Human umbilical cord mesenchymal stem cell chondrogenic induction differentiation culture medium, preparation method and application thereof

Abstract

The application discloses a human umbilical cord mesenchymal stem cell chondrogenic induction differentiation culture medium, a preparation method and application thereof. The culture medium comprises a basic culture medium, induction factors and additive factors; the induction factors comprise dexamethasone, TGF-beta 1, vitamin C phosphate and ITS+1; the additive factors comprise alisol B; the addition amount of alisol B is 50 micromoles / L or more. The application also provides a preparation method of the culture medium. The application further provides a method for in-vitro induction and directional differentiation of human umbilical cord mesenchymal stem cells into chondrocytes and quantification of the chondrocytes, and provides the chondrocytes obtained by the method. The application adds a certain amount of alisol B into a classical chondrogenic induction differentiation complete culture medium, and the obtained induction differentiation culture medium can significantly increase the differentiation number of chondrocytes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell technology, in particular to a human umbilical cord mesenchymal stem cell chondrogenic induction differentiation culture medium, a preparation method and applications thereof. BACKGROUND

[0002] Articular cartilage injury is a common clinical condition, which is not only high in the elderly population, but also has a high incidence in the young population in recent years. The lack of direct nutrition of blood vessels in articular cartilage, the lack of high-quality and effective migration ability of chondrocytes and stem cells, which leads to the limitation of the self-repairing ability of articular cartilage. The existing articular cartilage injury is treated by surgical methods, including subchondral drilling, microfracture, abrasion arthroplasty, joint replacement and autologous chondrocyte transplantation, etc. However, these methods have limitations due to technical difficulties, poor tissue integration and the influence of developing fibrocartilage, and cannot meet the needs of clinical cartilage tissue repair treatment. Therefore, seeking new treatment methods is an urgent problem to be solved for repairing cartilage tissue damage.

[0003] In recent years, the research on cartilage injury repair has gradually shifted to the direction of cell transplantation and cartilage tissue engineering, among which chondrocytes and mesenchymal stem cells have attracted more and more attention in the field of cartilage tissue engineering research.

[0004] Human umbilical cord mesenchymal stem cells (hUCMSCs) are a kind of mesenchymal stem cells, which have the potential of multi-directional differentiation and can differentiate into chondrocytes. Compared with bone marrow mesenchymal stem cells which are relatively easily affected by donor age factors and fetal-derived mesenchymal stem cells which are easily restricted by medical ethics, human umbilical cord mesenchymal stem cells have the characteristics of high proliferation efficiency, wide donor, low viral infection rate and low immunogenicity, and may become a better choice for clinical cartilage tissue engineering research.

[0005] Human umbilical cord mesenchymal stem cells have chondrogenic differentiation potential. In the process of inducing and culturing human umbilical cord mesenchymal stem cells into chondroblasts in vitro, the ability of human umbilical cord mesenchymal stem cells to differentiate into chondroblasts can be enhanced by adding exogenous growth factors. Among them, transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP) and insulin-like growth factor-1 (IGF-1) are several key factors for regulating the chondrogenic differentiation of stem cells. These key factors can induce the directional differentiation of stem cells into chondrocytes by regulating the expression of several key genes (COL1, Aggrecan and SOX9) that promote chondrocyte formation.

[0006] The ability of human umbilical cord mesenchymal stem cells to differentiate into chondroblasts can be improved by adding exogenous inducing factors in the complete culture medium for culturing human umbilical cord mesenchymal stem cells. However, the existing inducing medium for inducing human umbilical cord mesenchymal stem cells to differentiate into chondrocytes has the problems of less number of chondrocytes after directional induction, uneven distribution of cartilage lacunae and poor differentiation effect, which cannot effectively induce human umbilical cord mesenchymal stem cells to obtain the required chondrocytes, and hinders the development of various studies in the field of cell transplantation cartilage tissue engineering. SUMMARY

[0007] The present application provides a human umbilical cord mesenchymal stem cell chondrogenic induction and differentiation medium, a preparation method and applications thereof.

[0008] The first aspect of the present application provides a human umbilical cord mesenchymal stem cell chondrogenic induction and differentiation medium, which comprises a basic culture medium, an inducing factor and an additive factor.

[0009] The composition of the basic culture medium comprises penicillin, streptomycin, glutamine, FBS and high-glucose DMEM culture medium.

[0010] The inducing factor comprises dexamethasone, TGF-β1, vitamin C phosphate and ITS+1.

