Cartilage tissue induction differentiation culture medium, cartilage tissue and culture method

Abstract

This invention discloses a cartilage tissue induction differentiation culture medium, cartilage tissue, and a culture method. The culture medium is a basal culture medium supplemented with dexamethasone, linoleic acid, citrate, ascorbic acid, sodium pyruvate, trehalose, insulin-transferrin-selenose / selenoselate, BSA, proline, TGF-β3, etc. Using this culture medium to induce stem cell differentiation yields excellent cartilage tissue, overcoming the shortcomings of existing cartilage tissue induction differentiation media, such as inability to induce multiple stem cell types, long induction time, low cell proliferation coefficient, high requirements for the culture system, and low extracellular matrix content in the obtained cartilage tissue. This provides a new research method for cartilage tissue engineering and a new theoretical basis for the clinical treatment of osteoarthritis and cartilage damage.

Description

Technical Field

[0001] This invention belongs to the field of cartilage tissue engineering technology, specifically relating to a cartilage tissue induction differentiation culture medium, cartilage tissue, and culture method. Background Technology

[0002] In clinical practice, joint degeneration caused by damage to articular cartilage, particularly osteoarthritis, is quite common. Articular cartilage, primarily composed of chondrocytes and extracellular matrix (ECM), is a special type of connective tissue. Its function is to reduce the pressure of joint movement on the subchondral bone and decrease friction on the articular surfaces. Because cartilage lacks blood vessels and nerve tissue, it relies mainly on synovial fluid for metabolism, making cartilage degeneration or damage often difficult to reverse or repair. Traditional treatments, such as arthroscopic joint irrigation, microfracture repair, and arthroplasty, can improve patients' clinical symptoms in the short term, but their long-term efficacy is unsatisfactory. For arthritis caused by cartilage degeneration or damage, subsequent joint replacement becomes the only option for patients. While it can eliminate joint pain and restore function, it is expensive, has many complications, and its lifespan is limited. Younger patients often require revision surgery, causing significant physical and psychological distress and financial burden. Therefore, cartilage regeneration has become a major challenge for doctors in clinical practice. The development of cartilage tissue engineering has provided new solutions to these challenges. Cartilage tissue engineering involves seeding cells onto suitable bioscaffold materials to form a complex, which is then induced in vitro or directly implanted into articular cartilage defects to regenerate tissue structure and function. The selection of seed cells is crucial for repairing or replacing damaged tissue. To date, mesenchymal stem cells (MSCs), particularly bone marrow mesenchymal stem cells (BMSCs) and adipose-derived MSCs (ADSCs), have been extensively studied and are commonly used seed cells. MSCs possess strong proliferative capacity, multi-lineage differentiation potential, and immunomodulatory functions, making them a research hotspot in cartilage repair. Compared to autologous chondrocyte transplantation, MSCs have significant advantages: they are widely available, easily obtained, can be rapidly expanded, and are significantly less expensive. In conclusion, cartilage tissue engineering is currently the only promising treatment option for cartilage defects or injuries, and seed cells are a necessary factor for the success of bone tissue engineering.

[0003] However, patients with bone and joint injuries often exhibit decreased vitality and quantity of their own bone marrow mesenchymal stem cells (MSCs), impaired proliferation and differentiation potential, and reduced cartilage differentiation capacity. Because the directed induction of MSC differentiation is influenced by many different signaling pathways, and changes in the cellular microenvironment during MSC induction culture have a significant impact on MSC differentiation and its molecular structure, existing methods for MSC differentiation into chondrocytes require a long time, typically 3-5 weeks. Furthermore, after induction culture with most induction solutions, the expression efficiency of proteins such as type II collagen and glycosaminoglycans, the main components of the cartilage extracellular matrix, is low, making them unsuitable for clinical use in the repair and treatment of cartilage degeneration or injury. Some existing technologies have proposed solutions to these problems, such as the invention patent application number 201710631640.6, but compared with this invention, there are still many differences and defects, as shown in the table below:

[0004]

[0005]

[0006] While the aforementioned invention can shorten the chondrogenic cycle of human adipose-derived mesenchymal stem cells, the reduction in time is limited compared to previous induction culture methods. Furthermore, its applicability to chondrogenic induction of stem cells from other species (such as SD rats and New Zealand white rabbits commonly used in basic experiments) and stem cells from different sources within the same species has not been described. In addition, the expression of related extracellular matrix proteins such as type II collagen and glycosaminoglycans is low, which cannot meet the actual research needs of tissue engineering for repairing cartilage defects in implanted animals.

[0007] Therefore, the key to the successful research and clinical application of cartilage tissue engineering lies in developing a method that can rapidly induce stem cells to differentiate into chondrocytes and generate a large number of extracellular matrix functional proteins, such as type II collagen and glycosaminoglycans. This invention focuses on this issue and develops an induction culture medium and method that can rapidly induce various types of stem cells to proliferate and differentiate into cartilage tissue. Summary of the Invention

[0008] The primary objective of this invention is to provide a cartilage tissue induction and differentiation culture medium. Using this medium to induce stem cell differentiation yields superior cartilage tissue, overcoming the shortcomings of existing cartilage tissue induction and differentiation culture media, such as inability to induce multiple stem cell types, long induction time, low cell proliferation coefficient, high requirements for the culture system, and low extracellular matrix content in the obtained cartilage tissue. This invention is applicable to research in various stem cell tissue engineering fields.

