mL of 0.1 mol / L calcium chloride aqueous solution, 50 mL of 10 mmol / L sodium dodecyl sulfate aqueous solution, and 50 mL of 2 g / L tartaric acid aqueous solution; Divide the tartaric acid aqueous solution into two portions of 25 mL each, and pour them into the sodium carbonate solution and the calcium chloride solution respectively. Stir at 60 °C and 300 rpm for 25 min to obtain mixture A and mixture B respectively;

[0037] (2) Pour the sodium dodecyl sulfate aqueous solution described in step (1) into mixture A obtained in step (1), react in a water bath at 80 °C for 30 min, add mixture B obtained in step (1), continue the water bath reaction for 1.2 h, keep the stirring speed at 200 r / min, centrifuge at 5000 rpm for 10 min to collect the precipitate, wash with deionized water and absolute ethanol, and dry at 60 °C to obtain porous nanoparticles;

[0038] (3) Place the porous nanoparticles prepared in step (2) in a reaction kettle and evacuate. When the pressure in the reaction kettle is constant, stop evacuating, fill with oxygen. When the pressure in the reaction kettle reaches 0.35 MPa, stop filling with oxygen to obtain oxygen-carrying porous nanoparticles;

[0039] (4) Add 10 g of γ-cyclodextrin, 10 g of malic acid, and 8 g of sodium hypophosphite to 200 mL of deionized water, ultrasonicate at 400 W for 30 min, react at 100 °C for 3 h, react at 140 °C for 15 min, add 50 mL of distilled water and stir evenly, add 600 mL of absolute ethanol, let stand at 4 °C for 4 h, centrifuge at 8000 rpm for 5 min, wash the precipitate with absolute ethanol, and dry in vacuo at 50 °C to obtain malic acid-cyclodextrin;

[0040] (5) Add 10 g of the malic acid-cyclodextrin obtained in step (4), 15 g of resveratrol, and 0.5 g of aminosulfonic acid to 120 mL of benzene, stir at 100 rpm, heat under reflux at 110 °C for 6 h, filter while hot under vacuum, let the filtrate stand at 6 °C for 4 h, centrifuge at 5000 rpm for 20 min, recrystallize the precipitate with ethyl acetate, wash with absolute ethanol, and dry in vacuo at 60 °C to obtain modified cyclodextrin;

[0041] (6) Add 20 g of the modified cyclodextrin obtained in step (5) to 140 mL of deionized water, stir at 30 °C and 180 rpm for 30 min to obtain a modified cyclodextrin solution; Add the modified cyclodextrin solution to the reaction kettle described in step (3), and the mass ratio of the modified cyclodextrin solution to the oxygen-carrying porous nanoparticles in the reaction kettle is 5:1. Stir at 60 °C and 150 rpm for 3 h, let stand at 4 °C for 2 h, centrifuge at 10000 rpm for 8 min, wash the precipitate with absolute ethanol and deionized water, and dry in vacuo to obtain the oxygen slow-release material.

[0042] Preparation method of natural extract, comprising the following steps: drying 10 g of astragalus membranaceus and 10 g of dandelion at 100 °C, grinding and passing through a 100-mesh sieve to obtain a mixed powder; taking the mixed powder, adding deionized water with a weight 10 times that of the powder, heating and extracting at 100 °C for 1 h, filtering to obtain a filtrate, extracting 3 times, combining the filtrates, concentrating at 60 °C and 10 -3 MPa to 20% of the original volume, adding a 95 vol% ethanol solution until the final ethanol concentration reaches 60 wt%, standing at 4 °C for 8 h, centrifuging at 5000 rpm for 30 min, filtering the supernatant through a 0.22-μm filter membrane, and freeze-drying at -20 °C for 24 h to obtain the natural extract.

[0043] This example also provides a preparation method of the mesenchymal stem cell culture and expansion medium, comprising the following steps: filtering the DMEM / F12 medium through a 0.22-μm filter membrane, adding an oxygen-releasing material, a natural extract, a growth factor, nicotinamide, cholesterol, recombinant human transferrin, recombinant human insulin, vitamin C, L-glutamine, reduced glutathione, and sodium selenite, and stirring at 180 rpm for 40 min to obtain the mesenchymal stem cell culture and expansion medium.