[0011] The additive factor comprises alisol B. The addition amount (content in the human umbilical cord mesenchymal stem cell chondrogenic induction and differentiation medium) of alisol B is 50 μmol / L or more.

[0012] In some specific embodiments, the basic culture medium and the inducing factor can be prepared by adding the components in the proportions of the classic chondrogenic induction and differentiation complete culture medium.

[0013] In some embodiments, alisol B is selected at an upper limit value, for example 200 μmol / L, according to the requirements for formulating the chondrogenic induction complete medium of the present application.

[0014] In some embodiments, the ITS+1 (ITS+1 Liquid Media Supplement) used in the present application is a mixture prepared by adding the following components in the following proportions: 10 mg / ml recombinant human insulin, 0.55 mg / ml human transferrin, 0.5 μg / ml sodium selenite, 50 mg / ml BSA, 470 μg / ml linoleic acid, and 5 ml EBSS without phenol red.

[0015] In some embodiments, the medium of the present application is mainly composed of the above components (except as otherwise specified, the remaining components of the medium or mixture described in the present application are water).

[0016] To demonstrate that the medium provided by the present application is suitable for the directional differentiation induction of the stem cells, the prepared medium is set in 10 different groups according to the experimental requirements and is recorded as A-F5. Each group is cultured with the corresponding medium, and the human umbilical cord mesenchymal stem cells are cultured for 21-28 days. Then, the agar-embedded sections are dyed with Alcian blue staining solution, and the differentiation degree of the human umbilical cord mesenchymal stem cells induced by the different combinations of the medium is observed under an inverted microscope or a normal microscope.

[0017] After the above medium provided by the present application is used to induce the differentiation of chondrocytes by the conventional induction culture method in the prior art, the obtained cells are dyed with Alcian blue staining solution, and the chondrocyte lacunae are observed under a fluorescent inverted microscope. The results show that, compared with the chondrogenic cells cultured in the ordinary medium and the commercialized medium, the intracellular acid mucopolysaccharides of the chondrocytes cultured in the medium composition F3 of the present application are dyed blue by Alcian blue, the chondrocyte lacunae are dispersed and smooth, and the density and quantity of the chondrocytes are much higher than those obtained by using other conventional media and the medium with other concentrations of alisol B. It is proved that the medium provided by the present application has a good directional differentiation induction effect on the human umbilical cord mesenchymal stem cells, and is especially suitable for inducing the differentiation of the human umbilical cord mesenchymal stem cells into chondrocytes.

[0018] In some embodiments, preferably, the amount of alisol B added is 50 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, or 200 μmol / L. The addition of alisol B in these proportions can achieve the above culture and differentiation effects.

[0019] In some embodiments, preferably, the amount of alisol B added is 100 μmol / L. With this proportion of alisol B added, the number of chondrocytes obtained is the highest, 6 times that of the classic medium control group. The differentiation effect is optimal.

[0020] In some embodiments, preferably, the amounts of penicillin, streptomycin, fetal bovine serum, and glutamine are respectively:

[0021] Penicillin: 100 U / ml, streptomycin: 100 μg / ml, FBS: 5% of the total volume, glutamine: 2 mM / L.

[0022] In some embodiments, preferably, the amounts of dexamethasone, TGF-β1, vitamin C phosphate, and ITS+1 are respectively: dexamethasone: 10 μg / ml, TGF-β1: 10 ng / ml, vitamin C phosphate: 50 μg / ml, and ITS+1: 1%.

[0023] The inducing factors are added to the basic medium to prepare a classic chondrogenic induction and differentiation medium, which, in addition to the basic medium, contains 10 μg / ml dexamethasone, 10 ng / ml TGF-β1, 50 μg / ml vitamin C phosphate, and 1% ITS+1; the above-mentioned substances are mixed in this proportion, which can effectively assist alisol B in playing a role in the directional differentiation of the stem cells.

[0024] In some embodiments, preferably, the amounts of the components of ITS+1 are respectively: recombinant human insulin: 10 mg / ml, human transferrin: 0.55 mg / ml, sodium selenite: 0.5 μg / ml, BSA: 50 mg / ml, linoleic acid: 470 μg / ml, and EBSS without phenol red: 5 ml.