[0009] A cartilage tissue induction differentiation culture medium is a basal culture medium supplemented with the following components at concentrations: dexamethasone: 1uM-50uM, linoleic acid: 10-50ug / mL, citrate: 3ug / mL-15ug / mL, ascorbic acid: 30-150mg / L, sodium pyruvate: 150-210mg / L, trehalose: 100-250ug / mL, insulin-transferrin-selenium / selenite: 50-250mg / L, BSA (bovine serum albumin): 1200-1300mg / L, proline: 50-100mg / L, and TGF-β3: 20-30ng / mL.

[0010] Further, it includes the following preferred concentrations of components: dexamethasone: 20-30 μM, linoleic acid: 35-45 μg / mL, citrate: 5 μg / mL-15 μg / mL, ascorbic acid: 80-120 mg / L, sodium pyruvate: 150-200 mg / L, trehalose: 200-250 μg / mL, insulin-transferrin-selenite / selenite: 180-220 mg / L, BSA: 1200-1300 mg / L, proline: 60-90 mg / L, TGF-β3: 20-30 ng / mL.

[0011] Furthermore, it includes the following preferred concentrations of ingredients: dexamethasone: 25 μM, linoleic acid: 40 μg / mL, citrate: 10 μg / mL, ascorbic acid: 100 mg / L, sodium pyruvate: 180 mg / L, trehalose: 250 μg / mL, insulin-transferrin-selenite / selenite: 200 mg / L, BSA: 1250 mg / L, proline: 80 mg / L, TGF-β3: 20 ng / mL.

[0012] The citrate includes sodium citrate; the insulin-transferrin-selenium / selenite includes at least one of insulin-transferrin-selenium and insulin-transferrin-sodium selenite.

[0013] Furthermore, the proline used in this invention is preferably L-proline, and the ascorbic acid is preferably dextrorotatory ascorbic acid.

[0014] The citrate used in this invention can maintain a pH of around 3, which is very effective in stabilizing the initial dissolution of growth factor TGF-β3.

[0015] The insulin-transferrin-selenium / selenite and TGF-β used in this invention have a synergistic effect and can effectively promote induced differentiation.

[0016] Furthermore, the cartilage tissue induction differentiation medium includes a basal medium comprising DMEM high-glucose medium containing 10-12% FBS (by weight) but without HEPEs. Since HEPES affect the pH balance of the medium, DMEM high-glucose medium with HEPEs removed has been found to be very effective.

[0017] The second objective of this invention is to provide a method for inducing differentiation and culture of cartilage tissue, specifically using the aforementioned culture medium to induce stem cell differentiation. This method is simple to operate, low in cost, short in time, and highly efficient. Through preliminary studies on drug ratios and cell proliferation and differentiation, the optimal scientific ratio range of the induction solution components and the most common and simple cell culture method—the two-dimensional cell culture system—were identified. This shortens the traditional 3-5 week induction cycle for stem cells to chondrocytes to a minimum of 3-4 days, significantly reducing the stem cell induction cycle. This opens up new research methods for cartilage tissue engineering and provides a reference and theoretical basis for clinical cartilage injury repair.

[0018] The stem cells mentioned include mesenchymal stem cells, and further include bone marrow mesenchymal stem cells or adipose-derived mesenchymal stem cells. Examples include bone marrow mesenchymal stem cells (BMSCs) from SD rats or rabbits, and adipose-derived mesenchymal stem cells (ADSCs) from rabbits.

[0019] Furthermore, the Pellet culture system, a three-dimensional cell hydrogel scaffold culture system, or a two-dimensional cell culture system are used, especially the two-dimensional cell culture system.

[0020] Furthermore, the conditions for inducing differentiation are as follows: passaged P3-P4 generation bone marrow or adipose mesenchymal stem cells are added to a cell culture system including induction differentiation medium and cultured at 37°C in a 5% CO2 cell culture incubator. The induction differentiation medium is replaced every 2-3 days, and the cells are cultured for 3-4 days to obtain induced chondrocytes.

[0021] A third objective of this invention is to provide cartilage tissue obtained through the culture method described above. This cartilage tissue can form a large amount of extracellular matrix (ECM) of chondrocytes, and exhibits a high differentiation rate and a high cell proliferation coefficient. This provides a new method for animal models of cartilage defects and for the clinical treatment of cartilage defects.

[0022] Key points of the invention

[0023] 1. Different culture methods: In the numerous studies reported so far on inducing stem cell differentiation into chondrocytes, the pellet system and three-dimensional cell scaffold culture system are the main ones. Simple two-dimensional plate culture systems have not been reported. However, pellet culture systems are relatively complex compared to two-dimensional cell culture methods. This invention is applicable to two-dimensional cell culture systems. In addition, it is also applicable to pellet culture systems and three-dimensional scaffold culture systems.

[0024] 2. The types and amounts of inducers in the induction medium are different: Through extensive preliminary experiments, this invention has found the optimal scientific ratio of inducers in the induction medium. The components used are different from other induction formulas, and the content of the components used is unprecedented. The culture medium uses a high-glucose medium without HEPEs, and the serum FBS concentration is increased to 12%. These are all very different from the previously reported induction formulas.

[0025] 3. Adaptable to different species: Previous stem cell differentiation induction media were mostly effective for one species and one type of cell. However, the induction medium used in this invention has a strong differentiation-promoting effect on bone marrow mesenchymal stem cells of SD rats, and also has a strong differentiation-promoting effect on bone marrow mesenchymal stem cells (BMSCs) and adipose mesenchymal stem cells (ADSCs) of New Zealand white rabbits. This is an effect that has never been reported before.