[0044] Example 2: This example provides a mesenchymal stem cell culture and expansion medium, using the DMEM / F12 medium as the basic component, and the DMEM / F12 medium further comprises the following components: 8 g / L of oxygen-releasing material, 2 g / L of natural extract, 30 ng / mL of growth factor, 5 mM of nicotinamide, 8 μg / mL of cholesterol, 20 mg / L of recombinant human transferrin, 10 mg / L of recombinant human insulin, 4 mg / L of vitamin C, 2 mM of L-glutamine, 60 μg / L of reduced glutathione, and 20 mg / L of sodium selenite.

[0045] The growth factor is 10 ng / mL of basic fibroblast growth factor, 10 ng / mL of transforming growth factor-β, and 10 ng / mL of platelet-derived growth factor-BB.

[0046] Preparation method of the oxygen-releasing material, comprising the following steps:

[0047] (1) Prepare 100 mL of 0.1 mol / L sodium carbonate aqueous solution, 100 mL of 0.1 mol / L calcium chloride aqueous solution, 50 mL of 10 mmol / L sodium dodecyl sulfate aqueous solution, and 50 mL of 2 g / L tartaric acid aqueous solution; divide the tartaric acid aqueous solution into two portions of 25 mL, pour them into the sodium carbonate solution and the calcium chloride solution respectively, and stir at 60 °C and 200 rpm for 18 min to obtain mixed solution A and mixed solution B respectively;

[0048] (2) Pour the aqueous solution of sodium dodecyl sulfate described in step (1) into the mixed solution A obtained in step (1), react in a water bath at 80 °C for 20 min, add the mixed solution B obtained in step (1), continue the water bath reaction for 0.8 h, keep the stirring speed at 200 r / min, centrifuge at 5000 rpm for 10 min to collect the precipitate, wash with deionized water and absolute ethanol, and dry at 60 °C to obtain porous nanoparticles;

[0049] (3) Place the porous nanoparticles prepared in step (2) in a reaction kettle and evacuate it. When the pressure in the reaction kettle is constant, stop evacuating, fill it with oxygen. When the pressure in the reaction kettle reaches 0.35 MPa, stop filling oxygen to obtain oxygen-carrying porous nanoparticles;

[0050] (4) Add 10 g of γ-cyclodextrin, 10 g of malic acid, and 6 g of sodium hypophosphite to 200 mL of deionized water, ultrasonicate at 300 W for 30 min, react at 100 °C for 3 h, react at 140 °C for 10 min, add 50 mL of distilled water and stir evenly, add 500 mL of absolute ethanol, let it stand at 4 °C for 3 h, centrifuge at 8000 rpm for 5 min, wash the precipitate with absolute ethanol, and dry it under vacuum at 50 °C to obtain malic acid-cyclodextrin;

[0051] (5) Add 10 g of the malic acid-cyclodextrin obtained in step (4), 12 g of resveratrol, and 0.5 g of sulfamic acid to 100 mL of benzene, stir at 100 rpm, heat under reflux at 100 °C for 5 h, filter while it is hot under vacuum, let the filtrate stand at 4 °C for 3 h, centrifuge at 5000 rpm for 15 min, recrystallize the precipitate with ethyl acetate, wash with absolute ethanol, and dry it under vacuum at 60 °C to obtain modified cyclodextrin;

[0052] (6) Add 20 g of the modified cyclodextrin obtained in step (5) to 100 mL of deionized water, stir at 30 °C and 180 rpm for 30 min to obtain a modified cyclodextrin solution; add the modified cyclodextrin solution to the reaction kettle described in step (3), and the mass ratio of the modified cyclodextrin solution to the oxygen-carrying porous nanoparticles in the reaction kettle is 5:1. Stir at 40 °C and 100 rpm for 2 h, let it stand at 4 °C for 2 h, centrifuge at 8000 rpm for 5 min, wash the precipitate with absolute ethanol and deionized water, and dry it under vacuum to obtain the oxygen slow-release material.