[0025] Another aspect of the present application also provides a method for preparing the chondrogenic induction and differentiation medium for human umbilical cord mesenchymal stem cells described above, which comprises:

[0026] 1) adding 5% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM / L glutamine respectively to high-glucose DMEM medium, and mixing and stirring uniformly;

[0027] 2) then adding 10 μg / ml dexamethasone, 10 ng / ml TGF-β1, 50 μg / ml vitamin C phosphate, and 1% ITS+1 respectively, and mixing uniformly to obtain a mixed medium;

[0028] 3) adding alisol B to the mixed medium and mixing uniformly to obtain a chondrogenic induction and differentiation medium for human umbilical cord mesenchymal stem cells.

[0029] The preparation method of the present application is preferably operated in a sterile environment.

[0030] Preferably, alisol B is added into the chondrogenic induction medium of human umbilical cord mesenchymal stem cells at a final concentration of 50 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L or 200 μmol / L; further preferably, the concentration of alisol B is 100 μmol / L.

[0031] The medium is prepared by the above method, which is simple and efficient.

[0032] Another aspect of the present application also provides a method for inducing and differentiating human umbilical cord mesenchymal stem cells into chondrocytes in vitro, which comprises:

[0033] The human umbilical cord mesenchymal stem cells are inoculated into the chondrogenic induction medium of human umbilical cord mesenchymal stem cells and induced to culture to obtain chondrocytes.

[0034] In some specific embodiments, the method for inducing and differentiating human umbilical cord mesenchymal stem cells into chondrocytes in vitro further comprises the in vitro isolation, culture and confirmation of human umbilical cord mesenchymal stem cells:

[0035] 1) Isolation and culture of human umbilical cord mesenchymal stem cells in vitro:

[0036] Specifically, the umbilical cord is taken from a pregnant woman of 18-26 years old, healthy, first pregnancy, no genetic disease history and serious disease history, and cesarean section, the Wharton's jelly of the umbilical cord is separated under sterile conditions, the human umbilical cord mesenchymal stem cells are cultured and expanded with serum-free and phenol-free mesenchymal stem cell culture medium, the cells are digested with recombinant trypsin after the cells grow and multiply to a certain number, and the cells are subcultured for multiple times until the P10 generation is reached.

[0037] 2) Sampling and flow cytometry to identify cell surface antigens:

[0038] Specifically, the human umbilical cord mesenchymal stem cells are subjected to surface antibody labeling, and the proportion of the labeled antibodies is determined by flow cytometry to confirm the human umbilical cord mesenchymal stem cells; more specifically, the surface antibody labeling sites are CD90, CD105, CD73, CD34, CD19, CD45, CD11b and HLA-DR.

[0039] After that, the confirmed human umbilical cord mesenchymal stem cells are inoculated into the above-mentioned medium and induced to culture to obtain chondrocytes.

[0040] The obtained chondrocytes are counted after agar embedding, slicing by a slicing machine and staining with Alcian blue.

[0041] The method separates and cultures human umbilical cord mesenchymal stem cells from neonatal umbilical cords, and identifies the purity of the cultured mesenchymal stem cells by flow cytometry. The cells identified by flow cytometry have higher purity and higher reliability and accuracy for subsequent differentiation results. Then, the human umbilical cord mesenchymal stem cells are cultured by setting different induction groups of culture medium to induce them to differentiate into chondrocytes. After staining with Alcian blue staining solution, the differentiation effect of chondrocyte cells is observed under a fluorescence inverted microscope. The method is simple and can intuitively obtain the induction results of different induction groups, and the optimal medium combination scheme is obtained by comparison.

[0042] Another aspect of the present application also provides a chondrocyte induced and cultured by the above-mentioned culture medium or the above-mentioned induction and directional differentiation method. After staining with Alcian blue staining solution, the chondrocyte lacuna is observed under an inverted microscope or a upright microscope. The results show that, compared with the chondroblast cells cultured in ordinary culture medium and commercial culture medium, the intracellular acid mucopolysaccharide of the obtained chondrocyte is stained blue by Alcian blue, and the chondrocyte lacuna is dispersed and smooth. The chondrocyte with the morphological structure of the present application can better form chondrocyte spheres, and the chondrocyte lacuna is more uniformly dispersed.

[0043] The beneficial effects of the present application include:

[0044] 1) The human umbilical cord mesenchymal stem cell chondrogenic induction and differentiation culture medium, the preparation method and the application thereof provided by the present application induce the directional differentiation of human umbilical cord mesenchymal stem cells into chondrocytes by adding exogenous growth factors and alisol B in the ordinary culture medium for culturing human umbilical cord mesenchymal stem cells. The results show that the classic chondrogenic induction culture medium added with alisol B has better induction and differentiation effect than other combined culture media, which provides a more optimal scheme for culturing human umbilical cord mesenchymal stem cells into chondrocytes.