[0026] 4. Different Effects on Cell Phenotype: Most of the induced differentiation media used in currently reported patents and literature only promote the differentiation of the stem cells used; media that promote cell proliferation are rarely reported. Therefore, they often use the Pellet culture system, requiring a large number of cells. However, the induced differentiation media of this invention not only has a strong promoting effect on the differentiation of the stem cells used, but also a strong promoting effect on the proliferation of the induced stem cells. The growth rate after 6 days of induction is 1.89-2.42 times that of the control group. Furthermore, this invention can increase the cell proliferation rate from 1×10⁻⁶ cells within 7 days. 5 The fact that individual cells grow into cartilaginous tissue shaped like chondrocytes further illustrates the rapid growth rate.

[0027] 5. Different extracellular matrix production: Cartilage tissue is composed of chondrocytes and extracellular matrix (ECM). Most of the reported induction media induce the stem cells used to become chondrocytes, and have little effect on promoting the secretion of extracellular matrix (ECM). However, the induction media used in this invention can not only promote the differentiation of stem cells into chondrocytes, but also promote the secretion of a large amount of extracellular matrix, forming a large amount of extracellular matrix (ECM) of chondrocytes, and thus forming cartilage tissue rather than just differentiating into chondrocytes. This is something that other induction methods do not have.

[0028] This invention, through extensive preliminary basic research and scientific formulation of inducing agent content, has developed a cartilage induction culture medium applicable to the differentiation of various types of stem cells into chondrocytes. This culture medium is suitable for two-dimensional cell culture systems, is simple to operate, and the induction technique is easy to master, facilitating widespread application. Simultaneously, it promotes rapid cell proliferation and differentiation. This outstanding culture induction advantage can significantly shorten the induction and differentiation cycle of stem cells used in bone tissue engineering research (from the usual 3-5 weeks to 3-4 days), saving considerable manpower and research funding. Furthermore, because it strongly promotes the secretion of extracellular matrix (ECM), leading to the formation of cartilage tissue within 7 days, it can be used in animal cartilage defect models, providing a new research method for cartilage tissue engineering and a new theoretical basis for the clinical treatment of osteoarthritis and cartilage damage. Attached Figure Description

[0029] Figure 1 a) 48 hours after stem cell extraction in Example 1; b) 3 days after stem cell extraction in Example 1; c) 6 days after stem cell extraction in Example 1; d) P3 generation cells used in subsequent experiments in Example 1.

[0030] Figure 2 In Example 1, the cell proliferation rate was measured using the CCK-8 assay on days 1, 2, 3, 4, 5, and 6 after induction.

[0031] Figure 3 In Example 1, the cell proliferation rate was measured using the CCK-8 assay on days 1, 2, 3, 4, 5, and 6 after induction.

[0032] Figure 4 Expression of cartilage-related genes Collagen II, Aggrecan, and Sox9 in the negative control group, experimental group, and positive control group in Example 1;

[0033] Figure 5 a) Bone marrow mesenchymal stem cells (BMSCs) in the experimental group before induction in Example 1; b) Microscopic image taken after 4 days of induction culture in the experimental group in Example 1; c) Toluidine blue stained image taken after 4 days of induction culture in the experimental group in Example 1.

[0034] Figure 6 Toluidine blue staining images of the experimental group (a) and the positive control group (b) 7 days after induction in Example 1;

[0035] Staining details under a 50x (left) and 200x (right) microscope;

[0036] Figure 7Example 1: HE staining images of experimental group (a) and positive control group (b) sections after 7 days of induction, staining under a microscope at 50x (left) and 200x (right);

[0037] Figure 8 Example 1: Alcian blue stained images of sections from the experimental group (a) and the positive control group (b) 7 days after induction;

[0038] Staining details under a 50x (a left), 100x (b left), and 200x (right) microscope;

[0039] Figure 9 Example 1: Safranin O-stained slides of the experimental group (a) and the positive control group (b) 7 days after induction;

[0040] Staining details under a 50x (left), 100x (b right), and 200x (a right) microscope;

[0041] Figure 10 Example 1: Immunohistochemical staining results of Collagen II on experimental group (a) and positive control group (b) sections 7 days after induction; (magnification: 200x)

[0042] Figure 11 a) is the cartilage tissue "cartilaginous ball" formed in the experimental group of Example 1 after 7 days of induction; b) is the xiphoid cartilage tissue of SD rats.

[0043] Figure 12 Microscopic morphological changes of rabbit adipose-derived mesenchymal stem cells before and after induction in Example 2;

[0044] a, Rabbit adipose-derived mesenchymal stem cells before induction; b, Rabbit adipose-derived mesenchymal stem cells 7 days after induction.

[0045] Figure 13 In Example 2, the expression of relevant cartilage genes Collagen II, Aggrecan, and Sox9 in the samples was measured.

[0046] Figure 14 : The morphological changes of rabbit bone marrow mesenchymal stem cells before and after induction under a microscope in Example 3;

[0047] a, Rabbit bone marrow mesenchymal stem cell induction before; b, Rabbit bone marrow mesenchymal stem cell induction 7 days later;

[0048] Figure 15 In Example 3, the expression of relevant cartilage genes Collagen II, Aggrecan and Sox9 in the samples was measured.

[0049] In the diagram, Collagen 2 is Collagen II. Detailed Implementation

[0050] The following examples are intended to further illustrate the present invention, but not to limit it.

