Mesenchymal stem cell induced cartilage differentiation culture medium and preparation method thereof

Abstract

The invention relates to the technical field of biological cell culture, and the biological cell culture medium comprises a basic nutrient component, a signal regulation factor, a differentiation promoting factor and an auxiliary stable factor, and the unit of each component is mg / L according to the total volume of the culture medium; the basic nutrition component is prepared from the following substances: 200 to 800 L of glutamine, 150 to 600 L of serine, 300 to 1200 L of proline and 100 to 400 L of lysine hydrochloride. The invention relates to the technical field of biological cell culture. According to the mesenchymal stem cell induced chondrogenic differentiation culture medium and the preparation method thereof, by optimizing the composition of the culture medium, a specific activity enhancing component and a signal regulating component are selectively introduced, so that the efficiency of differentiating mesenchymal stem cells into chondrocytes is remarkably improved; an amino acid derivative in the activity enhancing component can effectively promote cell metabolism activity, and a growth factor simulant and a signal channel regulator in the signal regulating component synergistically act on a key signal channel in the cell differentiation process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological cell culture, in particular to a mesenchymal stem cell induced chondrogenic differentiation culture medium and a preparation method thereof. BACKGROUND

[0002] Mesenchymal stem cells are a kind of cells that can self-replicate and have multi-differentiation potential. In the human body, mesenchymal stem cells exist in various tissues such as umbilical cord, fat, bone marrow, dental pulp, placenta, etc. They can be collected and separated for in vitro expansion and culture of multiple passages. Under the culture of differentiation medium, mesenchymal stem cells can differentiate into various types of tissue cells such as fat, bone, cartilage, muscle, nerve, and skin. Therefore, mesenchymal stem cells have been widely used in clinical research of tissue and organ function damage repair. For example, in many early clinical studies, researchers used adipose-derived mesenchymal stem cells to repair cartilage tissue and achieved good efficacy, showing great potential for clinical transformation.

[0003] Referring to patent publication No. "CN117736980B", an adipose-derived mesenchymal stem cell induced differentiation medium for transforming into chondrocytes and its application are disclosed. The induced differentiation medium includes a DMEM high-sugar or DMEM / F12 component-based culture medium; the culture medium contains a nutrient solution and an inducer; wherein: the nutrient solution contains a final concentration of 3040 v / v% of blood substitute and a final concentration of 1.82.6 v / v% of non-essential amino acids.

[0004] As shown in the above-mentioned technology, the existing technology significantly improves the efficiency of adipose-derived mesenchymal stem cells differentiating into chondrocytes by up-regulating the expression of aggrecan and collagen II. The composition of the inducer used in the existing technical solution is relatively complex, especially the extraction and purification process of the extracellular matrix is difficult, which may increase the production cost and operational complexity. The existing mesenchymal stem cell induced chondrogenic differentiation culture medium and its preparation method still have certain deficiencies in terms of composition complexity, cost control, mechanism of action, differentiation efficiency and stability, etc. Therefore, the present application provides a novel mesenchymal stem cell induced chondrogenic differentiation culture medium and its preparation method, aiming to optimize the composition of the culture medium, simplify the preparation process, clarify the mechanism of action, and improve the efficiency and stability of mesenchymal stem cells differentiating into chondrocytes, so as to meet the needs of the field of regenerative medicine for efficient and low-cost cartilage tissue engineering. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a mesenchymal stem cell induced chondrogenic differentiation culture medium and a preparation method thereof, which solves the problem that the existing mesenchymal stem cell induced chondrogenic differentiation culture medium and its preparation method still have certain deficiencies in terms of composition complexity, cost control, mechanism of action, differentiation efficiency and stability, etc.