[0045] 2) The human umbilical cord mesenchymal stem cell chondrogenic induction and differentiation culture medium, the preparation method and the application thereof provided by the present application, after the human umbilical cord mesenchymal stem cells are induced and cultured into chondrocytes by using the culture medium, more chondroblasts are differentiated, the chondrocyte lacuna is more uniformly dispersed after Alcian blue staining, the differentiation degree is better, the cell differentiation degree is higher, the time for differentiation into chondrocytes is shorter, and the operation is simple. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The result graph of the mesenchymal stem cells separated and cultured from human umbilical cords.

[0047] Figure 2 The result graph of the surface markers of the mesenchymal stem cells separated from the umbilical cord identified by flow cytometry.

[0048] Figure 3 Photo of the chondrocyte pellet at the end of the chondrogenic induction of human umbilical cord mesenchymal stem cells into chondrocytes.

[0049] Figure 4 Alcian blue staining results of chondrogenic induction of human umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION

[0050] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, 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.

[0051] The materials used in the following examples are commercially available unless otherwise specified.

[0052] Example 1: Isolation and culture of human umbilical cord mesenchymal stem cells

[0053] The present example provides a method for isolating and culturing human umbilical cord mesenchymal stem cells, and the specific operation is as follows:

[0054] 1) Collect and clean fresh umbilical cord: place the collected fresh umbilical cord in a 145 culture dish, wash with PBS to remove blood stains on the surface of the umbilical cord, and immerse the umbilical cord in PBS containing 1% double-antibiotic (penicillin and streptomycin) for 10 min, and gently scrape the blood clots inside the umbilical cord with forceps along the umbilical cord;

[0055] 2) Isolate Wharton's jelly: immerse the cleaned umbilical cord in PBS, cut it into small pieces of about 2 cm with scissors, cut the umbilical cord along one side of the vein, and then peel off the inside vein and artery with forceps, and then separate the Wharton's jelly and place it in a new culture dish containing a small amount of PBS;

[0056] 3) Subculture of Wharton's jelly: cut the Wharton's jelly into 1 mm long pieces, resuspend them in a small amount of mesenchymal stem cell complete medium, and then plate them in a T75 culture flask, and incubate them in a 37°C, 5% CO2 incubator; add 5 ml of complete medium after 2 days of culture, and continue to observe during the period, and continue to add 5 ml of complete medium after the cells grow out, until the cells are sufficient for subculture;

[0057] 4) Collect and wash the primary cells: discard the culture supernatant, wash the cell surface with PBS once, remove the tissue pieces as much as possible, add 4 ml of recombinant trypsin and incubate at 37°C for 4 min, then add 5 ml of complete medium to terminate digestion, collect the cells, and continue to collect the cells by washing with PBS once;

[0058] 5) Passage culture: the collected cell suspension was passed through a 70 μm cell screen, the filtrate was collected, centrifuged at 400 x g / min, 20°C for 5 min, the supernatant was discarded, 100 μl of the cells were resuspended with complete culture medium and counted by trypan blue staining method, 8000 cells / cm 2 were inoculated at a density of 8000 cells / cm

[0059] The results of the mesenchymal stem cells isolated and cultured from the human umbilical cord are shown in Figure 1 Figures A, B, C and D, wherein Figure A is a result of the passage culture of the human umbilical cord mesenchymal stem cells to P2 generation, Figure B is a result of the passage culture of the human umbilical cord mesenchymal stem cells to P4 generation, Figure C is a result of the passage culture of the human umbilical cord mesenchymal stem cells to P5 generation, and Figure D is a result of the passage culture of the human umbilical cord mesenchymal stem cells to P10 generation. Figure 1 It can be seen that the mesenchymal stem cells were successfully isolated and cultured from the human umbilical cord according to the above method, and different generations of cells were obtained by multiple passage culture, and the cells grew well in the culture process and had a regular spindle structure.

[0060] Example 2: Identification of surface antigens of mesenchymal stem cells isolated from human umbilical cord by flow cytometry

[0061] This example provides a method for identifying the surface antigens of mesenchymal stem cells isolated from human umbilical cord by flow cytometry, which is specifically as follows:

[0062] 1) Prepare cell suspension: select P5 cells in Example 1, digest with trypsin to prepare cell suspension, and count 100 μl of cells by trypan blue staining method. Wash the cell suspension with PBS twice, centrifuge at 1500 rpm for 5 minutes, discard the supernatant. Resuspend the cell pellet with PBS, adjust the cell concentration to 1 x 10 7 6 cells / ml, and filter with a 200-mesh 70-μm cell screen to remove cell clumps that are not fully digested.