[0051] Example 1: Induction Experiment of Bone Marrow Mesenchymal Stem Cells (BMSCs) in SD Rats

[0052] Proliferative effect:

[0053] Extraction, culture, and passage of bone marrow mesenchymal stem cells from SD rats: Two-week-old male SD rats were euthanized by cervical dislocation and immersed in 75% ethanol for 5-10 minutes. They were then placed in a 10cm culture dish on a laminar flow hood. Ophthalmic scissors and forceps were used to peel off the skin from the rat's legs, carefully removing the muscles and other soft tissues from the bone surface. The femurs and tibias were then separated and immersed in sterile PBS. A 1ml syringe was used to draw high-glucose medium containing 12% FBS (fetal bovine serum) and 1% penicillin-antibody but without HEPEs (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and the mesenchymal cavity was slowly flushed into a new culture dish until the flushed femur and tibia turned white. The flushing was then stopped. The resulting mesenchymal cavity flushing material was filtered through a 200-mesh cell sieve and resuspended in a new centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes. The cells were then resuspended in low-glucose medium containing 10% FBS and 1% penicillin-antibody and transferred to a new 25cm culture dish. 2 The cells were cultured in flasks at 37°C in a 5% CO2 incubator. The medium was changed for the first time after 24-48 hours, and then every 1-2 days thereafter. When the cells reached 80%-90% confluence, they were digested with trypsin-EDTA. Under a microscope, when the cells appeared spherical, the digestion was stopped with serum-containing medium. The cells were then aspirated into centrifuge tubes, thoroughly mixed, and sampled and counted using a cell counter. The remaining cell suspension was centrifuged, the supernatant was discarded, and the cells were passaged at a ratio of 1:3. The cells were cultured to the third or fourth generation. After digestion and centrifugation, P3 or P4 generation bone marrow mesenchymal stem cells were obtained.

[0054] See results Figure 1 a) 48 hours after stem cell extraction; b) 3 days after stem cell extraction; c) 6 days after stem cell extraction; d) P3 generation cells used for subsequent experiments.

[0055] The experimental measurements included the effects of the induction solution on cell proliferation and differentiation.

[0056] First, the effect of differentiation induction solution on the proliferation of BMSCs in SD rats was determined—the CCK-8 proliferation experiment is as follows:

[0057] Two-dimensional cell culture system for inducing chondrocyte culture: First, P3 generation adipose-derived mesenchymal stem cells were resuspended and counted, and then seeded in 24-well plates with 1 × 10⁻⁶ cells per well. 4The experiment was divided into a control group, experimental group A, and experimental group B. The control group used high-glucose culture medium alone; experimental group A used Cyagen Biosciences (Guangzhou) Co., Ltd., catalog number: RASMX-90041, which is currently commercially available; and experimental group B used the chondrogenic solution prepared in this invention. Each group had four parallel replicates, and each group was treated as follows:

[0058] Control group: DMEM high-glucose medium containing 12% FBS but without HEPEs

[0059] Experimental Group A: Added finished induction solution (product number: RASMX-90041)

[0060] Experimental Group B: The inducing agent was dispersed in DMEM high-glucose medium containing 12% FBS but without HEPEs. The components of the inducing agent and their final concentrations in the induction differentiation medium are as follows: Dexamethasone: 50uM, Linoleic acid: 10ug / mL, Sodium citrate: 3ug / mL, Dextrorotatory ascorbic acid: 150mg / L, Sodium pyruvate: 210mg / L, Trehalose: 100ug / mL, Insulin-transferrin-selenium (Insulin-transferrin-selenium (ITS) from Beyotime, Shanghai—product number: C0341): 250mg / L, BSA: 1300mg / L, L-proline: 100mg / L, TGF-β3: 20ng / mL.

[0061] The above three groups were divided into four replicates per group. After cell adhesion, 2 mL of the corresponding culture medium was added to each well, and the cells were cultured at 37℃ in a 5% CO2 cell culture incubator. The induction differentiation medium was changed every 2-3 days. The cell proliferation rate was measured using the CCK-8 assay on days 1, 2, 3, 4, 5, and 6 after induction, as detailed below:

[0062] CCK-8 proliferation rate assay: After cell adhesion, 400 μL of CCK-8 stock solution (complete medium: CCK-8 stock solution = 10:1, where complete medium is a mixture of 10% FBS, 1% antibiotics, and basal medium) was added to each well on days 1, 2, 3, 4, 5, and 6 after induction. Cells were then incubated at 37°C for 1.5 h, and the OD value was measured at 450 nm using a microplate reader. The results are shown below. Figure 2 , Figure 3 As shown.

[0063] Measurement results:

[0064] Day 1: The proliferation rate of experimental group B was about 1.8 times that of the control group, while that of experimental group A was about 0.95 times that of the control group. This may be related to the initial stimulation of cells by the drug.

[0065] Day 2: The proliferation rate of experimental group B was about 1.78 times that of the control group, while that of experimental group A was about 0.96 times that of the control group. Compared with the proliferation rate of commercial induction solution, the proliferation rate of experimental group B showed a strong effect in promoting the proliferation of BMSCs.

[0066] Day 3: The proliferation rate of experimental group B was about 1.89 times that of the control group, while that of experimental group A was about 1.1 times that of the control group;

[0067] Day 4: The proliferation rate of experimental group B was approximately 2.23 times that of the control group, while that of experimental group A was approximately 1.17 times that of the control group;

[0068] Day 5: The proliferation rate of experimental group B was approximately 2.43 times that of the control group, while that of experimental group A was approximately 1.21 times that of the control group;

[0069] Day 6: The proliferation rate of experimental group B was approximately 2.42 times that of the control group, while that of experimental group A was approximately 1.14 times that of the control group. Due to the rapid proliferation and differentiation of experimental group B, cartilage-like tissue, i.e., chondrocyte structure, was formed by day 7. The proliferation experiment only measured the first 6 days.