[0006] To achieve the above object, the present application is implemented by the following technical solutions: a mesenchymal stem cell induced chondrogenic differentiation culture medium, comprising an activity enhancing component, a signal regulation component, a metabolic support component and an auxiliary stabilizing component, each component is calculated according to the total volume of the culture medium, and the unit is mg / L; The activity enhancing component is composed of the following components: 200-1000 L-serine methyl ester, 50-250 L-lysine ethyl ester, 300-1500 L-glutamine dipeptide, 100-500 L-arginine methyl ester, 20-100 L-tyrosine ethyl ester, 400-2000 L-asparagine dipeptide, 150-750 L-proline methyl ester, 50-250 L-isoleucine ethyl ester, 300-1500 L-phenylalanine methyl ester and 200-1000 L-threonine ethyl ester; The signal regulation component is composed of the following components: 10-50 chondroitin sulfate, 20-100 sodium hyaluronate, 5-25 polylactic acid-glycolic acid copolymer, 1-10 heparin fragment, 0.5-5 transforming growth factor β1 mimic peptide, 0.1-2 insulin-like growth factor 1 analogue, 0.05-0.5 interleukin-6 inhibitor, 0.01-0.2 fibroblast growth factor 18 fragment and 0.05-0.5 Wnt signaling pathway activator; The metabolic support component is composed of the following components: 500-2000 glycerophosphate calcium, 100-500 calcium gluconate, 200-1000 potassium dihydrogen phosphate, 100-500 magnesium chloride hexahydrate, 0.1-1 nickel nitrate hexahydrate, 0.05-0.5 ammonium molybdate tetrahydrate, 50-250 ethylene glycol bis-tetraacetic acid, 10-50 iron sodium salt of ethylenediaminetetraacetic acid, 5-25 sodium selenite and 100-500 sodium bicarbonate; The auxiliary stabilizing component is composed of the following components: 50-250 2-ethanesulfonic acid, 200-1000 polyvinylpyrrolidone K30, 10-50 pyruvic acid ethyl ester, 5-25 creatine monohydrate, 0.1-1 eicosapentaenoic acid ethyl ester, 2000-10000 anhydrous fruit sugar, 0.5-5 uracil, 0.1-1 putrescine monohydrochloride, 10-50 mercaptoethanol, 50-250 oxidized glutathione, 50-250 D-xylose, 1-10 choline chloride methyl ester, 0.01-0.1 ethyl lipoate, 19.92-99.61 S-adenosyl-L-methionine ethyl ester and 3.72-14.875-5-methylthioadenosine methyl ester.

[0007] Preferably, the activity enhancing component consists of 200 L-serine methyl ester, 50 L-lysine ethyl ester, 300 L-glutamine dipeptide, 100 L-arginine methyl ester, 20 L-tyrosine ethyl ester, 400 L-asparagine dipeptide, 150 L-proline methyl ester, 50 L-isoleucine ethyl ester, 300 L-phenylalanine methyl ester, and 1000 L-threonine ethyl ester; The signal regulation component consists of 10 chondroitin sulfate, 20 sodium hyaluronate, 5 polylactic-co-glycolic acid, 1 heparin fragment, 0.5 transforming growth factor beta 1 mimetic peptide, 0.1 insulin-like growth factor 1 analog, 0.05 interleukin-6 inhibitor, 0.01 fibroblast growth factor 18 fragment, and 0.05 Wnt signaling pathway activator; The metabolic support component consists of 500 glycerophosphate calcium, 100 calcium gluconate, 200 potassium phosphate monobasic, 100 magnesium chloride hexahydrate, 0.1 nickel nitrate hexahydrate, 0.05 ammonium molybdate tetrahydrate, 50 ethylene glycol bis-tetraacetic acid, 10 iron sodium ethylenediaminetetraacetate, 5 sodium selenite, and 100 sodium bicarbonate; The auxiliary stabilizing component consists of 502-ethanesulfonic acid, 200 polyvinylpyrrolidone K30, 10 pyruvic acid ethyl ester, 5 creatine monohydrate, 0.1 eicosapentaenoic acid ethyl ester, 2000 anhydrous fructose, 0.5 uracil, 0.1 putrescine monohydrochloride, 10 mercaptoethanol, 50 oxidized glutathione, 50 D-xylose, 1-10 choline chloride methyl ester, 0.01 thioctic acid ethyl ester, 19.92 S-adenosyl-L-methionine ethyl ester, and 3.725 deoxy-5-methylthioadenosine methyl ester.

[0008] Preferably, 600 L-serine methyl ester, 150 L-lysine ethyl ester, 900 L-glutamine dipeptide, 300 L-arginine methyl ester, 60 L-tyrosine ethyl ester, 1200 L-asparagine dipeptide, 450 L-proline methyl ester, 150 L-isoleucine ethyl ester, 900 L-phenylalanine methyl ester, and 600 L-threonine ethyl ester; The signal regulation component consists of 30 chondroitin sulfate, 60 sodium hyaluronate, 15 polylactic-co-glycolic acid, 5.5 heparin fragment, 2.75 transforming growth factor beta 1 mimetic peptide, 1.05 insulin-like growth factor 1 analog, 0.275 interleukin-6 inhibitor, 0.105 fibroblast growth factor 18 fragment, and 0.275 Wnt signaling pathway activator; The metabolic support component consists of: 1250 calcium glycerophosphate, 300 calcium gluconate, 600 potassium phosphate monobasic, 300 magnesium chloride hexahydrate, 0.55 nickel nitrate hexahydrate, 0.275 ammonium molybdate tetrahydrate, 150 ethylene glycol bis-tetraacetic acid, 30 iron sodium ethylenediaminetetraacetate, 15 sodium selenite and 300 sodium bicarbonate; The auxiliary stabilizing component consists of: 150 2-ethanesulfonic acid, 600 polyvinylpyrrolidone K30, 30 ethyl pyruvate, 15 creatine monohydrate, 0.55 eicosapentaenoic acid ethyl ester, 6000 anhydrous fructose, 2.75 uracil, 0.55 putrescine monohydrochloride, 30 mercaptoethanol, 150 oxidized glutathione, 150 D-xylose, 5.5 choline chloride methyl ester, 0.055 thioctic acid ethyl ester, 59.765 S-adenosyl-L-methionine ethyl ester and 9.2955-deoxy-5-methylthioadenosine methyl ester.