[0063] 2) Surface antibody labeling of cells: take 8 2-ml centrifuge tubes, label them 1-8 respectively, add 100 μl of filtered cell suspension to each centrifuge tube. Add the corresponding components of the human umbilical cord mesenchymal stem cell flow detection kit (BD) according to Table 1 to perform antibody labeling.

[0064] Table 1. Surface antibody labeling of human umbilical cord mesenchymal stem cells

[0065]

[0066] 3) Antibody incubation: After mixing the components of each tube well, incubate at 2-8°C for 30 min in the dark. After incubation, wash twice with PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cell pellet with 500 μl of PBS, and then use a BD Accuri C6 flow cytometer for sample loading and detection.

[0067] 4) Set the flow cytometer conditions: Before sample loading, open the BD Accuri C6 Plus software, and gently resuspend the cells in the flow tube. Place the sample tube at the sample loading needle. Set the gates according to the light scattering properties of the cells, and collect at least 20,000 cells in the gate.

[0068] Each sample tube has the following functions: tubes 1-4 are single-staining tubes for adjusting the thresholds and compensation of each channel of the flow cytometer and defining the positive and negative reference; tube 5 is a blank control for adjusting the cells and instrument background; tube 6 is a positive and negative isotype antibody control for determining the non-specific signal of the fluorescent antibody and defining the positive and negative reference; tubes 7 and 8 are the test sample detection tubes.

[0069] 5) Result analysis: After sample loading is complete, analyze the sample loading results. Create scatter plots and histograms based on the collected gates, and calculate the percentage of positive cells and the percentage of negative cells.

[0070] The results of surface antibody (CD90, CD105, CD73, CD34, CD19, CD45, CD11b, and HLA-DR) labeling are shown in Figure 2 . Figure 2 The results of flow cytometry for identifying the surface markers of mesenchymal stem cells isolated from umbilical cords are shown in the figure. Figure a is the result of surface antibody CD90 labeling. Figure b is the result of surface antibody CD34, CD19, CD45, CD11b, and HLA-DR labeling. Figure c is the result of surface antibody CD105 labeling. Figure d is the result of surface antibody CD73 labeling. As can be seen from Figure 2 , the cell contains specific antigen markers CD90, CD105, and CD73 of mesenchymal stem cells, and does not contain markers such as CD34, CD19, CD45, CD11b, and HLA-DR, which can confirm that it is a mesenchymal stem cell.

[0071] Example 3: Preparation of chondroblast cell induction and differentiation medium with different components

[0072] This example provides an induction and differentiation medium with different components.

[0073] According to the experimental requirements, A-F5, a total of 10 groups of culture medium containing different components are set. Group A is a negative control group, group B is a classic complete chondrogenic induction medium group, groups C-E are commercial chondrogenic induction medium culture groups, and groups F1-F5 are the culture medium provided by the present application as experimental groups (alisol B induction group). The components of groups F1-F5 are the same except that the content of alisol B is different, and the content of alisol B in each group F1-F5 is as follows: the content of alisol B in group F1 is 50 μmol / L, the content of alisol B in group F2 is 80 μmol / L, the content of alisol B in group F3 is 100 μmol / L, the content of alisol B in group F4 is 150 μmol / L, and the content of alisol B in group F5 is 200 μmol / L. The components and preparation methods of each group of culture medium are as follows:

[0074] (1) Group A: a negative control group (basic medium group), the components of which are shown in Table 2:

[0075] Table 2. Basic medium

[0076] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml

[0077] Specific operation steps:

[0078] 1) Prepare double-antibiotic stock solution: operate in a sterile environment. Dissolve 800,000 units of penicillin powder in 2 ml of normal saline and store at -20°C for standby. Weigh 1 g of streptomycin powder and dissolve it in 4 ml of normal saline. Store the aliquots at -20°C for standby.

[0079] 2) Prepare the basic medium: take 45 ml of high-sugar DMEM culture medium in a 50 ml centrifuge tube, then add the corresponding proportions of FBS, double-antibiotic and glutamine (the concentrations of each component described in the present application are the concentrations in the total volume) according to Table 2. Mix well with a pipette gun to obtain the basic medium.