[0070] Second, the differentiation effect of the induction solution:

[0071] The experiment was divided into a negative control group, an experimental group, and a positive control group. Each group received the following treatment:

[0072] Negative control group: DMEM high-glucose medium containing 12% FBS but without HEPEs;

[0073] Experimental group: The inducing agent was dispersed in DMEM high glucose medium containing 12% FBS but without HEPEs. The components of the inducing agent and their final concentrations in the induction differentiation medium are as follows: dexamethasone: 50uM, linoleic acid: 10ug / mL, sodium citrate: 3ug / mL, dextrorotatory ascorbic acid: 150mg / L, sodium pyruvate: 210mg / L, trehalose: 100ug / mL, insulin-transferrin-selenium: 250mg / L, BSA: 1300mg / L, L-proline: 100mg / L, TGF-β3: 20ng / mL;

[0074] Positive control group: xiphoid cartilage tissue from rats, stored at -80℃.

[0075] Two-dimensional cell culture system for inducing chondrocyte culture: Similarly, P3 generation adipose-derived mesenchymal stem cells were collected, resuspended, counted, and seeded in 6-well plates, with 1 × 10⁶ cells per well. 4The experiment was conducted in three groups: a negative control group, an experimental group, and a positive control group, with four replicates per group. After cell adhesion, 2 mL of the appropriate culture medium was added to each well. The negative control group was cultured in high-glucose medium alone. The experimental group was cultured in the cartilage induction medium prepared in this invention at 37°C in a 5% CO2 cell culture incubator. The positive control group consisted of xiphoid cartilage tissue from SD rats (stored at -80°C) which did not require cell seeding or induction. The medium in both the negative control group and the experimental group was changed every 2-3 days. On day 7, after the formation of "cartilage balls" in the experimental group, RNA was extracted from the negative control group cells using the cell extraction method, and RNA was extracted from the cartilage tissue of the experimental group and the positive control group using the tissue RNA extraction method. The expression of relevant cartilage genes Aggrecan, Collagen II, and Sox9 was measured using real-time quantitative PCR. Results Figure 4 As shown.

[0076] PCR test results showed that the expression of cartilage-related genes Aggrecan, Collagen II, and Sox9 was extremely low in the negative control group. In the experimental group, after 7 days of induction with the induction solution of this invention, BMSCs curled from a two-dimensional, flat state into a spherical shape, and the expression levels of related cartilage genes were almost equivalent to those in normal SD rat cartilage tissue, especially Aggrecan and Sox9 genes. This indicates that the induction medium and induction method of this invention can rapidly promote the differentiation of rat BMSCs into cartilage tissue.

[0077] also, Figure 5 This is a bright-field microscopic image of the cells in the experimental group of Example 1, taken on day 4 of two-dimensional induction culture after cell plating. Figure 5 'a' represents the cell morphology immediately after plating, before the addition of induction medium, after cell adhesion. Figure 5 b shows the cell morphology 4 days after induction with the induction solution, and the results of toluidine blue staining of the induced cells. Figure 5 c) The figure shows not only morphological changes in the cells, but also a positive result for toluidine blue staining, indicating that the cells have differentiated into chondrocytes.

[0078] Cells continued to be induced to differentiate in a two-dimensional culture system. By day 7, the cells automatically curled up into spheres from their flat two-dimensional plane state. This may be related to the formation of type II collagen in the extracellular matrix. There was a spontaneous elastic contraction in the matrix, which is called a cartilage ball. At this time, the cartilage balls were fixed, embedded, and stained accordingly, as well as immunohistochemical staining of Collagen II, to further identify the formation of chondrocytes and extracellular matrix (ECM).

[0079] Figure 6The toluidine blue staining results of cells in the experimental group after 7 days of induction are shown at 50x (left) and 200x (right) magnification. Under high magnification, it can be clearly seen that, compared with normal cartilage tissue (positive control group), the peripheral area of ​​the experimental group, i.e. the stem cell induction group, has formed a mesh-like structure similar to normal cartilage tissue, with a large amount of extracellular matrix collagen fibers. Although a large amount of extracellular matrix has formed in the central part of the sample, the overall amount of the cartilage-like mesh structure is relatively less than that of normal cartilage tissue, which may be related to the shorter induction time of the experimental group.

[0080] Figure 7 For HE staining, the cell nuclei are alkaline and easily stained purple-blue by hematoxylin, while the extracellular matrix is ​​acidic and easily stained red or pink by eosin. The image shows that in the experimental group (stem cell induction group) and normal cartilage tissue (positive control group), a large amount of pink or red extracellular matrix is ​​accumulated around the blue cell nuclei. The extracellular matrix in both groups exhibits a reticular structure. Figure 7 The low-power image clearly shows the staining difference between the peripheral and central regions of the sample. The central region is stained more deeply, with denser formation of extracellular matrix proteins. The peripheral region forms a fibrous mesh-like structure similar to normal cartilage tissue. The induced chondrocytes are dispersed in the mesh structure, and the nuclei are clearly visible, indicating that the mature, normal cartilage-like tissue structure in the peripheral region of the experimental group has been initially formed.

[0081] Figure 8 Alcian blue staining, a cationic dye, turns acidic extracellular matrix mucin blue and cell nuclei light blue. The stem cell induced group (experimental group) sample periphery and normal cartilage tissue (positive control group) showed similar tissue structure and the same staining results. Moreover, the amount of mucin formed in the extracellular matrix in the periphery of the experimental group sample was basically the same as that in normal cartilage tissue.

