Method for induced differentiation of blood-derived endometrial mesenchymal stem cells into cartilage cells

Abstract

The invention relates to the technical field of cell differentiation, in particular to a method for induced differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes, which comprises an endometrial tissue preservation solution and a novel induction reagent, the endometrial tissue preserving fluid is prepared from 500mL of DMEM / F-12 basal culture medium, penicillin, streptomycin and gentamicin; the novel induction reagent comprises a basic induction reagent, hydroxyapatite, patient serum and articular cavity tissue fluid. In some existing induced differentiation methods, the number, activity and purity of cells cannot meet the requirements of clinical application, compared with the prior art, a novel induction reagent is used, some substances are added on the basis of a basic induction reagent, and an endometrial tissue preservation solution is prepared to prevent bacteria from influencing the cells, so that the method has the advantages of simple operation and low cost. And the number, activity and purity of cartilage cells differentiated by induction are improved, so that the method can meet the requirements of clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of cell differentiation, and in particular to a method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes. Background Art

[0002] Bone and joint health is an important guarantee for people's quality of life. However, with the aging of the population and changes in lifestyle, the number of people suffering from osteoarthritis is increasing year by year. Among the elderly aged 70 and above, osteoarthritis is the seventh leading cause of disability and disability. Hip replacement is the highest-level orthopedic surgery, but most people are unwilling to accept it due to fear and dread of hip replacement surgery. In addition, hip replacement surgery requires the removal of some bones, which is an irreversible surgery with certain complications. Common complications include deep vein thrombosis of the lower limbs, pulmonary embolism, and local wound infection. Postoperative prosthesis loosening due to bone dissolution or other reasons causes hip pain. Long-term use and excessive movement of the joint prosthesis metal fatigue cause the stem to break or the artificial hip prosthesis to loosen. Dislocation of the artificial hip joint and femoral fracture after postoperative violence require reoperation. There is a certain service life after joint replacement, and reoperation may face the risk of failure.

[0003] In order to protect patients from secondary damage, with the advent of the century of life sciences, stem cell regenerative medicine has received more and more attention. The development of regenerative medicine has brought great impact on the treatment of many diseases, especially in the repair of various difficult diseases. Studies have shown that mesenchymal stem cells (MSCs) have the potential to differentiate into a variety of cells such as cartilage or osteoblasts. Moreover, this characteristic has been applied in tissue engineering research. At present, MSCs have become a gold standard for bone and cartilage tissue engineering research, but the current induction efficiency cannot meet the requirements of clinical treatment.

[0004] Inducing the differentiation of menstrual blood-derived endometrial mesenchymal stem cells (MMCs) into osteoblasts and using them to repair bone damage has always been a goal pursued by people. Domestic and foreign scholars have conducted research on the induction of menstrual blood-derived endometrial stem cells into osteoblasts using cytokines, compounds, natural active substances, etc. alone or in combination, and established some laboratory small-scale induction technologies. However, the methods used in the laboratories are different and the results obtained are not consistent. The existing differentiation induction technologies will result in insufficient cell quantity, activity and purity to meet the requirements of clinical application. Summary of the invention

[0005] In view of the technical problems of the prior art, the present invention provides a method for inducing the differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A reagent for inducing the differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes, comprising: endometrial tissue preservation fluid and a novel induction reagent; the endometrial tissue preservation fluid comprises: 500 mL DMEM / F-12 basic culture medium, penicillin, streptomycin, and gentamicin; the novel induction reagent comprises: a basic induction reagent, hydroxyapatite, patient serum, and joint cavity tissue fluid.

[0008] Furthermore, the penicillin in the endometrial tissue preservation solution is 100 U / mL, the streptomycin is 100 μg / mL, and the gentamicin is 25 mg / L. The hydroxyapatite in the new induction reagent is 3.0 mg / mL, and the added serum is 50 μL and the joint cavity tissue fluid is 25 μL.

[0009] Furthermore, the basic induction reagent is RPMI1640 culture medium, which contains: D-glucose, sodium bicarbonate, sodium pyruvate, HEPES and L-glutamine; dexamethasone, ascorbic acid, TGF-β1, and insulin-transferrin-selenous acid (ITS) are also added to the RPMI1640 culture medium.

[0010] A method for inducing the differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes comprises the following steps: preparing basic reagents and samples; processing various components in the samples and culturing the obtained cells; further processing the cultured cells; inducing differentiation of the treated cells using basic induction reagents and novel induction reagents; detecting some data of the cells induced and differentiated by the two reagents and comparing them.

[0011] Furthermore, the sample is 10 mL of menstrual blood, which contains mucosal tissue; the menstrual blood and mucosal tissue are processed separately; mononuclear cells of the menstrual blood are isolated and cultured; and the mucosal tissue is isolated, cultured and digested.

[0012] Furthermore, after processing the samples, menstrual blood-derived endometrial mesenchymal stem cells were obtained and cultured; after culture, the cells were fully replaced with fluid, cell passaged, and MMCs immunolabeling identified, and during these processes, the cells obtained after menstrual blood and mucosa treatment were processed separately, and the results obtained were compared.

