Umbilical cord mesenchymal stem cell cartilage induction differentiation medium and application thereof

Abstract

The present invention provides a chondrogenic differentiation medium for umbilical cord mesenchymal stem cells and its application, belonging to the field of stem cell technology. It includes: a basal culture medium, an induction factor and an additive factor; the basal culture medium includes: DMEM basal culture medium, fetal bovine serum and proline; the induction factor includes: ITS, TGF-β3, dexamethasone and vitamin C; the additive factor includes: actipride. By adding actipride to the chondrogenic differentiation medium for umbilical cord mesenchymal stem cells at a certain concentration, actipride and other components can effectively promote the formation of acidic mucopolysaccharides in umbilical cord mesenchymal stem cells, and at the same time can increase the amount of acidic mucopolysaccharides and the size of spontaneously formed chondrocytes, and accelerate the chondrogenic differentiation cycle, solving the problems of slow formation of acidic mucopolysaccharides in chondrogenic differentiation, extremely small amount of acidic mucopolysaccharides, and difficulty in forming the volume of chondrocytes in the three-way differentiation identification of umbilical cord mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells, and particularly relates to a chondrogenic differentiation medium for umbilical cord mesenchymal stem cells and an application thereof. Background Art

[0002] Drug development has gone through different stages of small molecule chemical drugs, large molecule biological drugs and biological drugs. Biological drugs are drugs extracted from organisms or synthesized by bioengineering methods. They are essentially different from traditional chemical drugs that can be completely synthesized artificially. Traditional chemical drugs are generally small molecules, and biological drugs are generally complex mixtures or biological macromolecules. They have good efficacy and safety and can be divided into several major categories, such as antibody drugs, cytokines, blood products, gene and cell therapy drugs, enzymes and hormones. Among them, cell therapy drugs are one of the most popular drug development areas in the world in recent years. Cell therapy drugs can be divided into stem cell therapy and immune cell therapy according to cell types. Stem cells are cells with multidirectional differentiation potential and self-renewal ability. They are the most primitive cells at the top of the origin of cell development lineage. They can differentiate into cells of a certain specific tissue type in the body. The types of stem cells used for clinical treatment mainly include bone marrow stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, pancreatic stem cells, adipose stem cells, etc. Stem cell therapy uses the differentiation and repair principles of human stem cells to transplant healthy stem cells into the body to repair diseased cells or rebuild normal functioning cells and tissues.

[0003] Immune cells are responsible for killing harmful invaders, including human T cells, NK cells, B cells, DC cells, etc. Immune cell therapy is to collect the body's own immune cells, culture them in vitro, or transform them, and then return them to the body to kill pathogens, cancer cells, and mutated cells in the blood and tissues, break immune tolerance, and activate and enhance the body's immune ability.

[0004] Mesenchymal stem cells (MSC) are stem cells with multi-directional differentiation potential. According to the definition of the minimum standard of mesenchymal stem cells by the International Society for Cellular Therapy, mesenchymal stem cells should have the following characteristics: ① When cultured under standard conditions, they must have the property of adhering to plastic substrates; ② The positive rate of CD105, CD73 and CD90 expression in the surface markers of mesenchymal stem cells detected by flow cytometry is ≥95%, and the positive rate of CD45, CD34, CD14 or CD11b, CD79a or CD19, HLA-DR expression is ≤2%; ③ After induction by standard methods in vitro, mesenchymal stem cells can be induced to differentiate into osteoblasts, adipocytes and chondrocytes. The three-way differentiation of mesenchymal stem cells is one of the minimum standards for identifying mesenchymal stem cells, but there are great differences in the three-way differentiation ability of mesenchymal stem cells from different species and different tissue sources. Especially in the induced cartilage differentiation, umbilical cord mesenchymal stem cells (UC-MSCs) have the characteristics of a long induced differentiation cycle, slow formation of endocytic mucopolysaccharides, extremely small amount of endocytic mucopolysaccharides and extremely small endocytic mucopolysaccharides compared with adipose derived mesenchymal stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs). In view of the characteristics of umbilical cord mesenchymal chondrogenic differentiation, the present invention proposes a culture medium that can promote the efficient chondrogenic differentiation of umbilical cord mesenchymal stem cells, which can effectively promote the formation of acidic mucopolysaccharides in umbilical cord mesenchymal stem cells, while increasing the amount and size of acidic mucopolysaccharides, and accelerating the chondrogenic differentiation cycle, thereby solving the problems of slow formation of acidic mucopolysaccharides in chondrogenic differentiation, extremely small amount of acidic mucopolysaccharides, and extremely small size of acidic mucopolysaccharides in the three-way differentiation identification of umbilical cord mesenchymal stem cells.