[0082] Figure 9 Safranin O staining, a basic dye, binds to the anionic groups of polysaccharides (chondroitin sulfate or keratin sulfate) in the extracellular matrix of cartilage, forming a red complex. Both groups showed similar reticular red staining results, indicating that the stem cell-induced group (experimental group) formed a large amount of extracellular matrix components similar to normal cartilage tissue (positive control group), such as chondroitin sulfate. Compared with normal cartilage tissue, the expression level in the stem cell-induced group was slightly lower. In addition, it can be observed under low magnification that the periphery of the experimental group sample first formed a cartilage-like structure, and the central part of the sample formed more extracellular matrix mass, but the cartilage-like structure was not as obvious as in the peripheral area. The overall formation was from the periphery to the center, gradually forming an extracellular matrix fibrous skeleton.

[0083] Figure 10The results of immunohistochemical staining of Collagen II are shown. As is well known, Collagen II is a major component of the extracellular matrix of cartilage tissue. This invention utilizes the principle of specific antibody-antigen interaction, and with the aid of labeled antibody chromogenic agents (fluorescein, enzymes, or metal ions), to display the expression of Collagen II in stem cell-induced groups (experimental group) and normal cartilage tissue (positive control group), and to study its localization, qualitative and quantitative properties. After Collagen II is bound by the antibody, the chromogenic agent appears brown, and the cell nuclei in the tissue are stained blue by hematoxylin, appearing as blue-brown in the image. It can be seen that both groups have high Collagen II protein expression, but the Collagen II protein expression in normal cartilage tissue is relatively higher, which is consistent with the Collagen II expression in this RT-PCR experiment.

[0084] Figure 11 a is a chondrocyte formed 7 days after induced differentiation. Figure 11 b is xiphoid cartilage tissue taken from normal SD rats, used as a positive control group for the experiment; the two are macroscopic comparisons of gross morphology.

[0085] The real-time quantitative PCR reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 34 s, 95℃ extension for 15 s, and 60℃ for 1 min; 20 μL reaction volume, repeated for 40 cycles. (Based on 2...) –ΔΔCt The relative expression level of the detection gene relative to the internal reference gene in the sample is calculated using the following algorithm: relative expression level = 2. –(Ct检测基因–Ct内参基因) The multiple of expression level in the experimental group relative to the control group = relative expression level in the experimental group / relative expression level in the control group.

[0086] Table 1 Primer sequences for quantitative real-time PCR of rat chondrogenic genes

[0087]

[0088] Example 2: Application of New Zealand White Rabbit Adipose-Derived Mesenchymal Stem Cells (ADSCs) Induction

[0089] Healthy female New Zealand white rabbits weighing 2.5-3 kg were selected. The harvested adipose tissue was rinsed three times with physiological saline containing 1% penicillin-streptomycin to thoroughly remove connective tissue. The adipose tissue was then minced into a paste and transferred to 50 mL centrifuge tubes. 0.2% type I collagenase was added at a 1:2 (v / v) ratio of the fat paste to the digestion solution. The mixture was placed in a 37°C shaker at 80 rpm for 60 min. Digestion was terminated with L-DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin), and the mixture was filtered through a 200-mesh cell sieve. The filtrate was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in L-DMEM complete medium. The cells were then cultured in a 5% CO2 incubator at 37°C and 95% relative humidity. The medium was changed every 2-3 days. Cells that were elongated spindle-shaped, in good condition, clustered, and covered the bottom of the flask were identified as adipose-derived mesenchymal stem cells (ADSCs).

[0090] Passaging of adipose-derived mesenchymal stem cells (ADSCs): Remove the cell culture flask from the cell culture incubator and rinse the bottom of the flask twice with PBS (2 mL / 10 cm). 2 Add trypsin digestion solution (1 mL / T25 culture flask), and digest at room temperature or 37°C for 4-5 minutes, then stop digestion. Gently pipette the bottom of the flask to collect the cell suspension into a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in 5 mL of complete culture medium, count the cells to estimate the cell concentration, and follow a 10⁻⁶ ratio. 4 / cm 2 The cells were passaged at a concentration of [specific concentration not specified] and cultured in a cell culture incubator at 37°C and 95% relative humidity (5% CO2). The culture medium was changed approximately every 2-3 days. Once the cells had spread to the bottom of the flask and reached confluence >80%, they were passaged and cultured continuously. The P3 generation was used for subsequent experiments.

[0091] Sampling of xiphoid cartilage in New Zealand white rabbits: After euthanizing the rabbits by air embolization of the ear vein, the fur of the xiphoid process at the center of the lower edge of the sternum was removed by surgical scissors, and the fascia and other soft tissues on the surface of the xiphoid process were removed by surgical scissors. The xiphoid cartilage was then cut off for later use.

[0092] RT-PCR detection of cartilage-related genes:

[0093] Using the most common two-dimensional cell culture method, cells were digested and counted using a cell counter. Cells were then seeded in 6-well plates, with 1 × 10⁶ cells per well. 5 The experiment was divided into three groups: a negative control group, an experimental group, and a positive control group. Each group had three replicates. The samples from the three groups were processed as follows:

[0094] Negative control group: DMEM high-glucose medium containing 12% FBS but without HEPEs;

[0095] Experimental group: The inducing agent was dispersed in DMEM high glucose medium containing 12% FBS but without HEPEs. The components of the inducing agent and their final concentrations in the induction differentiation medium are as follows: dexamethasone: 1uM, linoleic acid: 50ug / mL, sodium citrate: 15ug / mL, dextrorotatory ascorbic acid: 30mg / L, sodium pyruvate: 150mg / L, trehalose: 200ug / mL, insulin-transferrin-selenium: 150mg / L, BSA: 1250mg / L, L-proline: 50mg / L, TGF-β3: 30ng / mL;

[0096] Add 2 mL of the corresponding culture medium to each well of the negative control group and the experimental group in the 6-well plate.