[0013] Furthermore, when testing the cells at the induced differentiation site, Alcian blue staining analysis, chondroblast toluidine blue staining analysis and type II collagen immunohistochemical staining were performed respectively; when testing the induced differentiated cells, the morphology of the cartilage masses should also be observed, and the cell activity should be compared and analyzed.

[0014] Furthermore, the basic reagents that need to be configured are: endometrial tissue preservation solution, PBS buffer, and complete culture medium.

[0015] The beneficial effects of the present invention are: a new induction reagent is used, hydroxyapatite and the patient's serum and tissue fluid are added to the basic induction reagent, the effect of inducing differentiation is better, and endometrial tissue preservation fluid is used to prevent bacterial influence. After the two are used in the experiment, the obtained cell activity, quantity and purity are also higher, so that the induction method can meet the requirements of clinical application.

[0016] When collecting menstrual blood, mucosal tissue is also collected at the same time. Through experiments, it can be found that the cells obtained by the two processes are very different, and both can be used to induce differentiation, so that more samples can be used in the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Menstrual blood was collected by density gradient centrifugation 24 hours after inoculation of cells;

[0018] Figure 2 : Cells collected from mucosal tissue by tissue digestion;

[0019] Figure 3 : After the cells obtained by blood treatment were completely replaced with medium;

[0020] Figure 4 : After the cells obtained from mucosal tissue treatment were completely replaced with medium;

[0021] Figure 5 : When the cell confluence of cells obtained by blood treatment during cell passaging reaches 90%;

[0022] Figure 6 : When the cell confluence of cells obtained from mucosal tissue treatment reaches 90% during cell passaging;

[0023] Figure 7 :The results of MSCs surface antigen detection by flow cytometry of cells obtained by blood treatment;

[0024] Figure 8 :The results of flow cytometry detection of MSCs surface antigens by cells obtained from mucosal tissue processing;

[0025] Fig. 9 : Endometrial mesenchymal stem cells induced to differentiate into chondrocytes, Alcian blue staining 2D (100 times magnification);

[0026] Fig.10 : Endometrial mesenchymal stem cells induced to differentiate into chondrocytes, 3D staining with Alcian blue (100 times magnification);

[0027] Fig.11 :Characteristics of chondrogenic induction cell states;

[0028] Fig.12:Toluidine blue staining of chondrocytes (100 times magnification)

[0029] Fig.13 :Toluidine blue staining of chondrocytes (200 times magnification)

[0030] Fig.14 :Immunohistochemical staining of type II collagen in chondrocytes (200 times magnification)

[0031] Fig.15 : Chondrocyte spheres after 15 days of induction with two induction reagents;

[0032] Fig.16 : Test results of blank control culture after 1 week of induction;

[0033] Fig.17 : Test results of experimental group 1 after one week of induction;

[0034] Fig.18 : Test results of experimental group 2 after one week of induction;

[0035] Fig.19 : Test results of blank control culture after 25 days of induction;

[0036] Fig. 20 : Test results of experimental group 1 after 25 days of induction;

[0037] Fig.21 : Detection results of experimental group 2 after 25 days of culture induction. DETAILED DESCRIPTION

[0038] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0039] according to Figure 1-21 The present invention provides a method for inducing menstrual blood-derived endometrial mesenchymal stem cells to differentiate into chondrocytes.

[0040] First, prepare the following reagents:

[0041] 1. Endometrial tissue preservation solution: Add penicillin to 500mL DMEM / F-12 culture medium to make the concentration 100U / mL, and add 50mg streptomycin and 12.5mg gentamicin. After the preparation is completed, it is packaged and kept at -20℃ for use. After obtaining menstrual blood, it is not possible to conduct the test immediately. It needs to be processed before use. The culture medium contains a variety of substances required for cell growth to maintain cell activity. Penicillin is added to the culture medium, which plays an antibacterial role by inhibiting the synthesis of cell walls. Streptomycin and gentamicin are also added, which can act on bacterial ribosomes respectively, thereby inhibiting the synthesis of bacterial proteins and having an inhibitory effect on a variety of bacteria. Adding these antibiotics can prevent bacterial contamination without affecting the main cells, ensuring that the cells used in the test are active and accurate so that the test results are accurate.

[0042] 2. PBS buffer: Weigh 40g NaCl, 2g KCl, 0.3g KH2PO4, 1.75g ​​NaHCO3, and 0.75g Na2HPO4·12H2O respectively, pour into 5L deionized water, stir evenly (more than 5 minutes), and dispense into 500mL blue-capped bottles. Sterilize at 121℃ for 30 minutes. After sterilization, tighten the bottle cap, put it into the laboratory transfer window, and irradiate the outside of the bottle with ultraviolet light for standby use.