[0005] Zuk et al. (Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. [J]. Tissue Engineering Part A, 2001, 7(2): 211-228.) proposed a classic chondrogenic differentiation medium in 2001, which contained DMEM basal medium, 10% fetal bovine serum, 6.25 μg / ml insulin, 50 nM vitamin C, and 10 ng / ml TGF-β1. This chondrogenic differentiation medium formed more and larger endocytic mucopolysaccharides during the differentiation of adipose-derived mesenchymal stem cells, but formed very little and very small endocytic mucopolysaccharides during the differentiation of umbilical cord-derived mesenchymal stem cells into chondrogenic tissue. When the induction time was extended to 2-3 times that of the chondrogenic differentiation of adipose-derived mesenchymal stem cells, there was no significant difference between the induced group and the uninduced group after Alcian blue staining, and the induction effect was still not ideal. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a chondrogenic differentiation medium for umbilical cord mesenchymal stem cells and its application. By adding actipride at a certain concentration into the chondrogenic differentiation medium for umbilical cord mesenchymal stem cells, actipride and other components can synergistically promote the formation of acidic mucopolysaccharides in umbilical cord mesenchymal stem cells, and at the same time increase the amount of acidic mucopolysaccharides and the size of spontaneously formed chondrocytes, and accelerate the chondrogenic differentiation cycle, thereby solving the problems of slow formation of acidic mucopolysaccharides in chondrogenic differentiation, extremely small amount of acidic mucopolysaccharides, difficulty in spontaneously forming chondrocytes, and small size in the three-way differentiation identification of umbilical cord mesenchymal stem cells.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides an umbilical cord mesenchymal stem cell chondrogenic differentiation medium, comprising: a basal medium, an induction factor and an additive factor; the basal medium comprises: DMEM basal medium, fetal bovine serum and proline; the induction factor comprises: ITS, TGF-β3, dexamethasone and vitamin C; the additive factor comprises: actipidil.

[0009] Furthermore, in the basic culture medium, fetal bovine serum: 0.5-15% of the total volume, proline 10-400 μg / ml.

[0010] Furthermore, among the induction factors, ITS: 0.1-20×, TGF-β3: 0.1-30 ng / ml, dexamethasone: 0.01-20 μM, vitamin C: 10-200 μg / ml.

[0011] Further, aptadil: 1-200ng / ml.

[0012] Further, fetal bovine serum: 0.75-5% of the total volume, dexamethasone: 0.02-5 μM, vitamin C: 20-120 μg / ml, proline: 20-200 μg / ml, ITS: 0.2-10×, TGF-β3: 5-20 ng / ml, and actipidil 50-100 ng / ml.

[0013] Further, fetal bovine serum: 1-2% of the total volume, dexamethasone: 0.03-0.2 μM, vitamin C: 40-70 μg / ml, proline: 30-60 μg / ml, ITS: 0.5-3×, TGF-β3: 8-15 ng / ml, and actipidil: 70-80 ng / ml.

[0014] On the other hand, the present invention provides a method for preparing the above-mentioned umbilical cord mesenchymal stem cell chondrogenic differentiation medium, the method comprising: adding fetal bovine serum and proline to the DMEM basal medium according to a ratio, and mixing and stirring evenly; then adding ITS, TGF-β3, dexamethasone and vitamin C, and mixing and stirring evenly; finally adding actipidil, and mixing and stirring evenly to obtain the medium.

[0015] On the other hand, the present invention provides a method for inducing umbilical cord mesenchymal stem cells into cartilage in vitro, comprising: inoculating umbilical cord mesenchymal stem cells in the above culture medium and inducing culture to obtain chondrocytes.

