Differentiation-inducing culture medium for transformation of adipose-derived mesenchymal stem cells into chondrocytes, and use thereof

Abstract

Provided are a differentiation-inducing culture medium for the transformation of adipose-derived mesenchymal stem cells into chondrocytes and the use thereof. A basal culture medium of the differentiation-inducing culture medium comprises a nutrient solution and an inducer. The nutrient solution comprises a blood substitute, a non-essential amino acid, β-mercaptoethanol, penicillin / streptomycin, dexamethasone, sodium pyruvate, ascorbic acid, sodium selenite, transferrin, recombinant human insulin, β-glycerophosphate and TGF-β1; and the inducer comprises cyanamide, delphinidin chloride and an extracellular matrix derived from adipose-derived mesenchymal stem cells. The components of the inducer, i.e. cyanamide and delphinidin chloride, can stimulate chondrocyte generation by means of up-regulating the expression of aggrecan and collagen II; and the component of the inducer, i.e. extracellular matrix (ADSC-ECM), can increase the adhesion between chondrocytes, enhance the signaling communication between chondrocytes, and promote the growth and metabolism of chondrocytes, thereby greatly improving the differentiation efficiency of adipose-derived mesenchymal stem cells into chondrocytes.

Description

Adipose-derived mesenchymal stem cells transformed into chondrocytes and their application Technical Field

[0001] The present invention relates to the technical field of biological cell culture, and in particular to an induction differentiation culture medium for converting adipose mesenchymal stem cells into chondrocytes and an application thereof. Background Art

[0002] Articular cartilage is a specialized tissue with no blood vessels or nerves, a high extracellular matrix and a high cell-to-cell ratio. Chondrocytes have long been considered the only cell type present in articular cartilage. Consequently, lesions caused by articular cartilage damage are clinically common. Cartilage trauma is extremely difficult to repair because cartilage lacks blood vessels and nerves, meaning it cannot regenerate. Articular cartilage damage can lead to recurrent joint pain and limited mobility, severely impacting patients' daily lives. Technical issues

[0003] Mesenchymal stem cells (BMSCs) are an important member of the stem cell family in cell biology. They originate from the mesoderm and ectoderm of early developing embryos and were first discovered in the bone marrow of humans. Their main clinical characteristics are self-renewal, multidirectional differentiation, hematopoietic support, and immune regulation. Their clinical use is increasingly attracting attention from the public.

[0004] In most experiments using mesenchymal stem cells to treat articular cartilage damage, the differentiation of mesenchymal stem cells into chondrocytes requires a low generation of mesenchymal stem cells, and the number of differentiated chondrocytes is not large. In addition, the proliferation function of high-generation mesenchymal stem cells decreases during the differentiation process of chondrocytes, resulting in a decrease in the number of differentiated chondrocytes. Technical Solutions

[0005] Adipose-derived stem cells (ASCs) are superior to mesenchymal stem cells (BMSCs) because they are easily accessible, readily available, reproducible, and rapidly proliferating. Furthermore, ASCs can differentiate into chondrocytes, osteoblasts, adipocytes, and neurons in specific microenvironments, making them suitable biocompatible seed cells for tissue engineering. Therefore, the present invention provides a culture medium for inducing chondrocyte differentiation from adipose-derived mesenchymal stem cells. This culture medium is low-cost, exhibits excellent conversion efficiency, and produces a high number of differentiated chondrocytes.

[0006] One of the technical solutions of the present invention is as follows:

[0007] A culture medium for inducing differentiation of adipose-derived mesenchymal stem cells into chondrocytes, comprising a basic culture medium composed of DMEM high glucose or DMEM / F12; the basic culture medium contains a nutrient solution and an inducer; wherein:

[0008] The nutrient solution comprises a final concentration of 30-40 v / v% hemoglobin, a concentration of 1.8-2.6 v / v% non-essential amino acids, a final concentration of 0.1-0.4 v / v% β-mercaptoethanol, a final concentration of 0.5-2 v / v% penicillin / streptomycin, a final concentration of 8-18 μM dexamethasone, a final concentration of 2-4.5 mM sodium pyruvate, a final concentration of 0.4-2 mg / L ascorbic acid, a final concentration of 2-8 μg / L sodium selenite, a final concentration of 4-16 mg / L transferrin, a final concentration of 3-10 mg / L recombinant human insulin, a final concentration of 5-12 mM β-glycerophosphate, and a final concentration of 10-20 μg / L TGF-β1;

[0009] The inducer comprises cyanamide with a final concentration of 50 to 250 μM, delphinidin chloride with a final concentration of 30 to 1200 μM, and adipose mesenchymal stem cell-derived extracellular matrix with a final concentration of 10 to 20 μg / L.

[0010] In one embodiment, the differentiation induction medium comprises, in the basal medium:

[0011] The nutrient solution comprises hemoglobin at a final concentration of 25-35 v / v%, non-essential amino acids at a final concentration of 2-2.4 v / v%, β-mercaptoethanol at a final concentration of 0.2-0.3 v / v%, penicillin / streptomycin at a final concentration of 1 v / v%, dexamethasone at a final concentration of 10-15 μM, sodium pyruvate at a final concentration of 2.5-4 mM, ascorbic acid at a final concentration of 0.8-1.6 mg / L, sodium selenite at a final concentration of 3-7 μg / L, transferrin at a final concentration of 6-12 mg / L, recombinant human insulin at a final concentration of 4-8 mg / L, β-glycerophosphate at a final concentration of 7-10 mM, and TGF-β1 at a final concentration of 12-16 μg / L;

[0012] The inducer comprises cyanamide with a final concentration of 80 to 220 μM, delphinidin chloride with a final concentration of 100 to 800 μM, and adipose mesenchymal stem cell-derived extracellular matrix with a final concentration of 12 to 18 μg / L.