[0009] Preferably, the activity enhancing component consists of: 1000 L-serine methyl ester, 250 L-lysine ethyl ester, 1500 L-glutamine dipeptide, 500 L-arginine methyl ester, 100 L-tyrosine ethyl ester, 2000 L-asparagine dipeptide, 750 L-proline methyl ester, 250 L-isoleucine ethyl ester, 1500 L-phenylalanine methyl ester and 1000 L-threonine ethyl ester; The signal regulation component consists of: 50 chondroitin sulfate, 100 sodium hyaluronate, 25 polylactic acid-glycolic acid copolymer, 10 heparin fragment, 5 transforming growth factor β1 mimic peptide, 2 insulin-like growth factor 1 analogue, 0.5 interleukin-6 inhibitor, 0.2 fibroblast growth factor 18 fragment and 0.5 Wnt signaling pathway activator; The metabolic support component consists of: 2000 calcium glycerophosphate, 500 calcium gluconate, 1000 potassium phosphate monobasic, 500 magnesium chloride hexahydrate, 1 nickel nitrate hexahydrate, 0.5 ammonium molybdate tetrahydrate, 250 ethylene glycol bis-tetraacetic acid, 50 iron sodium ethylenediaminetetraacetate, 25 sodium selenite and 500 sodium bicarbonate; The auxiliary stabilizing component consists of: 250 2-ethanesulfonic acid, 1000 polyvinylpyrrolidone K30, 50 ethyl pyruvate, 25 creatine monohydrate, 1 eicosapentaenoic acid ethyl ester, 10000 anhydrous fructose, 5 uracil, 1 putrescine monohydrochloride, 50 mercaptoethanol, 250 oxidized glutathione, 250 D-xylose, 10 choline chloride methyl ester, 0.1 thioctic acid ethyl ester, 99.61 S-adenosyl-L-methionine ethyl ester and 14.875-Deoxy-5-methylthioadenosine methyl ester.

[0010] The application also discloses a preparation method of the mesenchymal stem cell-induced chondrogenic differentiation culture medium, and specifically comprises the following steps: S1, the active component, signal regulation component, metabolic support component and auxiliary stability component are weighed according to the weight of the above components, poured into a mixing device, mixed uniformly, then transferred into a grinding device for grinding, and the obtained dry powder medium is recorded as S1 after grinding is completed; S2, in a clean container, add deionized water with a volume of 70-80% of the final volume, start stirring when the initial water temperature is 15-25℃, and add the dry powder medium in S1 according to the mass concentration of 35.68g / L under stirring conditions, adjust the pH value to 8.50-8.70 after stirring for 20-30min, continue to stir for 10-15min, then adjust the pH value to 6.50-6.70, and stir for 10-15min again, then the obtained is recorded as a premix component; S3, add sodium bicarbonate powder to the premix component in S2 according to the mass concentration of 3g / L, stir for 10-20min until completely dissolved, adjust the pH value to 7.00-7.20, add deionized water to the final volume, and continue to stir for 5-20min, then filter and sterilize using a sterilization filter with a pore size of 0.22μm, and the obtained is mesenchymal stem cell induced chondrogenic differentiation medium.

[0011] Preferably, the pH value in S2 and S3 is adjusted using a potassium hydroxide solution with a concentration of 4-8mol / L and a nitric acid solution with a concentration of 4-8mol / L, and the stirring speed in S2 and S3 is 150-250r / min.

[0012] Preferably, during the storage of the dry powder medium in S1, the dry powder medium is divided into multiple layers of composite packaging, the outermost layer is a packaging bag made of a high-molecular material with elasticity and moisture-proof performance, each layer of packaging is sealed by heat sealing technology, desiccant is placed between two layers of packaging and vacuum treatment is applied to reduce water residue, and the desiccant is silica gel particles with a moisture absorption capacity of more than 30%.

[0013] Preferably, the grinding device in S1 is equipped with a high-precision ball milling system, which can grind the material to a particle size of less than 50 microns.