[0016] Furthermore, the umbilical cord mesenchymal stem cells are inoculated in any of the above-mentioned culture media; the culture media is replaced with new ones every 3-4 days; the induction differentiation cycle is 14-21 days, and chondrocytes are obtained after the induction differentiation is completed.

[0017] In another aspect, the present invention provides a chondrocyte, wherein the chondrocyte is obtained by inducing umbilical cord mesenchymal stem cells using the above-mentioned culture medium, or is prepared using the above-mentioned method.

[0018] The beneficial effects brought by the technical solution provided by the embodiment of the present invention include:

[0019] The present invention adds actipride to a chondrogenic differentiation medium of umbilical cord mesenchymal stem cells at a certain concentration. Actipride and other components can cooperate to effectively promote the formation of acidic mucopolysaccharides in umbilical cord mesenchymal stem cells, and can also increase the amount of acidic mucopolysaccharides and the size of spontaneously formed chondrocytes, and accelerate the chondrogenic differentiation cycle, thereby solving the problems of slow formation of acidic mucopolysaccharides in chondrogenic differentiation, extremely small amount of acidic mucopolysaccharides, difficulty in spontaneously forming chondrocytes, and small volume in the three-way differentiation identification of umbilical cord mesenchymal stem cells. Specifically, the mucopolysaccharides can be stained blue by alcian blue, and grayscale analysis of the stained area is performed by imageJ software. The present invention can enhance the grayscale by at least two times within the same induction time, and the relative viscosity of the prepared acidic mucopolysaccharides is increased by at least 45%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The chondrogenic spheres prepared by the F1 provided in the embodiment of the present invention after induction and differentiation;

[0022] Figure 2 The cell photos under an inverted microscope 20× objective lens after Alcian blue staining provided in the embodiment of the present invention, wherein A is the negative control group A, B is the control group B1, C is the control group B7, and D is the experimental group F6;

[0023] Figure 3 The photos of UC-MSCs cells after induced differentiation provided in the embodiments of the present invention, wherein A is the B1 control group and B is the F1 experimental group. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] The invention provides a chondrogenic differentiation medium for umbilical cord mesenchymal stem cells, comprising: a basic medium, an induction factor and an additive factor; the basic medium comprises: a DMEM basic medium, fetal bovine serum and proline; the induction factor comprises: ITS, TGF-β3, dexamethasone and vitamin C; the additive factor comprises: actipidil.

[0026] The present invention adds actipride to a chondrogenic differentiation medium of umbilical cord mesenchymal stem cells at a certain concentration. Actipride and other components can cooperate to effectively promote the formation of acidic mucopolysaccharides in umbilical cord mesenchymal stem cells, and can also increase the amount of acidic mucopolysaccharides and the size of spontaneously formed chondrocytes, and accelerate the chondrogenic differentiation cycle, thereby solving the problems of slow formation of acidic mucopolysaccharides in chondrogenic differentiation, extremely small amount of acidic mucopolysaccharides, difficulty in spontaneously forming chondrocytes, and small volume in three-way differentiation identification of umbilical cord mesenchymal stem cells. Specifically, mucopolysaccharides can be stained blue by alcian blue, and grayscale analysis of the stained area is performed by imagej software. The present invention can increase the grayscale by at least 23% within the same induction time, and in two-dimensional induction, the number of chondrocytes is increased by at least 40%, and the average diameter is increased by at least 31%.