[0013] The second technical problem of the present invention is to provide a culture method for converting adipose-derived mesenchymal stem cells into chondrocytes, comprising the following steps:

[0014] adding a mixture of polyethylene glycol 2000, collagen and hyaluronic acid to a PBS solution to obtain a PBS mixed solution; heating and stirring the PBS mixed solution at 40 to 50° C. for 30 to 60 minutes to obtain a coating gel;

[0015] Adding the coating gel into a culture vessel to form a gel film on the inner wall of the culture vessel, cooling at room temperature and solidifying the gel film;

[0016] Obtaining P2 to P5 adipose-derived mesenchymal stem cells, and resuspending the adipose-derived mesenchymal stem cells in the above-mentioned differentiation induction medium to obtain a cell suspension;

[0017] Adding the cell suspension dropwise onto the gel membrane in the incubator to form spherical cell beads;

[0018] dripping the coating medium onto the cell beads in a contact manner, and allowing the coating medium and the cell beads to fully fuse to obtain a cell mixture; wherein the volume ratio of the coating medium to the cell beads is 1:1;

[0019] The cell mixture was allowed to stand for 5 minutes and then the incubator was inverted to allow the cell beads to be suspended on the gel membrane in the incubator;

[0020] The culture vessel is placed in an incubator and cultured for 12 to 16 days to obtain induced differentiated chondrocytes.

[0021] In one embodiment, in the culture method, in the coating gel preparation step, the concentration of polyethylene glycol 2000 is 1 to 5 w / w%; in the mixture of collagen and hyaluronic acid, the total concentration of collagen and hyaluronic acid is 5 to 10 w / w%, and the mass ratio of collagen to hyaluronic acid is 1:5 to 10.

[0022] In one embodiment, in the culture method, the thickness of the gel film is 0.5-1 mm.

[0023] In one embodiment, in the culture method, when resuspending the adipose-derived mesenchymal stem cells, the cell density is maintained at 4×10 5 ~10×10 5 pieces / mL.

[0024] In one embodiment, in the culture method, the encapsulated culture medium is prepared by the following process:

[0025] First, methylcellulose is added to 1000 mL of DMEM high glucose or DMEM / F12 basal culture medium to a final concentration of 30 to 50 g / L; then, the mixture is heated and stirred at 70 to 90° C. for 20 to 40 minutes; then, heating is stopped and the mixture is cooled to 20 to 50° C. with continued stirring to prepare the encapsulation culture medium.

[0026] In one embodiment, in the culture method, the environment in the incubator is 37° C., 5 v / v% CO 2 and 2-8 v / v% O 2 .

[0027] In one embodiment, the culture method further comprises the following processing steps after obtaining the induced differentiated chondrocytes:

[0028] The chondrocytes are transferred into a culture system for cell expansion culture.

[0029] The third technical problem of the present invention is to provide the use of the chondrocytes prepared above in the preparation of drugs for treating osteoarthritis.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1. Cyanoamide, a component of the inducer, produces higher calcium deposition during the process of adipose-derived mesenchymal stem cells inducing chondrocyte transformation, which upregulates the expression of chondrocyte-specific genes BMP-2 and Runx-2, thereby inducing the secretion of more BMP-2 protein;

[0032] 2. The components of the inducer, cyanamide and delphinidin chloride, can stimulate chondrogenesis by upregulating the expression of aggrecan and collagen II;

[0033] 3. The extracellular matrix (ADSC-ECM) component of the inducer can increase the adhesion between chondrocytes, enhance the signal connection between chondrocytes, and promote the growth and metabolism of chondrocytes;

[0034] 4. Under the combined action of ADSC-ECM and gel membrane, a three-dimensional structural network will be formed during the induction process of adipose-derived mesenchymal stem cells. This structural network can promote the differentiation of adipose-derived mesenchymal stem cells into chondrocytes and provide a three-dimensional tissue space for chondrocyte proliferation, thereby improving the differentiation efficiency of adipose-derived mesenchymal stem cells;

[0035] 4. In the chondrogenic cell culture method, the hanging drop culture method, in which the induction culture medium is added dropwise into the culture flask in the form of drops, can maintain a high cell activity of adipose-derived mesenchymal stem cells without the need for fluid replenishment or fluid replacement, thereby saving the subsequent fluid replenishment and fluid replacement operations after cell inoculation, reducing the cell inoculation amount and the amount of culture medium used; at the same time, the hanging drop form makes it easier for cells to aggregate to form a three-dimensional structural network, greatly improving the efficiency of adipose-derived mesenchymal stem cells in differentiating into chondrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a picture of the cartilage ball after Alecian blue staining;

[0037] FIG2 is a bar graph showing the percentage of BMP-2 secreted by MSCs before and after chondrogenic differentiation in Examples 1 to 3 and Comparative Example 1;

[0038] FIG3 is a bar graph showing the expression rates of aggrecan for cartilage differentiation in Examples 1 to 3 and Comparative Example 1;

[0039] FIG4 is a bar graph showing the expression rates of cartilage differentiation collagen II in Examples 1 to 3 and Comparative Example 1;

[0040] FIG5 is a graph showing the relative gene expression of Examples 1 to 3 and Comparative Example 1, expressed as log2-fold change (-ΔΔCt);

[0041] FIG6 is a graph showing the relative gene expression Col-II of Examples 1 to 3 and Comparative Example 1 as log2 fold change (-ΔΔCt);

[0042] FIG7 is a graph showing the relative gene expression of Sox9 in Examples 1 to 3 and Comparative Example 1 as log2 fold change (-ΔΔCt). Best Mode for Carrying Out the Invention

[0043] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0044] The induction differentiation medium for converting adipose-derived mesenchymal stem cells into chondrocytes provided by the present invention includes DMEM high-glucose basal medium or DMEM / F12 basal medium, and the basal medium contains a nutrient solution and an inducer; wherein the basal medium and the nutrient solution are conventional culture components in the process of converting adipose-derived mesenchymal stem cells into chondrocytes, and the inducer stimulates and induces the rapid conversion of adipose-derived mesenchymal stem cells into chondrocytes.