[0014] The application provides a mesenchymal stem cell induced chondrogenic differentiation medium and a preparation method thereof. 1、The mesenchymal stem cell induced chondrogenic differentiation medium and the preparation method thereof, by optimizing the composition of the medium, selectively introducing specific active components and signal regulation components, significantly improving the efficiency of mesenchymal stem cells differentiating into chondrocytes, the amino acid derivatives in the active component can effectively promote cell metabolic activity, and the growth factor mimics and signal pathway regulators in the signal regulation component synergistically act on the key signal pathways in the cell differentiation process, ensuring the accuracy and stability of the differentiation direction.

[0015] 2、The mesenchymal stem cell induced chondrogenic differentiation medium and its preparation method provide essential trace elements and metabolic substrates through metabolic support components, and auxiliary stability components maintain the stability of the culture environment by adjusting the osmotic pressure and antioxidant capacity.

[0016] 3、The mesenchymal stem cell induced chondrogenic differentiation medium and its preparation method realize efficient and stable chondrocyte differentiation effect through reasonable collocation of components and optimization of preparation process, while reducing the preparation cost and operation complexity, providing important technical support for the field of regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 It is a schematic diagram of the preparation process of the medium of the present application; Fig. 2 It is a process flow chart of the preparation method of the present application. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0019] Please refer to Figs. 1-2 The present application discloses a preparation method of a mesenchymal stem cell induced chondrogenic differentiation medium: In the implementation process, first, a mixing device and a grinding device are needed to complete the preparation of the dry powder medium. According to the proportions described in the invention, the materials of the active enhancement component, the signal regulation component, the metabolic support component and the auxiliary stability component are weighed, and then these materials are poured into the mixing device for preliminary mixing. The mixing device adopts a double helix stirring structure to ensure that the materials are evenly distributed in the container. After preliminary mixing, the materials are transferred to the grinding device for further processing. The grinding device is equipped with a high-precision ball milling system, which can grind the materials to a particle size of less than 50 microns to ensure the solubility and stability of the subsequent process. The dry powder medium obtained after grinding needs to be immediately packaged and stored to avoid moisture absorption or contamination.

[0020] The sub-packaging and storage process of the dry powder medium is an important part of the present application. The dry powder medium is sub-packaged into multi-layer composite packaging. The outermost layer is a packaging bag made of a high-molecular material with elasticity and moisture-proof performance, such as a polyethylene-polyamide composite material. This material can effectively isolate external moisture and oxygen. Each layer of packaging is sealed using heat sealing technology. Desiccant, such as silica gel particles, is placed between two layers of packaging and vacuum treatment is applied. The moisture absorption capacity of the desiccant is more than 30%, which can further reduce the residual moisture. Through the above sub-packaging method, the shelf life of the dry powder medium can be significantly prolonged, and its stability in subsequent use can be ensured.

[0021] Next, the preparation of the premix component is entered. Deionized water with a volume of 70-80% of the final volume is added to a clean container. The initial water temperature is controlled within the range of 15-25°C. Under the action of the stirrer, stirring is started with a stirring speed of 150-250 r / min to ensure that the liquid is in a uniform flow state. Then, the dry powder medium prepared in S1 is added to the deionized water at a mass concentration of 35.68 g / L, and continuous stirring is performed for 20-30 minutes to fully dissolve the dry powder. In this process, the pH of the solution is monitored using a pH meter, and the pH value is adjusted to the range of 8.50-8.70 by adding a potassium hydroxide solution with a concentration of 4-8 mol / L. The purpose of this step is to promote the dispersibility of some difficult-to-dissolve components. Then, continuous stirring is performed for 10-15 minutes, and the pH value is adjusted to the range of 6.50-6.70 by adding a nitric acid solution with a concentration of 4-8 mol / L to maintain the chemical stability of the solution. After 10-15 minutes of final stirring, the premix component is obtained.

[0022] After the preparation of the premix component is completed, sodium bicarbonate powder is added thereto at a mass concentration of 3 g / L, and continuous stirring is performed for 10-20 minutes until complete dissolution. This process also requires the use of a pH meter 7 to monitor the pH of the solution, and the pH value is adjusted to the range of 7.00-7.20 by adding an appropriate amount of potassium hydroxide solution or nitric acid solution. Then, deionized water is added to the final volume, and continuous stirring is performed for 5-20 minutes to ensure the uniformity of the solution. The final solution needs to be treated by a sterilization filtration device. The sterilization filter 8 is connected to the outlet of the clean container 5, and the filter pore size is 0.22 μm, which can effectively remove microorganisms and particulate impurities in the solution. After sterilization filtration, the obtained is the mesenchymal stem cell induction into chondrogenic differentiation medium.