[0027] Dexamethasone is a long-acting adrenal glucocorticoid that can directly bind to the glucocorticoid receptor and is located upstream of bone morphogenetic protein 6. It can upregulate the expression of bone morphogenetic protein 6 and is associated with the upregulation of AGC1 and the downregulation of COL10A1. Bone morphogenetic protein 6 can also induce the expression of transforming growth factor β receptor. The combination of bone morphogenetic protein 6 and transforming growth factor β3 promotes the chondrogenic differentiation of mesenchymal stem cells. At the same time, the combination of bone morphogenetic protein 6 and activin promotes the chondrogenic differentiation of mesenchymal stem cells, upregulates the expression of aggrecan, dermatonectin and type Ⅺ collagen genes, and enhances the differentiation of mesenchymal stem cells into chondrocytes. Vitamin C is beneficial to the hydroxylation of lysproline during collagen formation and thus promotes the formation of a three-dimensional structure. The transforming growth factor (TGF) family is a family of multifunctional protein polypeptides that have the functions of promoting cell proliferation, regulating cell differentiation, and promoting the synthesis of extracellular matrix. TGF-β3 is the earliest discovered bioactive molecule that can induce cartilage formation. It can not only promote the proliferation of chondrocytes, but also increase the synthesis rate of proteins in the extracellular matrix such as proteoglycans and hyaluronidase, and improve the ability of mesenchymal stem cells to differentiate into cartilage. Insulin is also of great significance in cartilage tissue engineering. Exogenous insulin can promote the mitosis of chondrocytes and the synthesis of type II collagen and GAG in the extracellular matrix. Atidil is a vasoactive intestinal polypeptide (VIP) analogue, composed of 28 amino acid residues, belonging to the secretin family. It is arranged as a part of glucagon and secretin. It has the effect of vasodilation and lowering blood pressure. It can be used for the study of pulmonary fibrosis, pulmonary arterial hypertension (PAH), and respiratory failure caused by SARS-CoV-2. Atidil is extremely widely distributed in the body. Most systems and organs have Atidil nerve distribution, and neutrophils and mast cells contain Atidil immunoreactive substances. Atipidil can stimulate gastrointestinal endocrine cells to secrete insulin, glucagon, somatostatin and pancreatic polypeptide, and excite enteric neurons. The present application can solve the problems of slow formation of acidic mucopolysaccharides in cartilage-induced differentiation, extremely small amount of acidic mucopolysaccharides, difficulty in spontaneously forming chondrocytes, and small volume in the prior art three-way differentiation identification of umbilical cord mesenchymal stem cells by creatively combining the above substances.

[0028] It should be pointed out that the technical solution provided by the present invention has a great advantage in inducing umbilical cord mesenchymal stem cells into cartilage. At the same time, the technical solution is also applicable to adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, chorionic mesenchymal stem cells, etc. The umbilical cord mesenchymal stem cell high-efficiency chondrogenic differentiation medium provided by the present invention is not only for the problem of difficulty in chondrogenicity of human umbilical cord mesenchymal stem cells, but also for other species (rats, mice, rhesus monkeys, beagles, etc.) and mesenchymal stem cells from other tissue sources (fat, bone marrow, placenta, dental pulp, etc.) into cartilage induction differentiation. It is also applicable, and compared with the internal acidic mucopolysaccharides formed after induced differentiation of the umbilical cord, mesenchymal stem cells from other species and other tissue sources have better effects, that is, more and larger internal acidic mucopolysaccharides, and shorter induced differentiation test cycles. In addition, the preparation method provided by the present invention is simple and easy, and the prepared high-efficiency chondrogenic differentiation medium can achieve stability, efficiency, and repeatability of chondrogenic differentiation.

[0029] The DMEM basal culture medium can be high-sugar or low-sugar, both of which can achieve the purpose of the present invention.

[0030] Specifically, in the basic culture medium, fetal bovine serum: 0.5-15% of the total volume, proline 10-400 μg / ml.

[0031] Specifically, among the inducing factors, ITS: 0.1-20×, TGF-β3: 0.1-30 ng / ml, dexamethasone: 0.01-20 μM, vitamin C: 10-200 μg / ml.

[0032] Specifically, aptadil: 1-200ng / ml.

[0033] In the embodiment of the present invention, ITS is a mixture of insulin (10 mg / mL), transferrin (5.5 mg / mL), and sodium selenite (6.7 μg / mL), 100×, and 60708ES10 from YEASEN was purchased. DMEM culture medium and fetal bovine serum were purchased from Gibco, and other unspecified components were commercially available.

[0034] Preferably, fetal bovine serum: 0.75-5% of the total volume, dexamethasone: 0.02-5 μM, vitamin C: 20-120 μg / ml, proline: 20-200 μg / ml, ITS: 0.2-10×, TGF-β3: 5-20 ng / ml, and actipidil 50-100 ng / ml.