[0043] 3. Complete medium:

[0044] (1) High glucose medium containing 20% ​​fetal bovine serum (primary medium): Add 100 mL of fetal bovine serum to 400 mL of DMEM / HIGH GLUCOSE basal medium, and add penicillin to a concentration of 100 U / mL and streptomycin to a concentration of 100 μg / mL. Mix well and set aside for use.

[0045] (2) Subculture medium: Add 100 mL of Stemery serum replacement (Cat. No.: RC-002-100) to 500 mL of DMEM / F-12 medium, add penicillin to a concentration of 100 U / mL, add streptomycin to a concentration of 100 μg / mL, mix thoroughly and set aside.

[0046] When inducing differentiation, the following steps are required:

[0047] Sample preparation: Collect 10mL of menstrual blood (including mucosal tissue) and put it into a disposable sampling cup containing endometrial tissue preservation solution, and add 10mL of pre-prepared PBS buffer. Within 24 hours after obtaining the sample, place it at 4°C for transportation and preservation to the GMP laboratory for processing. The samples in a single collection process will contain menstrual blood and mucosal tissue, which will be processed separately. Menstrual blood is processed by gradient density centrifugation using lymphocyte separation solution Ficoll, and mucosal tissue is digested with (0.25% trypsin + EDTA 0.53mM). MMSc comes from menstrual blood. When collecting menstrual blood, mucosal tissue is collected at the same time. Since the environment of cells in mucosal tissue is different from that in menstrual blood, it may have different effects on the test results, so they are processed separately later.

[0048] Isolation and culture of menstrual blood mononuclear cells: Dilute menstrual blood with 10 mL of sterile phosphate buffered saline (PBS) and mix well. Use lymphocyte separation fluid and density gradient centrifugation to separate menstrual blood mononuclear cells by fast rise and slow fall (800g, 20min), then carefully aspirate the middle white film layer, add 20 mL of PBS buffer, centrifuge (500g, 5min), wash twice, discard the supernatant completely, add 2 mL of primary culture medium, blow, mix well, count, adjust to the appropriate cell density for cell culture, and inoculate 20 mL of cells in a T-75cm 2 In culture flask;

[0049] Isolation and culture of digestive mucosal tissue:

[0050] Receiving samples: Wipe the sample collection cup with 75% alcohol;

[0051] Transfer: Use hemostatic forceps to drain the blood from the mucosal tissue, remove it from the collection cup, and place it in a 100 mm culture dish;

[0052] Washing: Add an appropriate amount of physiological saline to the culture dish containing the mucosal tissue and wash it twice, drain as much water as possible, transfer it to a new centrifuge tube, and prepare for digestion;

[0053] Digestion of mucosal tissue:

[0054] First digestion: Add 3 mL of 0.25% trypsin containing EDTA to a 10 mL centrifuge tube (mucosal tissue occupies about 3 mL), place the centrifuge tube containing trypsin and mucosal tissue in a shaker at 37°C and 120 r / min for 15 min, and discard the supernatant;

[0055] Second digestion: Place the centrifuge tube containing pancreatic enzyme and mucosal tissue in a shaker at 37°C and 120 rpm for 40 min, collect the supernatant, and add 5 mL of primary culture medium to the supernatant to terminate the digestion;

[0056] Third digestion: transfer the mucosal tissue to a new 10mL centrifuge tube, add 3mL of 0.25% trypsin containing EDTA, place the centrifuge tube containing trypsin and mucosal tissue in a shaker at 37°C and 120r / min, digest for 1h, collect the supernatant, and add 5mL of primary culture medium to the supernatant to terminate the digestion.

[0057] Mucosal tissue digestion is carried out three times. The first time is to remove some extracellular matrix, red blood cells and other components to prevent these substances from affecting subsequent processes. The second and third times are for further purification to obtain cells that better meet the requirements.

[0058] Collect the supernatants of the second and third digestions, centrifuge at 500 g for 6 min, discard the supernatant, add 5 mL of primary culture medium to the centrifuge tube to resuspend the cells, and centrifuge at 4 °C per cm 2 / 10 5 The number of cells was inoculated into a 6-well plate, and each well contained 2.5 mL of primary complete medium. The 6-well plate was labeled with relevant information and then placed at 37°C and 5% CO. 2 Continue culturing in the incubator.

[0059] Figure 1 Cells seeded 24 hours after seeding were collected for density gradient centrifugation. Figure 2 The cells collected by tissue digestion were placed in a 37°C, 5% CO 2 Culture in an incubator with saturated humidity.

[0060] After processing menstrual blood and mucosal tissue, the cells cultured in the two processes were subjected to the following treatments:

[0061] Complete fluid change:

[0062] Observe whether the color of the cell culture medium is normal, and observe the cell growth (adhesion, cell morphology, and contamination) under an inverted microscope. 48 hours after inoculation, if there is no abnormality, gently pour out the culture medium in the culture bottle and observe under a microscope if there are many non-adherent suspended cells remaining. If necessary, rinse with 10mL PBS solution and add 20mL fresh primary culture medium. Figure 3 and Figure 4 Then place in 37°C, 5% CO 2 Continue culturing in the incubator.