[0023] In the above preparation process, the positional relationship and cooperation relationship between each part are crucial, for example, the connection of the mixing device and the grinding device is realized through a pipeline, which ensures that the material can be transmitted in a closed environment to avoid external pollution, the heat sealing part of the multi-layer composite package is designed as a double-layer sealing structure, each layer has a sealing width of not less than 5 mm to improve the sealing reliability, the drying agent is placed between the two layers of the package at a distance of at least 1 cm from the package edge to avoid leakage of the drying agent due to extrusion, the bottom of the clean container is designed as a conical structure to facilitate the formation of vortex when the stirrer operates at low speed, thereby improving the mixing efficiency, the probe of the pH meter is inserted to a depth of 3-5 cm below the liquid surface to ensure the accuracy of the measurement data, and the connection between the sterilization filter and the clean container 5 adopts a quick connector for easy disassembly and cleaning.

[0024] In addition, in order to verify the actual application effect of the present application, a group of mesenchymal stem cell samples were selected for experiment, and the experimental results showed that after using the culture medium provided by the present application, the cell differentiation efficiency was significantly improved, the differentiation direction accuracy reached more than 95%, and the differentiated chondrocyte morphology was complete and the function index met the expectation, which indicated that the culture medium of the present application not only could meet the laboratory research demand, but also had good industrial application prospect.

[0025] As can be seen from the above method, the present application realizes efficient and stable chondrocyte differentiation effect by optimizing the composition and preparation process of the culture medium. The reasonable layout and close cooperation between each part ensure the smooth progress of the entire preparation process, providing important technical support for the field of regenerative medicine.

[0026] The present application also provides a mesenchymal stem cell induction and chondrogenic differentiation culture medium, which is prepared according to the above method.

[0027] Hereinafter, the examples of the present application will be described. The examples described below are exemplary and are intended to explain the present application, but cannot be understood as limiting the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase on the market.

[0028] Example 1: The active enhancer component consists of the following ingredients: 200 L-serine methyl ester, 50 L-lysine ethyl ester, 300 L-glutamine dipeptide, 100 L-arginine methyl ester, 20 L-tyrosine ethyl ester, 400 L-asparagine dipeptide, 150 L-proline methyl ester, 50 L-isoleucine ethyl ester, 300 L-phenylalanine methyl ester, and 1000 L-threonine ethyl ester; The signal regulation component consists of: 10 chondroitin sulfate, 20 sodium hyaluronate, 5 polylactic-co-glycolic acid, 1 heparin fragment, 0.5 transforming growth factor beta 1 mimetic peptide, 0.1 insulin-like growth factor 1 analog, 0.05 interleukin-6 inhibitor, 0.01 fibroblast growth factor 18 fragment, and 0.05 Wnt signaling pathway activator; The metabolic support component consists of: 500 calcium glycerophosphate, 100 calcium gluconate, 200 potassium phosphate monobasic, 100 magnesium chloride hexahydrate, 0.1 nickel nitrate hexahydrate, 0.05 ammonium molybdate tetrahydrate, 50 ethylene glycol bis-tetraacetic acid, 10 iron sodium ethylenediaminetetraacetate, 5 sodium selenite, and 100 sodium bicarbonate; The auxiliary stabilization component consists of: 502-ethanesulfonic acid, 200 polyvinylpyrrolidone K30, 10 ethyl pyruvate, 5 creatine monohydrate, 0.1 eicosapentaenoic acid ethyl ester, 2000 anhydrous fructose, 0.5 uracil, 0.1 putrescine monohydrochloride, 10 mercaptoethanol, 50 oxidized glutathione, 50 D-xylose, 1-10 choline chloride methyl ester, 0.01 thioctic acid ethyl ester, 19.92 S-adenosyl-L-methionine ethyl ester, and 3.725 deoxy-5-methylthioadenosine methyl ester.

[0029] Example 2: 600 L-serine methyl ester, 150 L-lysine ethyl ester, 900 L-glutamine dipeptide, 300 L-arginine methyl ester, 60 L-tyrosine ethyl ester, 1200 L-asparagine dipeptide, 450 L-proline methyl ester, 150 L-isoleucine ethyl ester, 900 L-phenylalanine methyl ester, and 600 L-threonine ethyl ester; The signal regulation component consists of: 30 chondroitin sulfate, 60 sodium hyaluronate, 15 polylactic-co-glycolic acid, 5.5 heparin fragment, 2.75 transforming growth factor beta 1 mimetic peptide, 1.05 insulin-like growth factor 1 analog, 0.275 interleukin-6 inhibitor, 0.105 fibroblast growth factor 18 fragment, and 0.275 Wnt signaling pathway activator; The metabolic support component consists of: 1250 calcium glycerophosphate, 300 calcium gluconate, 600 potassium phosphate monobasic, 300 magnesium chloride hexahydrate, 0.55 nickel nitrate hexahydrate, 0.275 ammonium molybdate tetrahydrate, 150 ethylene glycol bis-tetraacetic acid, 30 iron sodium ethylenediaminetetraacetate, 15 sodium selenite, and 300 sodium bicarbonate; The auxiliary stabilizing component consists of the following ingredients: 1502-ethanesulfonic acid, 600 polyvinylpyrrolidone K30, 30 ethyl pyruvate, 15 monohydrate creatine, 0.55 eicosapentaenoic acid ethyl ester, 6000 anhydrous fructose, 2.75 uracil, 0.55 putrescine monohydrochloride, 30 mercaptoethanol, 150 oxidized glutathione, 150 D-xylose, 5.5 choline chloride methyl ester, 0.055 thioctic acid ethyl ester, 59.765 S-adenosyl-L-methionine ethyl ester, and 9.2955-deoxy-5-methylthioadenosine methyl ester.