[0035] Preferably, fetal bovine serum: 1-2% of the total volume, dexamethasone: 0.03-0.2 μM, vitamin C: 40-70 μg / ml, proline: 30-60 μg / ml, ITS: 0.5-3×, TGF-β3: 8-15 ng / ml, and actipidil: 70-80 ng / ml.

[0036] The present invention also provides a method for preparing the above-mentioned umbilical cord mesenchymal stem cell cartilage induction differentiation medium, the method comprising:

[0037] S10 Add fetal bovine serum and proline to the DMEM basal culture medium according to the ratio, and mix and stir evenly.

[0038] S11 then added ITS, TGF-β3, dexamethasone and vitamin C, and mixed them evenly.

[0039] Finally, add acetaminophen into S12, mix and stir evenly to obtain the product.

[0040] The present invention also provides a method for inducing umbilical cord mesenchymal stem cells into cartilage in vitro, wherein the umbilical cord mesenchymal stem cells are inoculated into the above culture medium and induced to culture to obtain chondrocytes.

[0041] Specifically, they include:

[0042] S20: inoculating the umbilical cord mesenchymal stem cells into the above-mentioned culture medium;

[0043] S21 replace new culture medium every 3-4 days;

[0044] The S22 induction differentiation cycle is 14-21 days, and chondrocytes are obtained after the induction differentiation is completed.

[0045] The embodiment of the present invention also provides a chondrocyte, wherein the chondrocyte is obtained by inducing umbilical cord mesenchymal stem cells using the above culture medium, or is prepared using the above method.

[0046] In order to better illustrate the embodiments of the present invention, the present invention is further described in detail below through specific examples.

[0047] Example 1

[0048] (1) This example provides umbilical cord mesenchymal stem cell chondrogenic differentiation culture medium with different components.

[0049] According to the experimental needs, three groups of culture medium preparation methods containing different components were set up, among which group A was a negative control group, group B was an experimental control group, and group F was the culture medium provided by the present invention as an experimental group.

[0050] Group A was the negative control group, which consisted of: fetal bovine serum (FBS): 1.5% of the total volume, proline 40 μg / ml.

[0051] Operate under sterile conditions. Add the above components according to the above proportions, mix well with a pipette and set aside.

[0052] Group B is the experimental control group, which includes B1-B8, and its culture medium components are as follows:

[0053] The B1 component consisted of: fetal bovine serum: 1% of the total volume, dexamethasone: 0.03 μM, vitamin C: 40 μg / ml, proline: 60 μg / ml, ITS: 0.5×, TGF-β3: 8 ng / ml.

[0054] The B2 component consisted of: fetal bovine serum: 2% of the total volume, dexamethasone: 0.2 μM, vitamin C: 70 μg / ml, proline: 60 μg / ml, ITS: 3×, TGF-β3: 15 ng / ml.

[0055] The B3 component consisted of: fetal bovine serum: 0.75 of the total volume, dexamethasone: 0.02 μM, vitamin C: 20 μg / ml, proline: 20 μg / ml, ITS: 0.2×, TGF-β3: 5 ng / ml.

[0056] The B4 component consisted of: fetal bovine serum: 5% of the total volume, dexamethasone: 5 μM, vitamin C: 120 μg / ml, proline: 200 μg / ml, ITS: 10×, TGF-β3: 20 ng / ml.

[0057] The B5 component consisted of: fetal bovine serum: 0.5% of the total volume, dexamethasone: 0.02 μM, vitamin C: 10 μg / ml, proline: 10 μg / ml, ITS: 0.1×, TGF-β3: 0.1 ng / ml.

[0058] The B6 component consisted of: fetal bovine serum: 15% of the total volume, dexamethasone: 20 μM, vitamin C: 200 μg / ml, proline: 400 μg / ml, ITS: 20×, TGF-β3: 30 ng / ml.

[0059] The difference between B7 and B6 culture medium is that 0.5 ng / ml acetaminophen was added to B7.

[0060] The difference between B8 and B6 culture medium is that 210 ng / ml of acetaminophen was added to B8.

[0061] The preparation method of group B is: operate under sterile environment, first mix the basic culture medium components by pipetting, then add the induction factor components by pipetting and mix, if there is acetaminophen, then add acetaminophen last and mix by pipetting.