[0053] The preparation method of the natural extract includes the following steps: Dry 10 g of astragalus membranaceus and 10 g of dandelion at 100 °C, grind them through a 100-mesh sieve to obtain a mixed powder; take the mixed powder, add 10 times the weight of deionized water, heat and extract at 100 °C for 1 h, filter to obtain a filtrate, extract 3 times, combine the filtrates, and at 60 °C, 10 -3Concentrate to 20% of the original volume in MPa, add 95 vol% ethanol solution until the final ethanol concentration reaches 60 wt%, let stand at 4 °C for 6 h, centrifuge at 5000 rpm for 30 min, filter the supernatant through a 0.22 μm filter membrane, and freeze-dry at -20 °C for 20 h to obtain the natural extract.

[0054] This example also provides a method for preparing the mesenchymal stem cell culture and expansion medium, which includes the following steps: Filter the DMEM / F12 medium through a 0.22 μm filter membrane, and add an oxygen-releasing material, natural extract, growth factors, niacinamide, cholesterol, recombinant human transferrin, recombinant human insulin, vitamin C, L-glutamine, reduced glutathione, and sodium selenite, and stir at 180 rpm for 30 min to obtain the mesenchymal stem cell culture and expansion medium.

[0055] Example 3: This example provides a mesenchymal stem cell culture and expansion medium, using DMEM / F12 medium as the basic component, and the DMEM / F12 medium also includes the following components: 8 g / L of oxygen-releasing material, 2 g / L of natural extract, 30 ng / mL of growth factors, 5 mM of niacinamide, 8 μg / mL of cholesterol, 20 mg / L of recombinant human transferrin, 10 mg / L of recombinant human insulin, 4 mg / L of vitamin C, 2 mM of L-glutamine, 60 μg / L of reduced glutathione, and 20 mg / L of sodium selenite.

[0056] The growth factors are 10 ng / mL of basic fibroblast growth factor, 10 ng / mL of transforming growth factor-β, and 10 ng / mL of platelet-derived growth factor-BB.

[0057] The preparation method of the oxygen-releasing material includes the following steps:

[0058] (1) Prepare 100 mL of 0.1 mol / L sodium carbonate aqueous solution, 100 mL of 0.1 mol / L calcium chloride aqueous solution, 50 mL of 10 mmol / L sodium dodecyl sulfate aqueous solution, and 50 mL of 2 g / L tartaric acid aqueous solution; Divide the tartaric acid aqueous solution into two portions of 25 mL, and pour them into the sodium carbonate solution and calcium chloride solution respectively, stir at 60 °C and 250 rpm for 20 min to obtain mixture A and mixture B respectively;

[0059] (2) Pour the sodium dodecyl sulfate aqueous solution in step (1) into the mixture A obtained in step (1), react in a water bath at 80 °C for 25 min, add the mixture B obtained in step (1), continue the water bath reaction for 1 h, keep the stirring speed at 200 r / min, centrifuge at 5000 rpm for 10 min to collect the precipitate, wash with deionized water and absolute ethanol, and dry at 60 °C to obtain the porous nanoparticles;

[0060] (3) Place the porous nanoparticles prepared in step (2) in a reaction kettle and evacuate it. When the pressure in the reaction kettle is constant, stop evacuating and fill it with oxygen. When the pressure in the reaction kettle reaches 0.35 MPa, stop filling with oxygen to obtain oxygen-carrying porous nanoparticles;

[0061] (4) Add 10 g of γ-cyclodextrin, 10 g of malic acid, and 7 g of sodium hypophosphite to 200 mL of deionized water, ultrasonicate at 350 W for 30 min, react at 100 °C for 3 h, react at 140 °C for 12 min, add 50 mL of distilled water and stir evenly, add 550 mL of absolute ethanol, let it stand at 4 °C for 3.5 h, centrifuge at 8000 rpm for 5 min, wash the precipitate with absolute ethanol, and dry it under vacuum at 50 °C to obtain malic acid-cyclodextrin;