[0045] The nutrient solution in the basal medium contains hemoglobin at a final concentration of 30-40 v / v%, non-essential amino acids at a final concentration of 1.8-2.6 v / v%, β-mercaptoethanol at a final concentration of 0.1-0.4 v / v%, penicillin / streptomycin at a final concentration of 0.5-2 v / v%, dexamethasone at a final concentration of 8-18 μM, sodium pyruvate at a final concentration of 2-4.5 mM, ascorbic acid at a final concentration of 0.4-2 mg / L, and ascorbic acid at a final concentration of 2-8 μg / L. L of sodium selenite, 4-16 mg / L of transferrin, 3-10 mg / L of recombinant human insulin, 5-12 mM of β-glycerophosphate, and 10-20 μg / L of TGF-β1; the inducer includes cyanamide at a final concentration of 50-250 μM, delphinidin chloride at a final concentration of 30-1200 μM, and extracellular matrix derived from adipose mesenchymal stem cells at a final concentration of 10-20 μg / L.

[0046] Preferably, in one embodiment, the nutrient solution in the basal culture medium comprises hemoglobin at a final concentration of 25-35 v / v%, non-essential amino acids at a final concentration of 2-2.4 v / v%, β-mercaptoethanol at a final concentration of 0.2-0.3 v / v%, penicillin / streptomycin at a final concentration of 1 v / v%, dexamethasone at a final concentration of 10-15 μM, sodium pyruvate at a final concentration of 2.5-4 mM, ascorbic acid at a final concentration of 0.8-1.6 mg / L, sodium selenite at a final concentration of 3-7 μg / L, transferrin at a final concentration of 6-12 mg / L, recombinant human insulin at a final concentration of 4-8 mg / L, β-glycerophosphate at a final concentration of 7-10 mM, and a final concentration of 12-16 μg / L. TGF-β1; the inducer includes cyanamide with a final concentration of 80-220 μM, delphinidin chloride with a final concentration of 100-800 μM, and adipose-derived mesenchymal stem cell-derived extracellular matrix with a final concentration of 12-18 μg / L.

[0047] The present invention provides a method for culturing adipose-derived mesenchymal stem cells into chondrocytes, comprising the following steps:

[0048] S1. Preparing a coating gel: First, adding a mixture of polyethylene glycol 2000, collagen, and hyaluronic acid to a PBS solution to obtain a PBS mixed solution; secondly, heating and stirring the PBS mixed solution at 40-50° C. for 30-60 minutes to obtain a coating gel;

[0049] S2. Shaping and solidifying the gel film: Add the coating gel to the incubator, slowly rotate the incubator, and form a thin film layer on the inner wall of the incubator. After cooling to room temperature, solidify and shape the gel film on the inner wall of the incubator for later use.

[0050] S3. Prepare cell suspension: freshly collected adipose-derived mesenchymal stem cells from passages P2 to P5 are taken and resuspended in the above-prepared differentiation induction medium to obtain a cell suspension;

[0051] S4. Preparation of cell beads: adding the cell suspension dropwise onto the gel membrane in the incubator to form spherical cell beads;

[0052] S5. Prepare a cell mixture: slowly drop the coating culture medium onto the cell beads in a contact manner, and allow the coating culture medium and the cell beads to fully blend to obtain a cell mixture;

[0053] S6. Obtaining chondrocytes: After the cell mixture is allowed to stand for 5 minutes, the culture vessel is inverted and placed in an incubator to culture the culture vessel in the form of suspended cell beads for 5 to 7 days to obtain induced differentiated chondrocytes;

[0054] S7. Chondrocyte expansion culture: Transfer the chondrocytes into the culture system for routine cell expansion culture.

[0055] In step S1 of the above-mentioned culture method, the concentration of polyethylene glycol 2000 is 1-5 w / w%; in the mixture of collagen and hyaluronic acid, the total concentration of collagen and hyaluronic acid is 5-10 w / w%, and the mass ratio of collagen to hyaluronic acid is 1:5-10.

[0056] In step S2 of the above-mentioned culture method, the thickness of the gel membrane is preferably 0.5 to 1 mm.

[0057] In step S3 of the above-mentioned culture method, when resuspending the adipose-derived mesenchymal stem cells, the cell density is maintained at 4×10 5 ~10×10 5 pieces / mL.

[0058] In step S5 of the above-mentioned culture method, the encapsulated culture medium is prepared by the following process:

[0059] Add 30-50 g / L methylcellulose to 1000 mL of basal culture medium, heat and stir at 70-90° C. for 20-40 min, stop heating and continue stirring while cooling to 20-50° C. to prepare the encapsulation culture medium.

[0060] Preferably, the encapsulated culture medium is prepared by the following process:

[0061] First, methylcellulose is added to 1000 mL of DMEM high glucose or DMEM / F12 basal culture medium to a final concentration of 30 to 50 g / L; then, the mixture is heated and stirred at 70 to 90° C. for 20 to 40 minutes; then, heating is stopped and the mixture is cooled to 20 to 50° C. with continued stirring to prepare the encapsulation culture medium.