[0030] Example 3: The active enhancing component consists of the following ingredients: 1000 L-serine methyl ester, 250 L-lysine ethyl ester, 1500 L-glutamine dipeptide, 500 L-arginine methyl ester, 100 L-tyrosine ethyl ester, 2000 L-asparagine dipeptide, 750 L-proline methyl ester, 250 L-isoleucine ethyl ester, 1500 L-phenylalanine methyl ester, and 1000 L-threonine ethyl ester; The signal modulating component consists of the following ingredients: 50 chondroitin sulfate, 100 sodium hyaluronate, 25 polylactic acid-glycolic acid copolymer, 10 heparin fragment, 5 transforming growth factor β1 mimetic peptide, 2 insulin-like growth factor 1 analog, 0.5 interleukin-6 inhibitor, 0.2 fibroblast growth factor 18 fragment, and 0.5 Wnt signaling pathway activator; The metabolic support component consists of the following ingredients: 2000 calcium glycerophosphate, 500 calcium gluconate, 1000 potassium phosphate monobasic, 500 magnesium chloride hexahydrate, 1 nickel nitrate hexahydrate, 0.5 ammonium molybdate tetrahydrate, 250 ethylene glycol bis-tetraacetic acid, 50 iron sodium ethylenediaminetetraacetate, 25 sodium selenite, and 500 sodium bicarbonate; The auxiliary stabilizing component consists of the following ingredients: 250 2-ethanesulfonic acid, 1000 polyvinylpyrrolidone K30, 50 ethyl pyruvate, 25 monohydrate creatine, 1 eicosapentaenoic acid ethyl ester, 10000 anhydrous fructose, 5 uracil, 1 putrescine monohydrochloride, 50 mercaptoethanol, 250 oxidized glutathione, 250 D-xylose, 10 choline chloride methyl ester, 0.1 thioctic acid ethyl ester, 99.61 S-adenosyl-L-methionine ethyl ester, and 14.875 5-deoxy-5-methylthioadenosine methyl ester.

[0031] The medium prepared in the above examples was used for chondrogenic differentiation experiments of mesenchymal stem cells, and the collagen expression level, the aggregate proteoglycan expression level, and the extracellular matrix formation ability were determined, and the results are shown in the following table: According to the experimental data, in Examples 1 to 3, by adding the natural complex peptide mixture, the cell proliferation rate, differentiation marker expression level and extracellular matrix formation ability are significantly improved, indicating that the culture medium has excellent performance, and the culture medium prepared in Example 2 has the best effect.

[0032] It should be noted that, in this text, relational terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device.

[0033] Although embodiments of the present application have been shown and described, it will be understood by those having ordinary skill in the art that various changes, modifications, alternatives and variations can be made thereto without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mesenchymal stem cell inducing chondrogenic differentiation medium, characterized by: The active enhancement component, the signal regulation component, the metabolic support component and the auxiliary stabilizing component, each component is mg / L according to the total volume of the medium; The active enhancement component consists of: 200-1000 L-serine methyl ester, 50-250 L-lysine ethyl ester, 300-1500 L-glutamine dipeptide, 100-500 L-arginine methyl ester, 20-100 L-tyrosine ethyl ester, 400-2000 L-asparagine dipeptide, 150-750 L-proline methyl ester, 50-250 L-isoleucine ethyl ester, 300-1500 L-phenylalanine methyl ester and 200-1000 L-threonine ethyl ester; The signal regulation component consists of: 10-50 chondroitin sulfate, 20-100 hyaluronic acid sodium, 5-25 polylactic acid-glycolic acid copolymer, 1-10 heparin fragment, 0.5-5 transforming growth factor β1 mimic peptide, 0.1-2 insulin-like growth factor 1 analogue, 0.05-0.5 interleukin-6 inhibitor, 0.01-0.2 fibroblast growth factor 18 fragment and 0.05-0.5 Wnt signaling pathway activator; The metabolic support component consists of: 500-2000 glycerol phosphate calcium, 100-500 calcium gluconate, 200-1000 potassium dihydrogen phosphate, 100-500 magnesium chloride hexahydrate, 0.1-1 nickel nitrate hexahydrate, 0.05-0.5 ammonium molybdate tetrahydrate, 50-250 ethylene glycol bis-tetraacetic acid, 10-50 iron sodium salt of ethylenediaminetetraacetic acid, 5-25 sodium selenite and 100-500 sodium bicarbonate; The auxiliary stabilizing component consists of: 50-250 2-ethanesulfonic acid, 200-1000 polyvinyl pyrrolidone K30, 10-50 pyruvic acid ethyl ester, 5-25 creatine monohydrate, 0.1-1 eicosapentaenoic acid ethyl ester, 2000-10000 anhydrous fruit sugar, 0.5-5 uracil, 0.1-1 putrescine monohydrochloride, 10-50 mercaptoethanol, 50-250 oxidized glutathione, 50-250 D-xylose, 1-10 choline chloride methyl ester, 0.01-0.1 ethyl lipoate, 19.92-99.61 S-adenosyl-L-methionine ethyl ester and 3.72-14.875-Deoxy-5-methylthioadenosine methyl ester.