[0063] Cell passaging:

[0064] Observe whether the color of the cell culture medium is normal, and observe the cell growth in the culture dish under an inverted microscope (adhesion, cell morphology, and whether it is contaminated). The cell fusion degree reaches 90%. Figure 5 and Figure 6 Perform the following subculture operation: aspirate the culture medium in the culture flask into a 50mL centrifuge tube, add PBS to the culture flask and wash twice. After aspirating the PBS, add 5mL of 0.25% trypsin to the culture flask and digest for about 5 minutes. Under the microscope, the adherent cells are observed to be round. Add the aspirated old culture medium to stop digestion, gently blow the adherent surface until most of the cells fall off, transfer the cell suspension to a new centrifuge tube, centrifuge at 500g for 6 minutes, and remove the supernatant; resuspend the cell pellet with physiological saline, take 0.5mL of the suspension for cell counting, and centrifuge at 500g for 6 minutes.

[0065] According to the seeding density of 12000 / cm 2 Calculate the number of culture bottles to be inoculated, add an appropriate amount of culture medium, gently blow to resuspend the cells, and dispense them into each culture bottle. Then add an appropriate amount of subculture medium to make the final volume of each culture bottle 20mL. Mark the cell generation number and culture time on the culture bottle, and then place it at 37℃, 5% CO 2 Continue culturing in the incubator.

[0066] Repeat the above operation process, and the next step of inducing differentiation can be carried out after culturing to the 4th generation.

[0067] Identification of MMCs by immunolabeling:

[0068] MMCs at passage 4 were collected and washed with PBS to prepare 5 × 10 6 cells / mL of cell suspension. 5 MSCs were divided into 5 μL PBS, and 5 μL of different fluorescein-labeled CD105, CD90, CD44, CD34, CD45 and HLA-DR monoclonal antibodies were added to mix gently, and incubated at room temperature for 30 min in the dark; PBS was washed twice, and 500 μL PBS was added to resuspend the cells, and the surface antigens of MSCs were detected by flow cytometry. The experimental results are shown in Figure 7 and Figure 8 .

[0069] Figure 7 and Figure 8 (In this experiment, FITC-A subset and FITC-A+ have the same meaning, and the corresponding other data are also the same), the first three peaks of each are suitable for positive detection of cell antigens, Figure 7 FITC-A subset: 98.9, perCP-A subset: 99.9, APC-A subset: 99.9; corresponding Figure 8In the figure, FITC-A+: 100, perCP-A+: 98.2, APC-A+: 100 (in this experiment, FITC-A subset and FITC-A+ are the same data, and the corresponding other data are also the same). The difference between the two is small, and both are above 95. The last peak graph is the negative detection of cell antigens. Figure 7 The PE-A subset in is 0.051, Figure 8 The PE-A+ in the test is 0.94, both of which are relatively small and are both below 2, that is, the properties of the two are relatively similar in this test, and the results obtained are also roughly the same, so they can be mixed for subsequent processing. Since the cells of the mucosal tissue have the same properties as the menstrual blood cells, they can also be used as induced differentiated cells in subsequent processing. Therefore, collecting menstrual blood and mucosal tissue at the same time can make more samples available for subsequent testing.

[0070] When inducing differentiation, using different induction reagents will have different effects on the results. The following is a comparison of two induction reagents and then select the best solution:

[0071] Basal induction reagent:

[0072] Serum-free basal medium (RPMI1640 medium) contains 4.5 g / L D-glucose, 1.5 g / L sodium bicarbonate, 1 mM sodium pyruvate, 10 mM HEPES and 300 mg / L L-glutamine; 10 -6 mol / L dexamethasone, 50 mg / L ascorbic acid, 10 ng / mL TGF-β1, take 5 mL insulin-transferrin-selenous acid (ITS) and add it to 500 mL RPMI basal medium, mix well, and keep at 4°C for use.

[0073] ITS (Insulin, Transferrin, Selenium) cell culture supplement (100-fold dilution) includes Insulin, Human Transferrin, and Selenous Acid. Each 10 mL contains: Human Recombinant insulin 6.25 mg; Human Holo Transferrin 6.25 mg; Selenous Acid 6.25 μg; BSA 1.25 g; Linoleic acid 5.35 mg.

[0074] The prepared complete differentiation induction medium needs to be stored in a 4°C refrigerator with a shelf life of 1 month until use.

[0075] Procedure for inducing chondrogenic differentiation of human mesenchymal stem cells:

[0076] 1. When the fusion degree of the 4th generation menstrual blood-derived endometrial mesenchymal stem cells cultured above reaches 80-90%, they can be digested with 0.25% trypsin.

[0077] 2. Count the digested cells and, based on the counting results, 3.5×

[0078] 10 5 Transfer the cells to be induced into ten 15 mL centrifuge tubes and centrifuge at 250 g for 5 minutes.