[0062] (5) Add 10 g of the malic acid-cyclodextrin obtained in step (4), 13 g of resveratrol, and 0.5 g of sulfamic acid to 110 mL of benzene, stir at 100 rpm, heat and reflux at 105 °C for 5.5 h, filter while it is hot under vacuum, let the filtrate stand at 5 °C for 3.5 h, centrifuge at 5000 rpm for 18 min, recrystallize the precipitate with ethyl acetate, wash it with absolute ethanol, and dry it under vacuum at 60 °C to obtain modified cyclodextrin;

[0063] (6) Add 20 g of the modified cyclodextrin obtained in step (5) to 120 mL of deionized water, stir at 30 °C and 180 rpm for 30 min to obtain a modified cyclodextrin solution; add the modified cyclodextrin solution to the reaction kettle described in step (3). The mass ratio of the modified cyclodextrin solution to the oxygen-carrying porous nanoparticles in the reaction kettle is 5:1. Stir at 50 °C and 120 rpm for 2.5 h, let it stand at 4 °C for 2 h, centrifuge at 9000 rpm for 6 min, wash the precipitate with absolute ethanol and deionized water, and dry it under vacuum to obtain an oxygen slow-release material.

[0064] The preparation method of the natural extract includes the following steps: Dry 10 g of astragalus membranaceus and 10 g of dandelion at 100 °C, grind them through a 100-mesh sieve to obtain a mixed powder; take the mixed powder, add 10 times the weight of deionized water, heat and extract at 100 °C for 1 h, filter to obtain a filtrate, extract 3 times, combine the filtrates, concentrate at 60 °C and 10 -3 MPa to 20% of the original volume, add a 95 vol% ethanol solution until the final ethanol concentration reaches 60 wt%, let it stand at 4 °C for 7 h, centrifuge at 5000 rpm for 30 min, filter the supernatant through a 0.22-μm filter membrane, and freeze-dry at -20 °C for 22 h to obtain the natural extract.

[0065] This embodiment also provides a method for preparing the mesenchymal stem cell culture and expansion medium, which includes the following steps: filtering the DMEM / F12 medium through a 0.22 μm filter membrane, adding an oxygen slow-release material, a natural extract, a growth factor, nicotinamide, cholesterol, recombinant human transferrin, recombinant human insulin, vitamin C, L-glutamine, reduced glutathione, and sodium selenite, and stirring at 180 rpm for 35 min to obtain the mesenchymal stem cell culture and expansion medium.

[0066] The difference between Comparative Example 1 and Example 1 is only that the oxygen slow-release material is not added.

[0067] The difference between Comparative Example 2 and Example 1 is only that γ-cyclodextrin is used to replace the modified cyclodextrin.

[0068] The difference between Comparative Example 3 and Example 1 is only that the natural extract is not added.

[0069] Experimental Example 1: Collect the umbilical cord aseptically, with a length of about 10 cm. After washing it 5 times with 0.9% physiological saline, mechanically cut the umbilical cord tissue into pieces with a size of 2 mm, and evenly place them in a 10 cm cell culture dish. The gap between the adherent tissue pieces is 5-10 mm. Add the mesenchymal stem cell culture and expansion medium described in Example 1, and place it in an incubator at 37°C and 5% CO 2 for culture. The obtained cells are P0 generation cells. Subsequently, change the medium once every 3-4 days. After the cell confluence reaches 80%, digest and harvest the cells with Tryspin-EDTA, and inoculate and passage the cells at a cell density of 8000 cells / cm 2 for subculture. One passage is the P1 generation cells, and so on, and observe the cell morphology. Use the DMEM + 10% FBS medium as the control group.

[0070] When subcultured to the P10 generation, photograph and record the cell morphology, and the results are as Figure 1 shown. Figure 1 The results show that the cell volume of the control group increases, showing obvious cell senescence characteristics. The cells in the Example 1 group still maintain a good spindle shape and have no obvious cell senescence characteristics, indicating that a mesenchymal stem cell expansion medium of the present invention is beneficial to the growth and proliferation of mesenchymal stem cells and reduces cell senescence.