[0062] Group F is the experimental group, and its components are as follows: when component B is prepared, it is prepared in duplicate, and different amounts of acetaminophen are added.

[0063] F1 is based on B1 with 70 ng / ml of acetaminophen added.

[0064] F2 is based on B2 with 80 ng / ml of acetaminophen added.

[0065] F3 is based on B3 with 50 ng / ml of acetaminophen added.

[0066] F4 is based on B4 with 100 ng / ml of acetaminophen added.

[0067] F5 is based on B5 with 1 ng / ml of acetaminophen added.

[0068] F6 is based on B6 with 200 ng / ml of acetaminophen added.

[0069] The preparation method of group F is as follows: operate under sterile environment, first mix the basic culture medium components by pipetting, then add the induction factor components by pipetting, and finally add actipidil and mix by pipetting.

[0070] (2) Methods of inducing chondrogenic differentiation of umbilical cord mesenchymal stem cells using different culture media.

[0071] S1 digestion and collection of human UC-MSCs were counted according to 5000-8000 cells / cm 2 The cells were inoculated at a density of 1.54 μg / mL in a 6-well cell culture plate with 2 mL / well of complete culture medium for human UC-MSCs and cultured in a 37°C, 5% CO2 incubator.

[0072] S2 When the cell fusion reaches 70-90%, discard the culture supernatant, add 2 mL of the prepared chondrogenic differentiation complete medium to each well, add 2 mL of the control group cell culture medium to the well, and culture in a 37°C, 5% CO2 incubator.

[0073] The S3 induction group was replaced with fresh chondrogenic differentiation complete medium every 3-4 days, and the control group was added with an equal volume of control medium and continued to be cultured in a 37°C, 5% CO2 incubator.

[0074] After 2-3 weeks of S4 induction, the induction culture was terminated when the cells in the experimental group lost their fibroblastic shape and grew densely compared with the control group under microscope. For the group with poor induction effect, the induction time was 3 weeks.

[0075] Taking the F1 experimental group as an example, the prepared chondrocytes are as follows: Figure 1 shown.

[0076] (3) Determination of the chondrogenic differentiation properties of umbilical cord mesenchymal stem cells using different culture media.

[0077] Staining: After the induction culture is completed, discard the culture supernatant, wash the cells twice with PBS, and fix the cells with 1 mL / well of 4% neutral paraformaldehyde solution at room temperature for 30 minutes (performed in a fume hood). Discard the fixative, wash twice with PBS, add 1 mL of Alcian blue staining solution to each well, and stain for 30 minutes at room temperature. Discard the Alcian blue staining solution, wash three times with PBS, and fully wash away the excess staining solution. Add 1 mL of PBS to each well, observe under a microscope and take pictures.

[0078] Grayscale analysis: After staining, the chondrocytes were observed in an inverted microscope imaging system and the images were collected. Four fields of view were randomly selected in each group to take pictures and collect pictures, and the grayscale analysis of the pictures was performed using imageJ software. The average value of each group was taken and normalized. The analysis results of each group are shown in Table 1.

[0079] Table 1 Number of chondrocytes in different groups

[0080]

[0081] For example, by Figure 2 It can be seen that the induction differentiation effect of B1 in the control group B is worse than that of B7, and the induction differentiation effect of B7 is worse than that of F6. The other groups not shown in the example have the same effect. The grayscale analysis of the stained area is performed by imageJ software. The present invention can increase the grayscale of F1-F6 by at least 2.23 times compared with B1-B6 within the same induction time. Figure 3 It can be seen that the effect of UC-MSCs cells induced and differentiated in the control group B1 is inferior to that in F1.

[0082] As shown in the random field of view pictures collected after random induction, B1-B6 in the experimental control group B could not effectively induce a large number of chondrocytes within 2 weeks, the induction effect and efficiency were limited, the amount of internal acid mucopolysaccharide was very small, the spontaneous formation of chondrocytes was difficult and the volume was small, and the induction differentiation effect of B7 and B8 in the control group B was improved compared with B1-B6, but the effect was still relatively poor; and the induction effect of F1-F6 in the embodiment of the present invention was significantly better than that of the experimental control group B, and the induction differentiation effect in the experimental control group was from best to worst: F1 and F2, F3 and F4, F5 and F6, and the number of chondrocytes induced and differentiated by the acetaminophen induction group was significantly more than the number of osteoblasts induced by the above groups. It can be seen from the counting results in Table 1 that F1 and F2 have about 6 times more chondrocytes differentiated than the induction group B. F3 and F4 have about 5 times more chondrocytes differentiated than the classical culture medium induction group B. The number of differentiated chondrocytes in F5 and F6 was about 4 times higher than that in induced group B, the amount of acidic mucopolysaccharide was high, and the spontaneously formed chondrocytes were large in volume and efficient.