[0062] In step S5 of the above-mentioned culture method, the volume ratio of the encapsulating culture medium to the cell beads is 1:1.

[0063] In step S6 of the above-mentioned culture method, the environmental atmosphere in the incubator is 37° C., 5 v / v% CO 2 , and 2-8 v / v% O 2 .

[0064] In step S7 of the above-mentioned culture method, the conventional expansion culture process of the chondrocytes transferred into the culture flask is as follows:

[0065] After inoculation, the inverted culture vessel was placed in a 37°C, 5% CO2 incubator for 48 hours, then taken out and placed upright, the lid was opened, 0.5 mL of fresh differentiation induction medium was added dropwise for each cell bead, the lid was covered, and the culture was placed upright in a 37°C, 5% CO2 incubator for culture. Subsequently, the culture vessel was observed under a microscope and the medium was changed every 2-3 days. The medium change operation was as follows: the upright culture vessel was taken out and the lid was opened, the liquid was gently tilted to collect the liquid at the bottom, the liquid was aspirated, an equal amount of differentiation induction medium was added, and the culture was gently shaken. After continuing to culture upright for 12-16 days, the culture vessel was tapped to make the chondrocytes fall off, the cells were collected, and set aside for processing such as fixation, paraffin embedding, and staining.

[0066] The chondrocytes prepared in the present invention can be used to prepare drugs for treating osteoarthritis, or used to prepare components of drugs for treating osteoarthritis, and can be made into biological preparations for clinical use. Modes for Carrying Out the Invention

[0067] 1. Preparation of Chondrocytes

[0068] Example 1

[0069] Preparation of differentiation induction medium: 1000 mL of DMEM high-glucose basal medium was prepared, and the following contents were added: a final concentration of 35 v / v% heparin, a final concentration of 2 v / v% non-essential amino acids, a final concentration of 0.3 v / v% β-mercaptoethanol, a final concentration of 1 v / v% penicillin / streptomycin, a final concentration of 12 μM dexamethasone, a final concentration of 3 mM sodium pyruvate, a final concentration of 1 mg / L ascorbic acid, a final concentration of 5 μg / L sodium selenite, a final concentration of 10 mg / L transferrin, a final concentration of 7 mg / L recombinant human insulin, a final concentration of 8 mM β-glycerophosphate, a final concentration of 15 μg / L TGF-β1, a final concentration of 100 μM cyanamide, a final concentration of 500 μM delphinidin chloride, and a final concentration of 14 μg / L adipose-derived mesenchymal stem cell-derived extracellular matrix to obtain differentiation induction medium;

[0070] Preparation of coating gel: Add a mixture of 3 w / w% polyethylene glycol 2000, 7 w / w% collagen (1 g), and hyaluronic acid (6 g) to 100 mL of PBS and heat at 45°C for 40 min to obtain a coating gel; wherein the mass ratio of collagen to hyaluronic acid is 1:7;

[0071] Preparation of encapsulation medium: Add 40 g of methylcellulose to 1000 mL of DMEM high-glucose basal medium to make the final concentration of methylcellulose 40 g / L, heat and stir at 80°C for 30 min, and cool to 40°C with stirring for use. Continue stirring to prepare the encapsulation medium.

[0072] The coated gel was added to the incubator and rotated to form a 0.6 mm thick gel film on the inner wall of the incubator, which was then cooled and solidified at room temperature.

[0073] Take freshly collected adipose-derived mesenchymal stem cells from the P2 generation, add 20 mL of differentiation induction medium to resuspend the adipose-derived mesenchymal stem cells, and maintain the cell density at 7 × 10 5 / mL, and obtain cell suspension;

[0074] The cell suspension was added dropwise onto the gel membrane on the inner wall of the culture vessel to form 0.2 mL spherical cell beads;

[0075] 0.2 mL of the coating medium was slowly added dropwise onto the cell beads to allow the coating medium and cell beads to fuse to obtain a cell mixture.

[0076] After the cell mixture was allowed to stand for 5 minutes, the incubator was inverted and placed in an incubator with cell beads suspended on the surface of the coated gel for 5 to 7 days. The environment in the incubator was 37° C., 5 v / v% CO2, and 5 v / v% O2 to obtain induced differentiated chondrocytes.

[0077] The obtained chondrocytes are transferred into the culture system for routine cell expansion culture.

[0078] Example 2

[0079] Preparation of differentiation induction medium: taking 1000 mL of DMEM / F12 basal medium, dispersing in DMEM / F12 medium a final concentration of 30 v / v% heparin, a final concentration of 1.8 v / v% non-essential amino acids, a final concentration of 0.1 v / v% β-mercaptoethanol, a final concentration of 2 v / v% penicillin / streptomycin, a final concentration of 18 μM dexamethasone, a final concentration of 2 mM sodium pyruvate, a final concentration of 1.6 mg / L ascorbic acid, a final concentration of 3 μg / L sodium selenite, a final concentration of 6 mg / L transferrin, a final concentration of 10 mg / L recombinant human insulin, a final concentration of 5 mM β-glycerophosphate, a final concentration of 12 μg / L TGF-β1, a final concentration of 250 μM cyanamide, a final concentration of 100 μM delphinidin chloride, and a final concentration of 18 μg / L adipose-derived mesenchymal stem cell-derived extracellular matrix to prepare a differentiation induction medium;

[0080] Preparation of coating gel: Add a mixture of 1 w / w% polyethylene glycol 2000, 5 w / w% collagen (1.7 g), and hyaluronic acid (3.4 g) to 100 mL of PBS and heat at 30°C for 60 min to obtain a coating gel; wherein the mass ratio of collagen to hyaluronic acid is 1:5;

[0081] Preparation of encapsulation medium: Add 30 g of methylcellulose to 1000 mL of DMEM / F12 basal medium to make the final concentration of methylcellulose 30 g / L, heat and stir at 70°C for 40 min, and cool to 20°C with stirring for use. Continue stirring to prepare the encapsulation medium.