[0079] 3. Discard the supernatant, add 1 mL of chondrogenic differentiation basal medium, resuspend the cells, and centrifuge at 150 g for 5 minutes.

[0080] 4. Discard the supernatant, add 0.5 mL of complete chondrogenic differentiation medium, resuspend the cells, and centrifuge at 150 g for 5 minutes.

[0081] 5. After centrifugation, do not shake or blow the cell pellet. Carefully loosen the cap of the centrifuge tube to facilitate gas exchange. Place in a 37°C, 5% CO 2 CO 2 Culture in an incubator. During culture, be careful not to shake the centrifuge tube within 24 hours and keep the centrifuge tube still.

[0082] 6. Replace the complete chondrogenic differentiation medium with fresh medium every 3 days, 0.5 mL per tube. After changing the medium, flick the cell mass so that it can detach from the bottom of the tube and suspend in the liquid. It should be noted that the action should be gentle when changing the medium to avoid sucking out the chondrocytes; after each change of the complete medium, the centrifuge tube should be flicked to detach the chondrocytes from the bottom of the tube and suspend in the liquid; after each change of the complete medium, be sure to loosen the centrifuge tube cap before placing it in the incubator.

[0083] 7. Continue induction. During the induction process, the diameter of the cell cluster will increase and the surface will become smooth. After 20-30 days of continuous induction, the diameter of the chondrocyte ball will reach 1.5-2.0mm.

[0084] New induction agents:

[0085] The introduction of the patient's autologous serum and joint cavity tissue effusion will increase the release of exosomes and integrins with anti-inflammatory properties due to inflammatory stimulation during the culture of umbilical cord mesenchymal stem cells. Inspired by this, we added these substances to the complete cartilage induction culture medium system, which can enhance the maturity and toughness of chondrocytes in vitro. By using patient serum and joint cavity tissue effusion, the growth environment of cells can be simulated, making it easier for cells to survive in this environment. Patients with symptoms such as rheumatoid arthritis will have effusion in the joint cavity tissue of such patients, and the effusion contains substances that are beneficial to promoting cell differentiation.

[0086] Hydroxyapatite was introduced into the culture system, and other components remained unchanged: 10 -6 mol / L dexamethasone, 50mg / L ascorbic acid, 10ng / mL TGF-β1, take 5mL insulin-transferrin-selenous acid (ITS), add 50μL patient's autologous serum and 25μL joint cavity tissue fluid, mix well, and keep at 4℃ for use.

[0087] The prepared induction differentiation complete medium needs to be stored in a 4°C refrigerator with a shelf life of 1 month until use.

[0088] Hydroxyapatite is introduced into the culture system for the following reasons:

[0089] Hydroxyapatite (HA) is currently one of the best biomaterials used for implantation in the human body as a hard tissue substitute. Because its chemical structure and physical properties are similar to the inorganic components of human bone tissue, it is widely used in orthopedics, plastic surgery, and oral surgery. There have been many reports on HA bone response. As a type of inorganic material, HA itself does not have the ability to generate bone, but it has a good osteoinductive effect and can induce new bone formation with the participation of related cells. HA and bone cells can interact and influence each other. The former can directly affect the biological characteristics and metabolic processes of the latter, and the latter can promote the former to play an osteoconductive role.

[0090] In terms of promoting cell growth: Since the chemical structure of hydroxyapatite is similar to the inorganic components in human bones, after implantation in the body, calcium and phosphorus will be released from the surface of the material and absorbed by the body tissues, and new tissues will grow. In cell culture research, this environment in the body can be simulated to provide a mineralization environment similar to that in the body for cell growth, and promote the growth and differentiation of certain specific cells (such as osteoblasts and other bone-related cells). For example, when studying cell culture related to bone tissue engineering, cells are inoculated in a culture system containing hydroxyapatite, and the cells may attach, proliferate and differentiate better. However, specific parameters such as inoculation density and culture time need to be optimized according to different cell types.

[0091] Dosage of Hydroxyapatite in Cell Culture:

[0092] Since different cell types may have different requirements and tolerance for hydroxyapatite, osteoblasts may require a relatively high concentration of hydroxyapatite to better simulate the bone formation environment in vivo. Studies have shown that the local concentration of HA microcrystals in the body can affect the division and proliferation of bone cells and the mineralization and deposition of cells. However, high concentrations of HA particles may have certain cytotoxicity, especially some very fine powdery substances. The purpose of the present invention is to find a suitable concentration of HA microcrystals that can maintain bone cell function and reduce toxic effects on cells; it can also promote calcium salt metabolism in cells and accelerate the mineralization of bone cells.

[0093] Procedure for inducing chondrogenic differentiation of human mesenchymal stem cells:

[0094] 1. When the fusion degree of the 4th generation menstrual blood-derived endometrial mesenchymal stem cells cultured above reaches 80-90%, they can be digested with 0.25% trypsin.

[0095] 2. Count the digested cells. According to the counting results, 3.5×10 5 Transfer the cells to be induced into ten 15 mL centrifuge tubes and centrifuge at 250 g for 5 minutes.