2. The mesenchymal stem cell-induced chondrogenic differentiation medium according to claim 1, characterized in that: The active enhancement component consists of: 200 L-serine methyl ester, 50 L-lysine ethyl ester, 300 L-glutamine dipeptide, 100 L-arginine methyl ester, 20 L-tyrosine ethyl ester, 400 L-asparagine dipeptide, 150 L-proline methyl ester, 50 L-isoleucine ethyl ester, 300 L-phenylalanine methyl ester and 1000 L-threonine ethyl ester; The signal regulation component consists of: 10 chondroitin sulfate, 20 sodium hyaluronate, 5 polylactic acid-glycolic acid copolymer, 1 heparin fragment, 0.5 transforming growth factor β1 mimic peptide, 0.1 insulin-like growth factor 1 analogue, 0.05 interleukin-6 inhibitor, 0.01 fibroblast growth factor 18 fragment and 0.05 Wnt signaling pathway activator; The metabolic support component consists of: 500 glycerophosphate calcium, 100 calcium gluconate, 200 potassium phosphate monobasic, 100 magnesium chloride hexahydrate, 0.1 nickel nitrate hexahydrate, 0.05 ammonium molybdate tetrahydrate, 50 ethylene glycol bis-tetraacetic acid, 10 iron sodium ethylenediaminetetraacetate, 5 sodium selenite and 100 sodium bicarbonate; The auxiliary stabilizing component consists of: 502-ethanesulfonic acid, 200 polyvinylpyrrolidone K30, 10 ethyl pyruvate, 5 creatine monohydrate, 0.1 eicosapentaenoic acid ethyl ester, 2000 anhydrous fruit sugar, 0.5 uracil, 0.1 putrescine monohydrochloride, 10 mercaptoethanol, 50 oxidized glutathione, 50 D-xylose, 1-10 choline chloride methyl ester, 0.01 thioctic acid ethyl ester, 19.92 S-adenosyl-L-methionine ethyl ester and 3.725-5-deoxy-5-methylthioadenosine methyl ester.

3. The mesenchymal stem cell induced chondrogenic differentiation medium of claim 1, wherein the activity enhancing component is comprised of the following components: 600 L-serine methyl ester, 150 L-lysine ethyl ester, 900 L-glutamine dipeptide, 300 L-arginine methyl ester, 60 L-tyrosine ethyl ester, 1200 L-asparagine dipeptide, 450 L-proline methyl ester, 150 L-isoleucine ethyl ester, 900 L-phenylalanine methyl ester and 600 L-threonine ethyl ester; The signal regulation component consists of: 30 chondroitin sulfate, 60 sodium hyaluronate, 15 polylactic acid-glycolic acid copolymer, 5.5 heparin fragment, 2.75 transforming growth factor β1 mimic peptide, 1.05 insulin-like growth factor 1 analogue, 0.275 interleukin-6 inhibitor, 0.105 fibroblast growth factor 18 fragment and 0.275 Wnt signaling pathway activator; The metabolic support component consists of: 1250 glycerophosphate calcium, 300 calcium gluconate, 600 potassium phosphate monobasic, 300 magnesium chloride hexahydrate, 0.55 nickel nitrate hexahydrate, 0.275 ammonium molybdate tetrahydrate, 150 ethylene glycol bis-tetraacetic acid, 30 iron sodium ethylenediaminetetraacetate, 15 sodium selenite and 300 sodium bicarbonate; The auxiliary stabilizing component consists of: 150 2-ethanesulfonic acid, 600 polyvinylpyrrolidone K30, 30 ethyl pyruvate, 15 creatine monohydrate, 0.55 eicosapentaenoic acid ethyl ester, 6000 anhydrous fruit sugar, 2.75 uracil, 0.55 putrescine monohydrochloride, 30 mercaptoethanol, 150 oxidized glutathione, 150 D-xylose, 5.5 choline chloride methyl ester, 0.055 thioctic acid ethyl ester, 59.765 S-adenosyl-L-methionine ethyl ester and 9.2955-5-deoxy-5-methylthioadenosine methyl ester.