[0082] The coated gel was added to the incubator and rotated to form a 0.5 mm thick gel film on the inner wall of the incubator, which was then cooled and solidified at room temperature.

[0083] Take freshly collected adipose-derived mesenchymal stem cells from the P2 generation, add 20 mL of differentiation medium to resuspend the adipose-derived mesenchymal stem cells, and maintain the cell density at 4 × 10 5 / mL, and obtain cell suspension;

[0084] The cell suspension was added dropwise onto the gel membrane on the inner wall of the culture vessel to form 0.2 mL spherical cell beads;

[0085] 0.2 mL of the coating medium was slowly added dropwise onto the cell beads to allow the coating medium and cell beads to fuse to obtain a cell mixture.

[0086] After the cell mixture was allowed to stand for 5 minutes, the incubator was inverted and placed in an incubator with cell beads suspended on the surface of the coated gel for 5 to 7 days. The environment in the incubator was 37° C., 5 v / v% CO2, and 5 v / v% O2 to obtain induced differentiated chondrocytes.

[0087] The obtained chondrocytes are transferred into the culture system for routine cell expansion culture.

[0088] Example 3

[0089] Preparation of differentiation induction medium: 1000 mL of DMEM high-glucose basal medium was prepared, and the following contents were added: a final concentration of 40 v / v% heparin, a final concentration of 2.6 v / v% non-essential amino acids, a final concentration of 0.4 v / v% β-mercaptoethanol, a final concentration of 2 v / v% penicillin / streptomycin, a final concentration of 15 μM dexamethasone, a final concentration of 4 mM sodium pyruvate, a final concentration of 2 mg / L ascorbic acid, a final concentration of 8 μg / L sodium selenite, a final concentration of 16 mg / L transferrin, a final concentration of 10 mg / L recombinant human insulin, a final concentration of 12 mM β-glycerophosphate, a final concentration of 20 μg / L TGF-β1, a final concentration of 80 μM cyanamide, a final concentration of 1200 μM delphinidin chloride, and a final concentration of 10 μg / L adipose-derived mesenchymal stem cell-derived extracellular matrix to obtain differentiation induction medium;

[0090] Preparation of coating gel: Add a mixture of 5 w / w% polyethylene glycol 2000, 10 w / w% collagen (0.9 g), and hyaluronic acid (1.1 g) to 100 mL of PBS and heat at 50°C for 30 min to obtain a coating gel; wherein the mass ratio of collagen to hyaluronic acid is 1:10;

[0091] Preparation of encapsulation medium: Add 50 g of methylcellulose to 1000 mL of DMEM high-glucose basal medium to make the final concentration of methylcellulose 50 g / L, heat and stir at 90°C for 20 min, and cool to 50°C with stirring for use. Continue stirring to prepare the encapsulation medium.

[0092] The coated gel was added to the incubator and rotated to form a 1mm thick gel film on the inner wall of the incubator, which was then cooled and solidified at room temperature.

[0093] Take freshly collected adipose-derived mesenchymal stem cells at P5, add 20 mL of differentiation medium to resuspend the adipose-derived mesenchymal stem cells, and maintain the cell density at 10 × 10 5 / mL, and obtain cell suspension;

[0094] The cell suspension was added dropwise onto the gel membrane on the inner wall of the culture vessel to form 0.2 mL spherical cell beads;

[0095] 0.2 mL of the coating medium was slowly added dropwise onto the cell beads to allow the coating medium and cell beads to fuse to obtain a cell mixture.

[0096] After the cell mixture was allowed to stand for 5 minutes, the incubator was inverted and placed in an incubator with cell beads suspended on the surface of the coated gel for 5 to 7 days. The environment in the incubator was 37° C., 5 v / v% CO2, and 5 v / v% O2 to obtain induced differentiated chondrocytes.

[0097] The obtained chondrocytes are transferred into the culture system for routine cell expansion culture.

[0098] Comparative Example 1 (medium does not contain inducer)

[0099] In this comparative example, the differentiation induction medium does not contain cyanamide, delphinidin chloride and extracellular matrix components.

[0100] Preparation of differentiation induction medium: 1000 mL of DMEM high-glucose basal medium was prepared by dispersing 40 v / v% heparin, 2.6 v / v% non-essential amino acids, 0.4 v / v% β-mercaptoethanol, 2 v / v% penicillin / streptomycin, 15 μM dexamethasone, 4 mM sodium pyruvate, 2 mg / L ascorbic acid, 8 μg / L sodium selenite, 16 mg / L transferrin, 10 mg / L recombinant human insulin, 12 mM β-glycerophosphate, and 20 μg / L TGF-β1 in the DMEM medium to prepare differentiation induction medium;

[0101] Take freshly collected adipose-derived mesenchymal stem cells at P5, add 20 mL of differentiation medium to resuspend the adipose-derived mesenchymal stem cells, and maintain the cell density at 10 × 10 5 / mL, and obtain cell suspension;

[0102] The cell suspension was added dropwise onto the gel membrane on the inner wall of the culture vessel to form 0.2 mL spherical cell beads;

[0103] The inoculated culture vessel was placed upright in a 37°C, 5% CO2 incubator for 48 hours. The vessel was opened and 0.5 mL of fresh differentiation induction medium was added dropwise to each cell bead. The vessel was covered and placed upright in a 37°C, 5% CO2 incubator for culturing. The culture medium was then observed under a microscope and replaced every 2-3 days. The replacement process was as follows: the culture vessel was taken out and opened, the lid was opened, and the liquid was collected at the bottom by gently tilting the vessel. The liquid was aspirated, being careful not to aspirate the cells. An equal amount of differentiation induction medium was added and the culture medium was gently shaken. The culture was continued upright for 12-16 days. The cells were then collected by tapping the culture vessel for fixation, paraffin embedding, and staining.