[0096] 3. Discard the supernatant, add 1 mL of chondrogenic differentiation basal medium, resuspend the cells, and centrifuge at 150 g for 5 minutes.

[0097] 4. Discard the supernatant, add 0.5 mL of chondrogenic differentiation complete medium and 3.0 mg / mL hydroxyapatite, resuspend the cells, and centrifuge at 150 g for 5 minutes.

[0098] 5. After centrifugation, do not shake or blow the cell pellet. Carefully loosen the cap of the centrifuge tube to facilitate gas exchange. Place in a 37°C, 5% CO 2 CO 2 Culture in an incubator.

[0099] Note: Do not shake the centrifuge tube within 24 hours and keep it still.

[0100] 6. Replace the complete chondrogenic differentiation medium with fresh medium every 3 days, 0.5 mL per tube. After changing the medium, flick the cell mass so that it can detach from the bottom of the tube and suspend in the liquid. It should be noted that the action should be gentle when changing the medium to avoid sucking out the chondrocytes; after each change of the complete medium, the centrifuge tube should be flicked to detach the chondrocytes from the bottom of the tube and suspend in the liquid; after each change of the complete medium, be sure to loosen the centrifuge tube cap before placing it in the incubator.

[0101] 7. Continue induction. During the induction process, the diameter of the cell cluster will increase and the surface will become smooth. After 20-30 days of continuous induction, the diameter of the chondrocyte ball will reach 1.5-2.0mm.

[0102] After the induction is completed, it is necessary to detect whether the induced cells are chondrocytes, and their quantity, activity, purity, etc. What is known during the test is that chondrocytes have no specific markers and can be identified by toluidine blue staining, Alcian blue staining, and immunohistochemical staining of type II collagen, the matrix component glycosaminoglycan (GAG) secreted by them, combined with the sampling site and culture observation. GAG is a proteoglycan polymer that can be metamorphosed when stained with toluidine blue and Alcian blue. Among them, Alcian blue staining mainly reacts with the mucopolysaccharide in GAG. Chondrocytes secrete different amounts of GAG, and the coloring depth varies. Immunohistochemical staining of type II collagen applies the principle of the ABC method to display the corresponding antigens of tissue cells through enzymatic reactions. Since the collagen of articular cartilage is mainly type II collagen, immunohistochemical staining can specifically reflect the content of type II collagen expressed by chondrocytes. In this experiment, it was observed that blue metachromatic granules were seen in chondrocytes during toluidine blue staining, and a small amount of metachromatic granules appeared around the cells; blue-purple granules were seen in chondrocytes during DMB staining, and a small amount of metachromatic granules appeared around the cells; during Alcian blue staining, Alcian blue reacted with the mucopolysaccharide in GAG, and the chondrocyte nucleus was light blue, and many light blue secretory granules and vesicles were seen in the cytoplasm; while during type II collagen immunohistochemical staining, the chondrocyte cytoplasm was stained brown-yellow, and the nucleus was not stained. Transmission electron microscopy analysis showed that the cell nucleus was large, mostly round, irregular in shape, with nucleoli fringe and obvious euchromatin. The cytoplasm was rich in cytoplasm, with more ribosomes and mitochondria, rich in rough endoplasmic reticulum and secretory vesicles, and the vesicles contained synthetic matrix components. A large number of clustered glycogen granules were also seen in the cytoplasm, confirming that the observed cells were chondrocytes.

[0103] The following are the tests for the identification of chondrocytes:

[0104] 1. Alcian blue staining analysis (I)

[0105] 1. At the end of the induction culture, discard the culture supernatant, wash the cells twice with DPBS, add 2 mL / tube of 4% neutral paraformaldehyde solution, and fix the cells at room temperature for more than 1 hour.

[0106] 2. Discard the fixative, wash twice with DPBS, dehydrate, embed in paraffin and slice. Be careful during embedding and slicing to avoid breaking or losing the cartilage balls.

[0107] 3. Alcian blue staining: After the sections are dewaxed, add Alcian blue staining solution to the dried sections and stain at room temperature for 1 hour. Rinse the slides with running water for 5 minutes, dry them, observe them under a microscope, and take pictures. Fig. 9 .

[0108] After 25 days of induction culture with mesenchymal stem cell chondrogenic differentiation and staining kit, chondrocyte spheres were made into paraffin sections and stained. Fig.10 ) The chondrocyte spheres were stained blue, proving the presence of proteins such as chondroitin sulfate and glial sulfate, showing typical chondrogenic differentiation.