[0080] (2) Group B: a classic complete chondrogenic induction medium group, the components of which are shown in Table 3:

[0081] Table 3. Classic complete chondrogenic induction medium

[0082] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v)

[0083] Operate in a sterile environment. First, prepare ITS+1 according to Table 4.

[0084] Table 4. Component table of ITS+1

[0085] Ingredients Amount Recombinant human insulin 10 mg / ml Human transferrin 0.55 mg / ml Sodium selenite 0.5 μg / ml BSA 50 mg / ml Linoleic acid 470 μg / ml EBSS without phenol red 5ml

[0086] Specific operation steps:

[0087] 1) In a sterile environment, 10 mg / ml recombinant human insulin, 0.55 mg / ml human transferrin, 0.5 μg / ml sodium selenite, 50 mg / ml BSA and 470 μg / ml linoleic acid were added into 5 ml EBSS without phenol red, respectively, to obtain ITS+1 stock solution.

[0088] 2) 5% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mM / L glutamine were added into high glucose DMEM medium, respectively, to obtain basal medium stock solution.

[0089] 3) Then, 10 μg / ml dexamethasone, 10 ng / ml TGF-β1, 50 μg / ml vitamin C phosphate and 1% ITS+1 were added into the basal medium, respectively, according to Table 3, and mixed to prepare a classic chondrogenic induction medium.

[0090] (3) Groups C-E were chondrogenic induction medium culture groups prepared by commercial kits.

[0091] Group C: chondrogenic induction medium group prepared by a kit purchased from Promcell (the obtained results are shown in Fig. C of Figure 4 );

[0092] Group D: chondrogenic induction medium group prepared by a kit purchased from Sciencell (the obtained results are shown in Fig. D of Figure 4 );

[0093] Group E: chondrogenic induction medium group prepared by a kit purchased from Sanyou Biotech (the obtained results are shown in Fig. E of Figure 4 );

[0094] Groups C-E were prepared according to the operation methods of the corresponding kit instructions.

[0095] (4) Group F1: the medium provided by the present application was used as an experimental group (alisol B induction group). The components are shown in Table 5:

[0096] Table 5. Medium of the experimental group

[0097] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v) Alisol B 50 μmol / L

[0098] Group F2: the medium provided by the present application was used as an experimental group (alisol B induction group). The components are shown in Table 6:

[0099] Table 6. Medium of the experimental group

[0100] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v) Alisol B 80 μmol / L

[0101] F3 group: the medium provided by the present application as an experimental group (alisol B induction group). Its components are shown in Table 7:

[0102] Table 7. Experimental group medium

[0103] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v) Alisol B 100 μmol / L

[0104] F4 group: the medium provided by the present application as an experimental group (alisol B induction group). Its components are shown in Table 8:

[0105] Table 8. Experimental group medium

[0106] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v) Alisol B 150 μmol / L

[0107] F5 group: the medium provided by the present application as an experimental group (alisol B induction group). Its components are shown in Table 9:

[0108] Table 9. Experimental group medium

[0109] Ingredients Amount Fetal bovine serum (FBS) 5% (v / v) Penicillin 100 U / ml Streptomycin 100 μg / ml Glutamine 2 mM / L High glucose DMEM medium 45ml Dexamethasone 10 μg / ml TGF-β1 10 ng / ml Vitamin C phosphate 50 μg / ml ITS+1 1% (v / v) Alisol B 200 μmol / L

[0110] F1-F5 groups are experimental groups in this example, and the preparation method of the medium is as follows: first, prepare the classic complete chondrogenic induction and differentiation medium according to the preparation method of group B, and then add 50 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L alisol B to each group, respectively, and mix the components with a pipette gun for standby.

[0111] Example 4: Chondrogenic cell induction and differentiation of human umbilical cord mesenchymal stem cells in different media

[0112] In this example, the 10 groups of media prepared in Example 3 were used for chondrogenic cell induction and differentiation of human umbilical cord mesenchymal stem cells. The specific operation is as follows:

[0113] (1) Cell passage and grouping: when the human umbilical cord mesenchymal stem cells cultured to P5 generation reached 80-90% confluence, trypsin was used for digestion treatment, washed twice with mesenchymal stem cell culture medium, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The precipitate was resuspended with mesenchymal stem cell culture medium, and 100 μl was taken for cell counting by trypan blue staining method.