[0104] 0.2 mL of the coating medium was slowly added dropwise onto the cell beads to allow the coating medium and cell beads to fuse to obtain a cell mixture.

[0105] The culture vessel and the cell mixture were placed in an incubator for static culture. The environment in the incubator was 37° C., 5 v / v% CO 2 , and 5 v / v% O 2 ; chondrocytes at the differentiation stage were obtained.

[0106] 2. Chondrocyte Detection Test

[0107] 1. Alcian blue staining analysis

[0108] In order to facilitate observation and characterization, the chondrocytes in Examples 1 to 3 and Comparative Example 1 were stained with Alcian blue for observation.

[0109] 1.1. The specific implementation steps of Alcian blue staining observation are as follows:

[0110] 1) The cartilage balls were embedded in paraffin and then sliced;

[0111] 2) Dyeing steps:

[0112] a) Dewaxing and dehydration;

[0113] b) Staining with Alcian blue solution for 30 minutes;

[0114] c) Rinse with deionized water for 2 minutes.

[0115] 1.2. Observe the effect of Alcian blue staining under a microscope

[0116] Microscope: inverted microscope; Model: XD; Manufacturer: Ningbo Sunny Optical.

[0117] As shown in Figure 1, pictures of the positive control sample, Examples 1 to 3 and the chondrocytes in Comparative Example 1 observed under a microscope after alcin blue staining are shown; wherein, Picture a in Figure 1 is a 200-fold magnification picture of the bovine articular cartilage tissue cells of the positive control sample; Picture b in Figure 1 is a 200-fold magnification picture of the chondrocytes prepared in Example 1; Picture c in Figure 1 is a 200-fold magnification picture of the chondrocytes prepared in Example 2; Picture d in Figure 1 is a 200-fold magnification picture of the chondrocytes prepared in Example 3; Picture e in Figure 1 is a 200-fold magnification picture of the chondrocytes prepared in Comparative Example 1.

[0118] Alcian blue dye was used to identify the presence of glycosaminoglycans (GAGs) in chondrocyte-loaded microcarriers (CL+MCs). Histological preparation of the example samples was performed as described above. On day 12 of chondrogenesis induction, the formation of cartilage nodules was assessed on prepared slides.

[0119] As shown in Figures b, c, and d in Figure 1, on day 12 of culture, the chondrogenic cells obtained in Examples 1-3 exhibited cartilage formation and stained positive with calcium blue. Figure 1a shows bovine articular cartilage cells as a positive control, and Figure 1e shows that the chondrogenic cells obtained in Comparative Example 1 stained essentially negative with calcium blue, indicating undifferentiated cartilage tissue. This demonstrates that the cyanamide and delphinidin chloride components of the differentiation induction medium and the coated hanging drop method for culturing chondrogenic cells provided by the present invention can effectively promote the differentiation of adipose-derived mesenchymal stem cells into chondrogenic cells and shorten the induction culture time.

[0120] 2. Detection of BMP-2 secretion

[0121] BMP-2 ELISA kits were used to measure BMP-2 secretion by MSCs before and after chondrogenic differentiation. Supernatants from chondrogenic cells were collected on day 12 of culture, and absorbance was measured at 450 nm using a Tecan Infinite 200 PRO plate reader and i-control software. The data are shown in Figure 2.

[0122] The results in Figure 2 show that the BMP-2 concentrations in Examples 1 to 3 (differentiated into cartilage) are between 120 and 140 pg / mL, which is about 6 times that of Comparative Example 1 (undifferentiated into cartilage). This also confirms from the factor level that the addition of cyanamide and delphinidin chloride and the coated hanging drop culture method effectively promote the differentiation of adipose-derived mesenchymal stem cells into chondrocytes.

[0123] 3. Detection of Aggrecan and Collagen II Content

[0124] Chondrogenic cells were harvested on days 6, 9, 12, and 15 of culture, washed twice with PBS, and fixed with 4% formaldehyde in PBS for 15 minutes at room temperature. After three washes with PBS, cells were permeabilized with 0.5% saponin and 1% BSA in PBS for 15 minutes at room temperature and then blocked with 3% BSA in PBS for 30 minutes. Cells were then incubated with primary antibodies against chondroblast-derived aggrecan and collagen II overnight at 4°C. The following day, cells were rinsed with PBS for 5 minutes and incubated with secondary antibodies (donkey anti-goat antibody conjugated with FITC) for 1 hour at room temperature in the dark. Samples were loaded and analyzed using a CytoFLEX flow cytometer, with each sample run in duplicate, with 10,000 particles per run. Data were gated between forward scatter (FSC-H) values ​​of 200 and 800 to exclude cell debris and cell aggregates, respectively. Control samples were stained with secondary antibodies alone. The expression of aggrecan is shown in Figure 3, and the expression of collagen II is shown in Figure 4. Figures 3 and 4 show that the expression rates of aggrecan and collagen II in Examples 1-3 were higher than those in Comparative Example 1 during the culture process, and the test results of Comparative Example 1 on day 15 were lower than those of Examples 1-3 on day 6. The expression rates of Examples 1-3 on days 12 and 15 were essentially the same, indicating that the cartilage differentiation levels of stem cells in Examples 1-3 reached a high level after day 12, much higher than those in Comparative Example 1.