[0083] Determination of the size of endo-acid mucopolysaccharide: The size of endo-acid mucopolysaccharide is determined by its degree of polymerization. The higher the degree of polymerization, the larger the endo-acid mucopolysaccharide is, and the higher the viscosity is; the lower the degree of polymerization, the smaller the endo-acid mucopolysaccharide is, and the lower the viscosity is. This application conducts a qualitative measurement of the viscosity to determine the size of the endo-acid mucopolysaccharide prepared in different test examples. The viscosity is measured using an existing measurement method, preferably, a rotary viscometer is used for measurement. The control group is taken as 1, and the analysis results of the other groups are shown in Table 2.

[0084] Table 2 Relative viscosity of different groups

[0085]

[0086] In the present invention, F1-F6 can increase the relative viscosity by at least 45% compared with B1-B6 within the same induction time, so that the internal acid mucopolysaccharide prepared by the technical solution of the present application is larger and has a high degree of polymerization.

[0087] In conclusion, human umbilical cord mesenchymal stem cells cultured in the induction medium supplemented with acetaminophen were better differentiated into chondrocytes than the other groups.

[0088] 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 chondrogenic differentiation medium for umbilical cord mesenchymal stem cells, characterized in that: include: Basal medium, induction factors and supplementary factors; The basic culture medium comprises: DMEM basic culture medium, fetal bovine serum and proline; The induction factors include: ITS, TGF-β3, dexamethasone and vitamin C; The added factors include: actipidil.

2. The umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to claim 1, characterized in that: In the basic culture medium, fetal bovine serum accounts for 0.5-15% of the total volume, and proline accounts for 10-400 μg / ml.

3. The umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to claim 2, characterized in that: Among the inducing factors, ITS: 0.1-20×, TGF-β3: 0.1-30 ng / ml, dexamethasone: 0.01-20 μM, vitamin C: 10-200 μg / ml.

4. The umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to claim 3, characterized in that: Atridil: 1-200ng / ml.

5. The umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to claim 4, characterized in that: Fetal bovine serum: 0.75-5% of total volume, dexamethasone: 0.02-5μM, vitamin C: 20-120μg / ml, proline: 20-200μg / ml, ITS: 0.2-10×, TGF-β3: 5-20ng / ml, 50-100ng / ml.

6. The umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to claim 5, characterized in that: Fetal bovine serum: 1-2% of total volume, dexamethasone: 0.03-0.2μM, vitamin C: 40-70μg / ml, proline: 30-60μg / ml, ITS: 0.5-3×, TGF-β3: 8-15ng / ml, acetaminophen: 70-80ng / ml.

7. A method for preparing the umbilical cord mesenchymal stem cell chondrogenic differentiation medium according to any one of claims 1 to 6, characterized in that: The method comprises: Add fetal bovine serum and proline to the DMEM basal medium according to the ratio, and mix and stir evenly; Then add ITS, TGF-β3, dexamethasone and vitamin C, mix and stir evenly; Finally, add acetaminophen and mix well to obtain the product.

8. A method for inducing umbilical cord mesenchymal stem cells into cartilage in vitro, characterized in that: include: Umbilical cord mesenchymal stem cells are inoculated in the culture medium as claimed in any one of claims 1 to 6 and induced to obtain chondrocytes.

9. The method according to claim 8, characterized in that include: Inoculating umbilical cord mesenchymal stem cells in the culture medium according to any one of claims 1 to 6; Replace the culture medium as described in any one of claims 1 to 6 with a new one every 3-4 days; The induction differentiation cycle is 14-21 days, and chondrocytes are obtained after the induction differentiation is completed.

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