[0114] (2) Cell induction culture: 15 ml centrifuge tubes were used for each of the 10 groups of media prepared in Example 3, and three biological replicates were set up for each group. The human umbilical cord mesenchymal stem cells treated in (1) were inoculated at 5×10 5The cells were inoculated in 15 ml centrifuge tubes at a cell density of 500 cells / tube, centrifuged at 500 g for 5 min, the supernatant was removed, 0.5 ml of the corresponding medium of each group was added to the centrifuge tube to resuspend the cell pellet, and the tube was centrifuged at 500 g for 5 min again. The cap of the centrifuge tube was loosened to facilitate gas exchange, and the tube was placed in a 37°C incubator with 5% CO2. Then, the medium in each centrifuge tube was removed every 3 days, and fresh medium of the corresponding group was added. After induction for 21-28 days, the cell morphology and growth were observed, and the photographs were taken and saved. The photographs of each group are shown in Figure 3

[0115] Figure 3 The photographs of the chondrocyte pellets at the end of the chondrocyte differentiation culture of the human umbilical cord mesenchymal stem cells. Photograph A is the photograph of the chondrocyte pellets at the end of the culture of the negative control group; photograph B is the photograph of the chondrocyte pellets at the end of the culture of the classic chondrocyte induction medium; photograph C is the photograph of the chondrocyte pellets at the end of the culture of the chondrocyte induction medium prepared from the kit purchased from Promcell; photograph D is the photograph of the chondrocyte pellets at the end of the culture of the chondrocyte induction medium prepared from the kit purchased from Sciencell; photograph E is the photograph of the chondrocyte pellets at the end of the culture of the chondrocyte induction medium prepared from the kit purchased from Saiye Biological; and photographs F1-F5 are the photographs of the chondrocyte pellets at the end of the culture of the chondrocyte induction medium provided by the application.

[0116] As shown in Figure 3 , except that the cells of the negative control group were not spherical, the cells of the other induction groups were spherical.

[0117] Example 5: Alcian blue staining identification

[0118] This example provides a method for staining with an alcian blue staining solution after agar embedding sectioning after the end of chondrocyte induction and differentiation, and analysis of the induction and differentiation results of 10 groups of culture media.

[0119] The staining steps with the alcian blue staining solution are as follows:

[0120] (1) Fixing: After the end of chondrocyte induction and differentiation, the medium in the 15 ml centrifuge tube was removed, and 0.5 ml of 4% neutral formaldehyde solution was added to each tube for fixation treatment of the cells.

[0121] (2) Agar embedding and staining observation: After fixation, the chondrocyte pellets were agar embedded and sectioned, and then stained with the alcian blue solution.

[0122] Example 6

[0123] The stained chondrocyte pellets were made into special countable slides, and the chondrocyte staining effect was observed under an inverted microscope or a upright microscope and counted. The chondrocytes in 8 large grids of the upper left, upper right, lower left and lower right of two regions were counted, and the average number was taken. ​

[0124] Figure 4 The images show the results of chondrogenic differentiation of 10 groups of human umbilical cord mesenchymal stem cells after induction with Alixin Blue staining. Image A shows the results of the negative control group of human umbilical cord mesenchymal stem cells cultured in ordinary culture medium; Image B shows the results of the group induced with classic chondrogenic induction medium; Image C shows the results of the group induced with chondrogenic induction medium prepared using a kit purchased from Promcell; Image D shows the results of the group induced with chondrogenic induction medium prepared using a kit purchased from Sciencell; Image E shows the results of the group induced with chondrogenic induction medium prepared using a kit purchased from Cyagen Biosciences; and Images F1-F5 show the results of the group induced with the chondrogenic induction medium provided by this invention.

[0125] Depend on Figure 4 It can be seen that, except for the group of human umbilical cord mesenchymal stem cells cultured in the ordinary culture medium (group A) which could not differentiate into chondrocytes, the other groups (group BF) could all differentiate human umbilical cord mesenchymal stem cells into chondrocytes, but the degree of differentiation varied.

[0126] Among them, by Figure 4 As shown in image B, the classical induction culture medium can induce human umbilical cord mesenchymal stem cells to differentiate into chondrocytes, but the degree of differentiation is generally low, with a low proportion of chondrocytes after differentiation, and a large number of stem cells failing to undergo directed differentiation; through Figure 4 A comparison of the results obtained from images F1-F5 with those in this figure shows that the culture medium provided by this invention has a significant advantage in inducing the directed differentiation of these stem cells into chondrocytes, with a more ideal induction effect. Group F1 showed a 4-fold increase in relative counts compared to Group B using a specially designed slide. Group F2 showed a 4.5-fold increase. Group F3 showed a 6-fold increase, Group F4 a 5.5-fold increase, and Group F5 a 5.5-fold increase. Group F3 showed the most ideal effect in inducing the directed differentiation of these stem cells into chondrocytes, with a 6-fold increase.