[0125] 4. Fluorescence quantitative PCR analysis

[0126] Chondrocytes were collected on day 12, and cDNA was synthesized from the extracted RNA using a cDNA synthesis kit. Primers were designed and synthesized based on the sequences of human GAPDH (an internal reference gene), Col-II, AGG, and Sox9 genes. Quantitative gene expression analysis was performed using a fluorescent quantitative PCR kit using real-time RT-PCR according to the manufacturer's instructions.

[0127] The relative mRNA expression levels of Col-Ⅱ, AGG and Sox9 in chondrocytes on day 12 were detected. The Ct values ​​were normalized to the average Ct value of the internal reference gene GAPDH. The relative gene expression of Examples 1 to 3 and Comparative Example 1 was expressed as a log2-fold change (-ΔΔCt) and compared with the expression of the control group, where the relative gene expression was set to zero, to obtain the relative mRNA expression levels of Col-Ⅱ, AGG and Sox9 in each group, as shown in Figures 5, 6 and 7. Figures 5, 6 and 7 show that the relative mRNA expression levels of Col-Ⅱ, AGG and Sox9 in Examples 1 to 3 on day 12 were all higher than those in Comparative Example 1, and the results of Col-Ⅱ and AGG corresponded to the results of the detection of aggrecan and collagen II content. Industrial Applicability

[0128] The induction differentiation medium for converting adipose-derived mesenchymal stem cells into chondrocytes provided by the present invention includes DMEM high-glucose basal medium or DMEM / F12 basal medium, and the basal medium contains a nutrient solution and an inducer; wherein the basal medium and the nutrient solution are conventional culture components in the process of converting adipose-derived mesenchymal stem cells into chondrocytes, and the inducer stimulates and induces the rapid conversion of adipose-derived mesenchymal stem cells into chondrocytes; this allows the adipose-derived mesenchymal stem cells to form a three-dimensional structural network during the induction process, which in turn can promote the differentiation of adipose-derived mesenchymal stem cells into chondrocytes and provide a three-dimensional tissue space for chondrocyte proliferation, thereby improving the differentiation efficiency of adipose-derived mesenchymal stem cells.

[0129] In the chondrogenic cell culture method, the hanging drop culture method, in which the induction culture medium is added drop by drop into the culture bottle in the form of drops, can maintain a high cell activity of adipose-derived mesenchymal stem cells without the need for fluid replenishment and fluid replacement, thereby saving the subsequent fluid replenishment and fluid replacement operations of cell inoculation, reducing the cell inoculation amount and the amount of culture medium used; at the same time, the hanging drop form makes it easier for cells to aggregate to form a three-dimensional structural network, greatly improving the efficiency of adipose-derived mesenchymal stem cells in differentiating into chondrocytes.

[0130] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the appended claims.

Claims

1. A culture medium for inducing differentiation of adipose-derived mesenchymal stem cells into chondrocytes, characterized in that: The differentiation induction medium comprises a basic medium composed of DMEM high glucose or DMEM / F12 components; the basic medium contains a nutrient solution and an inducer; wherein: The nutrient solution comprises hemoglobin at a final concentration of 30-40 v / v%, non-essential amino acids at a final concentration of 1.8-2.6 v / v%, β-mercaptoethanol at a final concentration of 0.1-0.4 v / v%, penicillin / streptomycin at a final concentration of 0.5-2 v / v%, dexamethasone at a final concentration of 8-18 μM, sodium pyruvate at a final concentration of 2-4.5 mM, ascorbic acid at a final concentration of 0.4-2 mg / L, sodium selenite at a final concentration of 2-8 μg / L, transferrin at a final concentration of 4-16 mg / L, recombinant human insulin at a final concentration of 3-10 mg / L, β-glycerophosphate at a final concentration of 5-12 mM, and TGF-β1 at a final concentration of 10-20 μg / L; The inducer comprises cyanamide with a final concentration of 50 to 250 μM, delphinidin chloride with a final concentration of 30 to 1200 μM, and adipose-derived mesenchymal stem cell-derived extracellular matrix with a final concentration of 10 to 20 μg / L.

2. The differentiation induction medium according to claim 1, characterized in that In the basal medium: The nutrient solution comprises hemoglobin at a final concentration of 25-35 v / v%, non-essential amino acids at a final concentration of 2-2.4 v / v%, β-mercaptoethanol at a final concentration of 0.2-0.3 v / v%, penicillin / streptomycin at a final concentration of 1 v / v%, dexamethasone at a final concentration of 10-15 μM, sodium pyruvate at a final concentration of 2.5-4 mM, ascorbic acid at a final concentration of 0.8-1.6 mg / L, sodium selenite at a final concentration of 3-7 μg / L, transferrin at a final concentration of 6-12 mg / L, recombinant human insulin at a final concentration of 4-8 mg / L, β-glycerophosphate at a final concentration of 7-10 mM, and TGF-β1 at a final concentration of 12-16 μg / L; The inducer comprises cyanamide with a final concentration of 80 to 220 μM, delphinidin chloride with a final concentration of 100 to 800 μM, and adipose-derived mesenchymal stem cell-derived extracellular matrix with a final concentration of 12 to 18 μg / L.