[0097] Positive control group: xiphoid cartilage tissue from New Zealand white rabbits, which was stored at -80℃ for later use without cell seeding and induction culture.

[0098] Figure 12 a) Before induction, adipose-derived mesenchymal stem cells (ADSCs) are polygonal and elongated with rounded edges. b) After 6 days of induction, adipose-derived mesenchymal stem cells (ADSCs) proliferate in large numbers, growing in a "fish school" pattern, with elongated shapes, distinct cell edges, and increased secretion of extracellular matrix.

[0099] RNA extraction from xiphoid cartilage tissue: The xiphoid process was separated using ophthalmic scissors and immersed in sterile PBS. The soft tissue on the surface of the xiphoid process was further removed using ophthalmic scissors and moist saline gauze. It was washed again with sterile PBS and then cut into small pieces of approximately 1 mm using ophthalmic scissors. 3 Cartilage fragments of a certain size were added with a certain amount of Trizol and then the xiphoid cartilage was thoroughly crushed using a grinder. RNA was extracted, reverse transcribed, and stored at -20℃ for later use.

[0100] Seven days after induction, RNA was extracted from samples of the negative control group, experimental group, and positive control group according to the cell and tissue RNA extraction methods. The expression levels of relevant cartilage genes Collagen II, Aggrecan, and Sox9 in the samples were measured using RT-PCR (primers are shown in Table 2; the procedure was the same as in Example 1). Results... Figure 13 As shown in the PCR results, CollagenII gene expression was approximately 149 times higher than that of the negative control group and 0.014 times higher than that of the positive control group; agrecan gene expression in the experimental group was approximately 10.6 times higher than that of the negative control group and 0.91 times higher than that of the positive control group, with expression levels being similar; sox-9 gene expression was approximately 2.5 times higher than that of the negative control group and approximately 0.77 times higher than that of the positive control group. These results indicate that the induction of the experimental group can effectively promote stem cell differentiation and rapidly and significantly promote the high expression of cartilage-related genes.

[0101] Table 2 Primer sequences for quantitative real-time PCR of rabbit chondrogenic genes.

[0102]

[0103] Example 3: Application of New Zealand White Rabbit Bone Marrow Mesenchymal Stem Cells (BMSCs) Induction

[0104] Extraction of bone marrow mesenchymal stem cells (BMSCs) from New Zealand white rabbits: Healthy female New Zealand white rabbits weighing 2.5-3 kg were selected. 1 mL of acyclovir 50 injection was injected into the gluteus maximus muscle. When the rabbits were sluggish and the corneal reflex disappeared, indicating a state of deep anesthesia, 10 mL of air was injected via the marginal ear vein, and the rabbits were euthanized. The femur and tibia were separated, and the soft tissue on the surface of the tibia and femur, as well as the epiphyses on both sides, were removed. The resulting tissues were then immersed in sterile PBS. Bone marrow mesenchymal stem cells were extracted using the whole bone marrow adherent method. Specifically, a 5ml syringe was used to draw low-glucose DMEM medium containing 10% FBS (volume fraction) and 1wt.% penicillin-antibody. The needle was inserted into the femoral end to slowly flush the femoral and tibial mesenchymal cavities. The flushing was stopped when the femoral and tibial bone turned white. The flushing material was filtered through a 200-mesh cell sieve and collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in fresh medium in a 10cm culture dish and cultured in a constant temperature cell culture incubator at 37℃ and 95% relative humidity with 5% CO2. The medium was changed every 5-7 days. When the cells were observed to be elongated spindle-shaped, in good condition, clustered, and covered the bottom of the flask, these cells were identified as New Zealand white rabbit bone marrow mesenchymal stem cells (R-BMSCs). When the cells reached 80% confluence, they were digested with trypsin and passaged. The P3 generation cells were used for subsequent differentiation-induced PCR gene expression assays.

[0105] Extraction of xiphoid cartilage tissue from New Zealand white rabbits: As above, after euthanizing the rabbits with air embolism, the skin and fur of the central xiphoid process at the lower edge of the sternum are peeled off with surgical scissors, the fascia and other soft tissues on the surface of the xiphoid process are peeled off, and the xiphoid cartilage is cut off with surgical scissors for later use.

[0106] Cell plating induction: The experiment consisted of a negative control group, an experimental group, and a positive control group (the positive control group consisted of xiphoid cartilage tissue, which did not require induction; RNA was extracted directly from the tissue using a grinder). P3 generation BMSCs were used in both the negative control and experimental groups, centrifuged and resuspended, counted, and plated in 6-well plates, with approximately 1 × 10⁶ cells per well. 5 Cells were collected, and when the cells reached 80% confluence, each group of cells was treated as follows:

[0107] Negative control group; supplemented with high-sugar DMEM containing 12% FBS but no HEPEs;

[0108] Experimental group: The inducing agent was dispersed in DMEM high-glucose medium containing 12% FBS but without HEPEs. The components of the inducing agent and their final concentrations in the induction differentiation medium were as follows: dexamethasone: 25 μM, linoleic acid: 35 μg / mL, sodium citrate: 10 μg / mL, dextrorotatory ascorbic acid: 90 mg / L, sodium pyruvate: 200 mg / L, trehalose: 250 μg / mL, insulin-transferrin-selenium: 50 mg / L, BSA: 1200 mg / L, L-proline: 80 mg / L, TGF-β3: 20 ng / mL.

[0109] Add 2 mL of the corresponding culture medium to each well of the control group and experimental group in the 6-well plate.