[0071] Experimental Example 2: Adjust the cell density of the umbilical cord mesenchymal stem cells P5 (obtained by using the media prepared in Example 2 and Comparative Examples 1-3 respectively according to the method of Experimental Example 1) with the same source to 1×10 4 cells / mL, and add them to a 96-well cell culture plate, 100 μL / well. Place it at 37°C and 5% CO 2Cultured in an incubator, after incubating for 24, 48, 72, 96, and 168 hours respectively, 10 μL of CCK8 solution was added to each well, incubated in the incubator for 3 hours, the absorbance value of OD450 was read by an enzyme-linked immunosorbent assay (ELISA) reader, and the growth curve of cells in different media was plotted. The results are as Figure 2 shown.

[0072] Figure 2 The results showed that the cells cultured in the Example 2 group grew faster, the number of cells cultured in Example 2 was larger at the same time, the cell proliferation rate in the Example 2 group was faster, and they could stably proliferate and passage. While Comparative Examples 1-3 showed a slowdown in proliferation, indicating that the culture medium of the present invention could promote cell proliferation.

[0073] Experimental Example 3: Umbilical cord mesenchymal stem cells P10 with the same source (prepared by using the culture media obtained in Examples 1-3 and Comparative Examples 1-2 respectively according to the method of Experimental Example 1) were adjusted to a cell density of 1×10 5 cells / mL, and flow cytometry identification of cell phenotypes was carried out; fluorescein-labeled CD73, CD90, CD105, CD34, and CD45 antibodies and isotype controls were added, incubated at room temperature in the dark for 30 minutes, and the expression levels of cell surface markers were detected by flow cytometry. The results are shown in Table 1.

[0074] Table 1:

[0075] CD73 (%) CD90 (%) CD105 (%) CD34 (%) CD45 (%) Example 1 99.41 99.68 99.12 0.081 0.543 Example 2 99.45 99.67 99.18 0.085 0.539 Example 3 99.47 99.62 99.14 0.082 0.543 Comparative Example 1 96.12 96.38 95.15 1.231 1.612 Comparative Example 2 97.42 97.68 97.14 1.011 1.212 Comparative Example 3 96.43 96.68 96.15 1.191 1.498

[0076] The results in Table 1 showed that the expression levels of CD73, CD90, and CD105 in the P10-generation cells cultured with the mesenchymal stem cell expansion culture medium of the present invention were all ≥95%, and the positive rates of CD45 and CD34 were lower than 2%, which met the phenotypic characteristics of umbilical cord mesenchymal stem cells, showed the properties of umbilical cord mesenchymal stem cells, had the characteristics of MSCs, and had a high purity. Compared with the results of Comparative Examples 1-3, the flow cytometry results of Examples 1-3 were better.

[0077] Experimental Example 4: Umbilical cord mesenchymal stem cells P3 with the same source (prepared by using the culture media obtained in Example 3 and Comparative Example 2 respectively according to the method of Experimental Example 1) were adjusted to a cell density of 1×10 5 cells / mL, inoculated into a 12-well plate at 1 mL / well, and cultured under the conditions of 37°C and 5% CO 2 . After culturing for 1 day, 2 days, 4 days, 7 days, and 12 days, the oxygen concentration changes were measured. The results are as Figure 3 shown.

[0078] Figure 3The results showed that the oxygen concentration in Comparative Example 2 increased significantly after 1 day of culture and then began to decrease on the 2nd day, while in Example 3, it increased smoothly and remained at a certain level after 4 days, indicating that the culture medium prepared by the present invention has good stability, the change of oxygen concentration is relatively stable, and the overall oxygen concentration is maintained at a certain level, which is beneficial to the growth of mesenchymal stem cells.