4. The mesenchymal stem cell-induced chondrogenic differentiation medium according to claim 1, wherein: The active enhancement component is composed of 1000 L-serine methyl ester, 250 L-lysine ethyl ester, 1500 L-glutamine dipeptide, 500 L-arginine methyl ester, 100 L-tyrosine ethyl ester, 2000 L-asparagine dipeptide, 750 L-proline methyl ester, 250 L-isoleucine ethyl ester, 1500 L-phenylalanine methyl ester and 1000 L-threonine ethyl ester; The signal regulation component is composed of 50 chondroitin sulfate, 100 sodium hyaluronate, 25 polylactic acid-glycolic acid copolymer, 10 heparin fragment, 5 transforming growth factor β1 mimic peptide, 2 insulin-like growth factor 1 analogue, 0.5 interleukin-6 inhibitor, 0.2 fibroblast growth factor 18 fragment and 0.5 Wnt signaling pathway activator; The metabolism support component is composed of 2000 glycerophosphate calcium, 500 calcium gluconate, 1000 potassium dihydrogen phosphate, 500 magnesium chloride hexahydrate, 1 nickel nitrate hexahydrate, 0.5 ammonium molybdate tetrahydrate, 250 ethylene glycol bis-tetraacetic acid, 50 iron sodium salt of ethylenediaminetetraacetic acid, 25 sodium selenite and 500 sodium bicarbonate; The auxiliary stability component is composed of 250 2-ethanesulfonic acid, 1000 polyvinylpyrrolidone K30, 50 pyruvic acid ethyl ester, 25 creatine monohydrate, 1 eicosapentaenoic acid ethyl ester, 10000 anhydrous fructose, 5 uracil, 1 putrescine monohydrochloride, 50 mercaptoethanol, 250 oxidized glutathione, 250 D-xylose, 10 choline chloride methyl ester, 0.1 ethyl lipoate, 99.61 S-adenosyl-L-methionine ethyl ester and 14.875- deoxy-5-methylthioadenosine methyl ester.

5. A method for preparing a mesenchymal stem cell-induced chondrogenic differentiation medium according to any one of claims 1 to 4, characterized in that: Specifically comprising the following steps: S1, according to the weight of the components, the active enhancement component, the signal regulation component, the metabolism support component and the auxiliary stability component are weighed and poured into a mixing device, and after being uniformly mixed, they are transferred into a grinding device for grinding. After grinding is completed, the obtained product is recorded as dry powder medium; S2, in a clean container, add deionized water with a volume of 70-80% of the final volume. Start stirring when the initial water temperature is 15-25℃, and add the dry powder medium in S1 according to the mass concentration of 35.68g / L under stirring conditions. After stirring for 20-30min, adjust the pH value to 8.50-8.70, continue stirring for 10-15min, then adjust the pH value to 6.50-6.70, and stir again for 10-15min. The obtained product is recorded as the premix component; S3, according to the mass concentration of 3g / L, add sodium bicarbonate powder to the premix component in S2, stir for 10-20min until completely dissolved, then adjust the pH value to 7.00-7.20, add deionized water to the final volume, and continue stirring for 5-20min. After filtering and sterilizing using a sterilization filter with a pore size of 0.22μm, the obtained product is the mesenchymal stem cell induced chondrogenic differentiation medium.

6. The method for preparing a culture medium for inducing mesenchymal stem cell differentiation into chondrocytes according to claim 5, characterized in that: The potassium hydroxide solution with a concentration of 4-8 mol / L and the nitric acid solution with a concentration of 4-8 mol / L are used to adjust the pH value in S2 and S3, and the stirring speed in S2 and S3 is 150-250 r / min.

7. The method for preparing a culture medium for inducing mesenchymal stem cell differentiation into chondrocytes according to claim 5, characterized in that: In the process of storing the dry powder medium in S1, the dry powder medium is divided into multiple layers of composite packaging, the outermost layer is a packaging bag made of a high-molecular material with elasticity and moisture-proof performance, each layer of packaging is sealed by using a heat sealing technology, a drying agent with a moisture absorption capacity of more than 30% is placed between two layers of packaging, and vacuum treatment is applied to reduce the residual moisture.

8. The method for preparing a culture medium for inducing mesenchymal stem cell differentiation into chondrocytes according to claim 5, characterized in that: The grinding device in S1 is equipped with a high-precision ball milling system, and the material is ground to a particle size of less than 50 microns.

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