3. The differentiation induction medium according to claim 1, characterized in that In the DMEM basal medium: The nutrient solution comprises a final concentration of 35 v / v% hemoglobin, a final concentration of 2 v / v% non-essential amino acids, a final concentration of 0.3 v / v% β-mercaptoethanol, a final concentration of 1 v / v% penicillin / streptomycin, a final concentration of 12 μM dexamethasone, a final concentration of 3 mM sodium pyruvate, a final concentration of 1 mg / L ascorbic acid, a final concentration of 5 μg / L sodium selenite, a final concentration of 10 mg / L transferrin, a final concentration of 7 mg / L recombinant human insulin, a final concentration of 8 mM β-glycerophosphate, and a final concentration of 15 μg / L TGF-β1; The inducer comprises cyanamide at a final concentration of 100 μM, delphinidin chloride at a final concentration of 500 μM, and adipose-derived mesenchymal stem cell-derived extracellular matrix at a final concentration of 14 μg / L.

4. The differentiation induction medium according to claim 1, characterized in that In the DMEM / F12 medium: The nutrient solution contains a final concentration of 30 v / v% hemoglobin, a final concentration of 1.8 v / v% non-essential amino acids, a final concentration of 0.1 v / v% β-mercaptoethanol, a final concentration of 2 v / v% penicillin / streptomycin, a final concentration of 18 μM dexamethasone, a final concentration of 2 mM sodium pyruvate, a final concentration of 1.6 mg / L ascorbic acid, a final concentration of 3 μg / L sodium selenite, a final concentration of 6 mg / L transferrin, and a final concentration of 10 mg / L recombinant human insulin, final concentration 5 mM β-glycerophosphate, final concentration 12 μg / L TGF-β1; The inducer contains cyanamide at a final concentration of 250 μM, delphinidin chloride at a final concentration of 100 μM, and adipose-derived mesenchymal stem cell-derived extracellular matrix at a final concentration of 18 μg / L.

5. The differentiation induction medium according to claim 1, characterized in that In the DMEM basal medium: The nutrient solution comprises a final concentration of 40 v / v% hemoglobin, a final concentration of 2.6 v / v% non-essential amino acids, a final concentration of 0.4 v / v% β-mercaptoethanol, a final concentration of 2 v / v% penicillin / streptomycin, a final concentration of 15 μM dexamethasone, a final concentration of 4 mM sodium pyruvate, a final concentration of 2 mg / L ascorbic acid, a final concentration of 8 μg / L sodium selenite, a final concentration of 16 mg / L transferrin, a final concentration of 10 mg / L recombinant human insulin, a final concentration of 12 mM β-glycerophosphate, and a final concentration of 20 μg / L TGF-β1; The inducing agent comprises cyanamide at a final concentration of 80 μM, delphinidin chloride at a final concentration of 1200 μM and extracellular matrix derived from adipose mesenchymal stem cells at a final concentration of 10 μg / L, and is used to prepare an induction differentiation medium.

6. A method for culturing adipose-derived mesenchymal stem cells into chondrocytes, characterized in that: The steps include: adding a mixture of polyethylene glycol 2000, collagen and hyaluronic acid to a PBS solution to obtain a PBS mixed solution; heating and stirring the PBS mixed solution at 40 to 50° C. for 30 to 60 minutes to obtain a coating gel; Adding the coating gel into a culture vessel, rotating the culture vessel, and solidifying the coating gel on the inner wall of the culture vessel to form a gel film; taking adipose-derived mesenchymal stem cells of passages P2 to P5, and resuspending the adipose-derived mesenchymal stem cells in the differentiation induction medium according to claim 1 to obtain a cell suspension; Adding the cell suspension dropwise onto the gel membrane in the incubator to form spherical cell beads; dripping the coating medium onto the cell beads in a contact manner, and allowing the coating medium and the cell beads to fully fuse to obtain a cell mixture; wherein the volume ratio of the coating medium to the cell beads is 1:1; The cell mixture was allowed to stand for 5 minutes and then the incubator was inverted to allow the cell beads to be suspended on the gel membrane in the incubator; The culture vessel is placed in an incubator for 12 to 16 days to obtain induced differentiated chondrocytes.

7. The culture method according to claim 6, characterized in that In the coating gel preparation step, the concentration of polyethylene glycol 2000 is 1 to 5 w / w%.

8. The culture method according to claim 6, characterized in that In the coating gel preparation step, in the mixture of collagen and hyaluronic acid, the total concentration of collagen and hyaluronic acid is 5-10 w / w%, and the mass ratio of collagen to hyaluronic acid is 1:5-10.

9. The culture method according to claim 6, characterized in that The thickness of the gel film is 0.5-1 mm.

10. The culture method according to claim 6, characterized in that When resuspending the adipose-derived mesenchymal stem cells, the cell density was maintained at 4×10 5 ~10×10 5 pieces / mL.

11. The culture method according to claim 6, characterized in that The encapsulated culture medium is prepared by the following process: First, methylcellulose is added to 1000 mL of a basal culture medium containing DMEM high glucose or DMEM / F12 components to a final concentration of 30 to 50 g / L of methylcellulose; then, the mixture is heated and stirred at 70 to 90° C. for 20 to 40 minutes; then, heating is stopped and the mixture is cooled to 20 to 50° C. with continued stirring to prepare the encapsulation culture medium.

12. The culture method according to claim 6, characterized in that In the incubator, the environmental atmosphere is 37° C., 5 v / v% CO 2 and 2-8 v / v% O 2 .

13. Use of chondroblasts obtained by the culture method according to any one of claims 6 to 12 in the preparation of a drug for treating osteoarthritis.

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