[0110] Positive control group: xiphoid cartilage tissue, which was stored at -80℃ for later use without cell seeding and induction culture.

[0111] After 7 days of induction culture, Figure 14 The images show the morphological changes of rabbit bone marrow mesenchymal stem cells before and after induction under a microscope. a) Before induction, rabbit bone marrow mesenchymal stem cells were photographed under a bright field microscope. The cells were polygonal, with short and rounded outlines and plump cell morphology. b) After 7 days of induction, rabbit bone marrow mesenchymal stem cells proliferated in large quantities, growing in a vortex-like pattern like a school of fish. The cells were elongated and had angular outlines, resembling chondrocytes in morphology.

[0112] Seven days after induction, RNA was extracted from three groups of samples using Trizol, isopropanol, and chloroform. Reverse transcription was performed, and in vitro amplification was conducted using quantitative real-time PCR. The primer sequences for the relevant cartilage genes are shown in Table 2. The procedure was the same as in Example 1. The test results are as follows: Figure 15 As shown, after 7 days of induction, the expression level of Aggrecan gene in the experimental group was approximately 14.83 times that of the negative control group and 0.91 times that of the positive control group; the expression level of CollagenII in the experimental group was approximately 145 times that of the negative control group and 1.43 times that of the positive control group; and the expression level of sox-9 gene in the experimental group was approximately 1.3 times that of the negative control group and approximately 0.71 times that of the positive control group. This indicates that the induction medium of this invention can induce stem cells to differentiate into chondrocytes in a short period of time, with a significant increase in gene expression levels, especially Aggrecan and sox-9, which are almost comparable to the gene expression levels of normal cartilage tissue. sequence list <110> Zhang Jie <120> Cartilage tissue induction differentiation culture medium, cartilage tissue and culture method <150> 2021111674008 <151> 2021-10-07 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 gcaagttcaa cggcacag 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 gccagtagac tccacgacat 20 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 agagcggaga ctactggatt g 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 tctggacgtt agcggtgtt 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 aaaggaggtg gtactgttgg 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 ggagcgaagg ttctggatt 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 gcacatcaag acggagcaa 19 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <400> 8 aggtgaaggt ggagtagagc c 21 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 gtttgtgatg ggcgtgaa 18 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 gaggcaggga tgatgttct 19 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <400> 11 tcctggcaag cctggtga 18 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 agccctgggt agcctctgtg 20 <210> 13 <211> twenty two <212> DNA <213> Artificial Sequence <400> 13 agaactttgg tagaatccgt aa 22 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <400> 14 cccagactgt agcccactt 19 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 agtacccgca cctgcacaac 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 cgcctgccca ttcttcacc 19

Claims

1. A cartilage tissue induction differentiation culture medium, characterized in that, The basal culture medium is supplemented with the following components at the following concentrations: dexamethasone: 1uM-50uM, linoleic acid: 10-50ug / mL, citrate: 3ug / mL-15ug / mL, ascorbic acid: 30-150mg / L, sodium pyruvate: 150-210mg / L, trehalose: 100-250ug / mL, insulin-transferrin-selenite / selenite: 50-250mg / L, BSA: 1200-1300mg / L, proline: 50-100mg / L, TGF-β3: 20-30ng / mL; The basal culture medium is DMEM high-glucose medium containing 10-12% FBS but not HEPEs; The insulin-transferrin-selenium / selenite mentioned is insulin-transferrin-selenium (ITS) from Shanghai Beyotime Biotechnology Co., Ltd., product number: C0341.

2. The cartilage tissue induction differentiation culture medium according to claim 1, characterized in that, The product contains the following concentrations of ingredients: Dexamethasone: 20-30 μM, Linoleic acid: 35-45 μg / mL, Citrate: 5 μg / mL-15 μg / mL, Ascorbic acid: 80-120 mg / L, Sodium pyruvate: 150-200 mg / L, Trehalose: 200-250 μg / mL, Insulin-transferrin-selenium / selenite: 180-220 mg / L, BSA: 1200-1300 mg / L, Proline: 60-90 mg / L, and TGF-β3: 20-30 ng / mL.

3. The cartilage tissue induction differentiation culture medium according to claim 1, characterized in that, The ingredients include the following concentrations: dexamethasone: 25 μM, linoleic acid: 40 μg / mL, citrate: 10 μg / mL, ascorbic acid: 100 mg / L, sodium pyruvate: 180 mg / L, trehalose: 250 μg / mL, insulin-transferrin-selenite / selenite: 200 mg / L, BSA: 1250 mg / L, proline: 80 mg / L, and TGF-β3: 20 ng / mL.

4. The cartilage tissue induction differentiation culture medium according to claim 1, characterized in that, Citrates include sodium citrate.

5. A method for inducing differentiation and culture of cartilage tissue, characterized in that, Mesenchymal stem cells were induced to differentiate using the cartilage tissue induction differentiation culture medium as described in any one of claims 1-4.

6. The method according to claim 5, characterized in that, The mesenchymal stem cells mentioned are bone marrow mesenchymal stem cells or adipose-derived mesenchymal stem cells.

7. The method according to claim 5, characterized in that, The Pellet culture system, three-dimensional cell hydrogel scaffold culture system, or two-dimensional cell culture system are used.

8. The method according to claim 5, characterized in that, A two-dimensional cell culture system was used.

9. The method according to claim 5, characterized in that, Passaged P3-P4 generation bone marrow or adipose-derived mesenchymal stem cells were added to a cell culture system including induction differentiation medium and cultured at 37°C in a 5% CO2 cell culture incubator. The induction differentiation medium was replaced every 2-3 days to obtain induced chondrocytes.

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