[0109] The following groups Fig.11 From day 0 to day 4 (AB), the cell morphology changed significantly, the intercellular gaps were obvious, and some cells became round or even floated. On day 5 (C), small cell clusters appeared in some areas. From day 6 to day 14 (DEF), the number of cell clusters increased, the volume gradually increased, and larger cell clusters were rare. On day 15 (GH), cells in some areas detached from the wall and aggregated into larger cell clusters, especially at the edge of the hole, where a large area of ​​cell detachment and aggregation appeared. From day 16 to day 23 (IJ), the number of cell clusters detached from the wall gradually increased, and the number of larger cell clusters formed also gradually increased. The staining results showed (KLMN) that the cell parts of the entire field of view appeared blue, and the depth of blue staining was slightly different, and the blue staining was heavier at the cell cluster position.

[0110] 2. Analysis of chondrocytes by toluidine blue staining

[0111] 1. After the cell differentiation is induced, carefully discard the cell culture supernatant, rinse with PBS 1-2 times, and fix at room temperature for 30 minutes. (The cell fixative is 4% neutral formaldehyde solution)

[0112] 2. Aspirate the cell fixative and rinse twice with PBS. Slowly add toluidine blue staining solution along the well wall and stain at room temperature for 30 minutes. (Note: There may be precipitation at the bottom of the staining solution. Try not to touch the bottom when aspirating. If there is precipitation after staining, wash it off with PBS.)

[0113] 3. Aspirate the staining solution, rinse with PBS, and remove the floating color. Observe the cell staining effect under a microscope. The background is light blue and the chondrocytes are purple-red.

[0114] The results are as follows Fig.12 shown.

[0115] 3. Immunohistochemical staining of type II collagen

[0116] Type II collagen: It is the main component of the extracellular matrix and a characteristic marker of chondrocytes. After immunohistochemical staining of type II collagen, the cell cytoplasm appears brown-red or brown-yellow, and the cytoplasm is stained in different shades. The darker the staining, the higher the expression of type II collagen.

[0117] Experimental steps of immunohistochemical staining of type II collagen in chondrocytes:

[0118] 1. Preparation and fixation of tissue cell sections: The tissue cells were fixed in formalin, then embedded in paraffin, and adhered to glass slides after sectioning.

[0119] 2. Baking and dewaxing: Bake the slices in a constant temperature oven to prevent tissue cells from falling off, and then dewax them with xylene.

[0120] 3. Dewaxing and hydration: Soak the paraffin sections in fresh xylene for 15 minutes, repeat three times. After removing the excess liquid, soak in anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol in turn, soak for 5 minutes each, rinse with distilled water (or tap water) for 5 minutes. Rinse the sections with PBS buffer for 5 minutes, repeat three times.

[0121] 4. Antigen repair: Immerse the slices in boiling 0.01M citric acid buffer solution (pH 6.0), cover the pot, add a pressure valve, continue heating until it sprays, leave the heat source after 2 minutes, and rinse with tap water to room temperature.

[0122] 5. Block endogenous peroxidase: incubate the sections with 3% H2O2 at room temperature for 10 minutes, rinse with PBS for 5 minutes, and repeat 3 times.

[0123] 6. Serum blocking: wipe the slide dry with absorbent paper, add 50 μL of normal goat serum working solution, and block at 37°C for 20 minutes to reduce nonspecific staining.

[0124] 7. Primary antibody incubation: Use antibody diluent to dilute the primary antigen solution into working solution, wipe the liquid around the slide tissue with absorbent paper, add appropriate amount of antibody working solution, incubate at 37℃ for 1h-2h, rinse the slices with PBS buffer for 5 minutes, and repeat three times.

[0125] 8. Secondary antibody incubation: After wiping the sections dry with absorbent paper, add 50 μL of biotin-labeled goat anti-rabbit IgG, incubate at 37°C for 20 minutes, rinse the sections with PBS buffer for 5 minutes, and repeat three times.

[0126] 10. Triple antibody incubation: After wiping the sections dry with absorbent paper, add 50 μL of horseradish enzyme-labeled streptavidin and incubate at 37°C for 20 minutes. Rinse the sections with PBS buffer for 5 minutes and repeat three times.

[0127] 11. Color development: shake off the PBS buffer, wipe the slices dry with absorbent paper, and add 50 μL of freshly prepared DAB working solution to each slice.

[0128] 12. Re-staining: Add appropriate amount of hematoxylin staining solution for re-staining, incubate for 15 minutes, rinse with tap water for 5 minutes, add hydrochloric acid alcohol differentiation solution for differentiation for 30 seconds, and rinse with tap water.

[0129] 13. Dehydration and sealing: Place the slides in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and anhydrous ethanol in turn, soak for 5 minutes each, then soak the slides in xylene for 15 minutes, repeat three times. Take out the slides, dry them slightly, and seal them with neutral gum.

[0130] Result judgment:

[0131] The stained sections were observed and judged under an optical microscope. Hematoxylin stained the cell nucleus blue, and DAB stained the positive expression brown-yellow. Fig.13 shown.

[0132] In summary, the present invention obtains menstrual blood-derived mesenchymal stem cells by collecting female menstrual blood (including shed endometrial tissue), and stably proliferates in a given culture system. In addition, it can be known from literature that mesenchymal stem cells have the ability to differentiate into different cells. The present invention can induce them to differentiate into chondrocytes through a variety of induction factors, and the differentiated cells induced by this method are identified as chondrocytes through toluidine blue staining, Alcian blue staining and immunohistochemical staining of type II collagen.