[0079] Those of ordinary skill in the art should understand that the discussion of any above embodiments is only exemplary and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A mesenchymal stem cell culture and expansion medium, characterized in that: The DMEM / F12 culture medium is used as a basic component, and the DMEM / F12 culture medium also includes the following components: 8 g / L of oxygen slow-release material, 2 g / L of natural extract, 30 ng / mL of growth factor, 5 mM of nicotinamide, 8 μg / mL of cholesterol, 20 mg / L of recombinant human transferrin, 10 mg / L of recombinant human insulin, 4 mg / L of vitamin C, 2 mM of L-glutamine, 60 μg / L of reduced glutathione, and 20 mg / L of sodium selenite; The growth factors are basic fibroblast growth factor, transforming growth factor-β and platelet-derived growth factor-BB; The method for preparing the natural extract comprises the following steps: drying and grinding astragalus and dandelion to obtain a mixed powder; taking the mixed powder, adding deionized water, heating for extraction, filtering, concentrating under reduced pressure, adding an ethanol solution, standing, centrifuging, filtering the supernatant, and freeze-drying to obtain the natural extract; The mass ratio of dandelion to astragalus is 1:1; The preparation method of the oxygen slow-release material comprises the following steps: (1) preparing a 0.1 mol / L sodium carbonate aqueous solution, a 0.1 mol / L calcium chloride aqueous solution, a 10 mmol / L sodium dodecyl sulfate aqueous solution, and a 2 g / L tartaric acid aqueous solution; dividing the tartaric acid aqueous solution into two equal volumes, pouring the two into the sodium carbonate solution and the calcium chloride solution, respectively, and stirring to obtain a mixed solution A and a mixed solution B, respectively; (2) pouring the sodium dodecyl sulfate aqueous solution obtained in step (1) into the mixed solution A obtained in step (1), reacting, adding the mixed solution B obtained in step (1), continuing the reaction, centrifuging, washing the precipitate, and drying to obtain porous nanoparticles; (3) placing the porous nanoparticles obtained in step (2) in a reaction kettle, evacuating the air, and filling the reactor with oxygen to obtain oxygen-carrying porous nanoparticles; (4) adding γ-cyclodextrin, malic acid, and sodium hypophosphite to deionized water, ultrasonicating, reacting at 100° C., reacting at 140° C., adding distilled water, adding anhydrous ethanol, standing, centrifuging, washing the precipitate, and drying to obtain malic acid-cyclodextrin; (5) adding the malic acid-cyclodextrin, resveratrol and aminosulfonic acid obtained in step (4) to benzene, stirring, heating to reflux, filtering with suction, letting the filtrate stand, centrifuging, recrystallizing the precipitate with ethyl acetate, washing, and drying to obtain modified cyclodextrin; (6) adding the modified cyclodextrin obtained in step (5) into deionized water and stirring to obtain a modified cyclodextrin solution; adding the modified cyclodextrin solution into the reaction kettle described in step (3), stirring, standing, centrifuging, washing the precipitate, and drying to obtain an oxygen sustained-release material.

2. The mesenchymal stem cell culture and expansion medium according to claim 1, characterized in that In step (1), the volume ratio of the prepared 0.1 mol / L sodium carbonate aqueous solution, 0.1 mol / L calcium chloride aqueous solution, 10 mmol / L sodium dodecyl sulfate aqueous solution and 2 g / L tartaric acid aqueous solution is 2:2:1:

1.

3. The mesenchymal stem cell culture and expansion medium according to claim 2, characterized in that: In step (4), the dosage ratio of γ-cyclodextrin, malic acid, sodium hypophosphite and deionized water is 1g:1g:0.6-0.8g:20mL; the volume ratio of distilled water to deionized water is 1:4; and the volume ratio of the added anhydrous ethanol to distilled water is 10-12:

1.

4. The mesenchymal stem cell culture and expansion medium according to claim 3, characterized in that: In step (5), the dosage ratio of malic acid-cyclodextrin, resveratrol, aminosulfonic acid and benzene is 1g:1.2-1.5g:0.05g:10-12mL.

5. The mesenchymal stem cell culture and expansion medium according to claim 4, characterized in that: In step (6), the dosage ratio of the modified cyclodextrin to deionized water is 1 g:5-7 mL; the mass ratio of the modified cyclodextrin solution to the oxygen-carrying porous nanoparticles in the reactor is 5:

1.

6. The mesenchymal stem cell culture and expansion medium according to claim 5, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

7. A method for preparing a mesenchymal stem cell culture and expansion medium according to any one of claims 1 to 6, characterized in that: The following steps are involved: The DMEM / F12 medium is filtered, and oxygen sustained-release materials, natural extracts, growth factors, nicotinamide, cholesterol, recombinant human transferrin, recombinant human insulin, vitamin C, L-glutamine, reduced glutathione, and sodium selenite are added and stirred to obtain a mesenchymal stem cell culture and expansion medium.

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