[0127] The induction effect using existing commercial culture media is comparable. (See also...) Figure 4 Images C-E show that the induction effect of the commercially available culture medium was better than that of the classic culture medium induction group, but due to... Figure 4 It is evident that the induction results of existing commercial culture media are still inferior to those of the culture media provided by this invention.

[0128] See Figure 4 Image F3 shows that the experimental group with culture medium F3 provided by this invention has the best induction effect, with a large number of chondrocytes, uniform dispersion of chondrocytes in the lacunae, and significantly better differentiation effect than any other group.

[0129] Depend on Figure 4 Figure 4It can be seen that, compared with chondroblasts cultured by ordinary medium and commercial medium, the intracellular acid mucopolysaccharide in chondrocytes cultured by the medium composition provided by the application is dyed blue by Alcian blue, the chondrocyte density and quantity are much higher than those obtained by other existing culture media. It is proved that the medium provided by the application has a good directional differentiation induction effect on human umbilical cord mesenchymal stem cells, and is especially suitable for inducing and culturing chondrocytes from human umbilical cord mesenchymal stem cells.

[0130] In conclusion, the number of human umbilical cord mesenchymal stem cells cultured by the medium added with 100 umol / L alisol B is 6 times that of chondroblasts differentiated from the classic commercial medium.

[0131] Although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions recorded in the foregoing embodiments or make equivalent replacements to some technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A chondrogenic induction medium for human umbilical cord mesenchymal stem cells, which is composed of the following raw materials: a basic medium, an inducing factor and an additive factor; wherein the basic medium comprises penicillin, streptomycin, glutamine, FBS and high-sugar DMEM medium; the inducing factor is dexamethasone, TGF-β1, vitamin C phosphate and ITS+1; the additive factor is alisol B, and the amount of alisol B added is 50-200 μmol / L; the amounts of dexamethasone, TGF-β1, vitamin C phosphate and ITS+1 are respectively: dexamethasone: 10 μg / ml, TGF-β1: 10 ng / ml, vitamin C phosphate: 50 μg / ml, and ITS+1: 1% of the total volume.

2. The human umbilical cord mesenchymal stem cell chondrogenic induction differentiation medium according to claim 1, wherein, the amount of alisol B added is 100 μmol / L.

3. The human umbilical cord mesenchymal stem cell chondrogenic induction differentiation medium according to claim 1, wherein, the amounts of penicillin, streptomycin, fetal bovine serum and glutamine are respectively: penicillin: 100 U / ml, streptomycin: 100 μg / ml, FBS: 5% of the total volume, and glutamine: 2 mM / L.

4. The human umbilical cord mesenchymal stem cell chondrogenic induction differentiation medium according to claim 1, wherein, the amounts of components of the ITS+1 are respectively: recombinant human insulin: 10 mg / ml, human transferrin: 0.55 mg / ml, sodium selenite: 0.5 μg / ml, BSA: 50 mg / ml, linoleic acid: 470 μg / ml, and EBSS without phenol red: 5 ml.

5. A method for preparing the chondrogenic induction medium for human umbilical cord mesenchymal stem cells according to any one of claims 1-4, the method comprising: adding 5% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mM / L glutamine to high-sugar DMEM medium, and mixing and stirring uniformly; then adding 10 μg / ml dexamethasone, 10 ng / ml TGF-β1, 50 μg / ml vitamin C phosphate and 1% ITS+1, and mixing uniformly to obtain a mixed medium; adding alisol B to the mixed medium and mixing uniformly to obtain the chondrogenic induction medium for human umbilical cord mesenchymal stem cells.

6. A method for inducing and differentiating human umbilical cord mesenchymal stem cells into chondrocytes in vitro, the method comprising: seeding human umbilical cord mesenchymal stem cells in the medium according to any one of claims 1-4 and inducing and culturing to obtain chondrocytes.

7. The method according to claim 6, further comprising an in-vitro isolation, culture and confirmation process of human umbilical cord mesenchymal stem cells: carrying out in-vitro isolation and culture of human umbilical cord mesenchymal stem cells; identifying cell surface antigens by sampling flow cytometry: taking any subcultured human umbilical cord mesenchymal stem cells, labeling the surface with antibodies, and determining the proportion of labeled antibodies by flow cytometry to confirm the human umbilical cord mesenchymal stem cells.

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