[0133] The method of inducing fluid replacement is consistent with the above method. The comparative experimental results are as follows:

[0134] 1. From the perspective of cartilage masses, the induced experimental group 1 (the experimental group using the basic induction reagent) is flat and small, while the induced experimental group 2 (the experimental group using the new induction reagent) is round and full with a three-dimensional sense. Since the initial inoculation amount of cells is the same, it is considered that the addition of serum and tissue to the induction reagent may cause different cell morphologies, which is conducive to ensuring the activity and storage of cells and subsequent clinical applications. Add pictures under the microscope to observe the growth status of cells. As follows Fig.15 As shown, Figure A is the induction of experimental group 1, and Figure B is the induction of experimental group 2.

[0135] 2. Comparative analysis of cell activity:

[0136] The cells were tested by Countstar IC1000 automatic cell counter. The cells were tested one week after induction and on the 25th day after induction. The experimental test results were exported from the system software Countstar. The parameters such as cell activity and number were grouped as follows: Figures 16 to 21 As shown:

[0137] Results: The blank control used serum-free basal medium (RPMI1640 medium) containing 4.5 g / L L-glucose, 1.5 g / L sodium bicarbonate, 1 mM sodium pyruvate, 10 mM HEPES and 300 mg / L L-glutamine, which only provided basic nutrition for cells and did not produce chondrocytes. The number of cells decreased by nearly 50% and the cell activity decreased by 10 percentage points. Compared with experimental group 1 and experimental group 2, the results of experimental group 2 were better than those of experimental group 1. Specifically, after one week of induction, the number and activity of cells in experimental group 1 were 96.08% and 7.88×10 5 / mL, the cell number and activity of experimental group 2 were 99.06% and 1.09×10 6 / mL; after 25 days of induction, the number and activity of cells in experimental group 1 were 90.11% and 1.36×10 6 / mL, the cell number and activity of experimental group 2 were 97.95% and 1.74×10 6 / mL, both the number of cells and the activity of cells were significantly improved. Conclusion The induction results of experimental group 2 were better, and the method was better than that of experimental group 1. That is, the results of using the new induction reagent were significantly better than those of the basic induction reagent, and the number, activity and purity of the obtained cells were higher, which could meet the requirements of clinical application.

[0138] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A reagent for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes, characterized in that: include: Endometrial tissue preservation solution, new induction reagents; Endometrial tissue preservation solution includes: 500 mL DMEM / F-12 basal medium, penicillin, streptomycin, and gentamicin; The new induction reagents include: basic induction reagents, hydroxyapatite, patient serum and joint cavity tissue fluid.

2. The reagent for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 1, characterized in that: The endometrial tissue preservation solution contains 100 U / mL of penicillin, 100 μg / mL of streptomycin, and 25 mg / L of gentamicin. The hydroxyapatite in the novel induction reagent is 3.0 mg / mL, and the added serum is 50 μL and the added joint cavity tissue fluid is 25 μL.

3. The reagent for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 1, characterized in that: The basic induction reagent is RPMI1640 culture medium, which contains: D-glucose, sodium bicarbonate, sodium pyruvate, HEPES and L-glutamine; Dexamethasone, ascorbic acid, TGF-β1, and insulin-transferrin-selenous acid (ITS) are also added to the RPMI1640 culture medium.

4. A method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes, characterized in that: A reagent for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes as claimed in claim 1; The following steps are involved: Prepare basic reagents and samples; Process the components in the sample and culture the resulting cells; further processing the cultured cells; Inducing differentiation of treated cells by basic induction agents and novel induction agents; Some data of cells differentiated by the two reagents were detected and compared.

5. The method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 4, characterized in that: The sample is 10 mL of menstrual blood, which contains mucosal tissue; Separately handle menstrual blood and mucosal tissue; Isolation and culture of mononuclear cells from menstrual blood; The mucosal tissue was isolated, cultured and digested.

6. The method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 4, characterized in that: After processing the samples, menstrual blood-derived endometrial mesenchymal stem cells were obtained and cultured. After culture, the cells were fully replaced with medium, cell passaged, and MMCs immunolabeling was performed. During these processes, the cells obtained after menstrual blood and mucosa treatment were processed separately, and the results were compared.

7. The method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 4, characterized in that: When testing the cells at the induced differentiation site, Alcian blue staining analysis, chondroblast toluidine blue staining analysis and type II collagen immunohistochemical staining were performed; When testing the induced differentiated cells, it is also necessary to observe the morphology of the cartilage masses and conduct a comparative analysis of the cell activity.

8. The method for inducing differentiation of menstrual blood-derived endometrial mesenchymal stem cells into chondrocytes according to claim 4, characterized in that: The basic reagents that need to be configured are: endometrial tissue preservation solution, PBS buffer, and complete culture medium.

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