3D cartilage tissue and preparation method and application thereof

Abstract

The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a 3D cartilage tissue and a preparation method and application thereof. In order to solve the problems that in the prior art, the source of mesenchymal stem cells is limited, induced cartilage is prone to fibrosis and the like, skin fibroblasts are reprogrammed into cartilage precursor cells by combining transcription factor transfection with multi-stage small molecule regulation, and formation and function maintenance of cartilage particles are optimized by utilizing a dynamic 3D culture system. Experiments prove that the cartilage tissue marker gene induced by the method is normal in expression and is negative in COL10, the cartilage transparency characteristic of the cartilage tissue marker gene is verified through alcian blue staining and immunohistochemistry, and in-vivo repair experiments show that the curative effect is remarkable. According to the scheme, raw materials are easy to obtain, the cost is controllable, and an efficient and stable technical platform is provided for cartilage defect repair and organoid research.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering and regenerative medicine, and particularly to a 3D cartilage tissue and its preparation method and application. Background Art

[0002] Cartilage is a translucent and elastic tissue that forms the skeleton of vertebrate embryos and very young larvae. In higher vertebrates, most of it is transformed into bone, but primitive species (such as sturgeons and elasmobranchs) retain it throughout their lives and it is the main component of the skeleton. Cartilage is composed of chondrocytes, fibers, and a matrix. The matrix contains 70% water, and the organic components are mainly various proteins, such as chondromucoid, collagen, and chondrocalcin, etc.

[0003] Cartilage tissue engineering is an important field of regenerative medicine, but current mainstream cartilage induction methods highly rely on mesenchymal stem cells (MSCs), such as bone marrow mesenchymal stem cells (BMSCs), umbilical cord mesenchymal stem cells (UMSCs), or adipose mesenchymal stem cells (ADSCs). These cell sources have significant limitations: BMSCs need to be obtained by invasive bone marrow aspiration, UMSCs are limited to neonatal umbilical cord samples, and ADSCs need to be extracted by liposuction surgery, all of which have ethical controversies, strong donor dependence, and low clinical application accessibility. In addition, existing induction techniques generally rely on high concentrations of TGF-β and BMP family factors, resulting in the induced cartilage tissue being prone to fibrosis (manifested by high expression of type X collagen) and unable to maintain the physiological functions of hyaline cartilage (such as joint shock absorption and lubrication).

[0004] Although serum-free media and 3D culture techniques have partially improved the culture environment, the limited cell source and fibrosis problem are still the core bottlenecks restricting the clinical translation of cartilage repair.

[0005] Therefore, there is an urgent need for an alternative solution with a wide source, controllable cost, and capable of stably generating hyaline cartilage. Summary of the Invention

[0006] The purpose of the present invention is to provide a 3D cartilage tissue and its preparation method and application, which provide an efficient and stable solution for cartilage defect repair and organoid research.

[0007] In order to achieve the above invention purpose, the present invention provides the following technical solutions: The present invention provides a preparation method of a 3D cartilage tissue, comprising the following steps: Digest skin fibroblasts to obtain cell pellets; Transfect a plasmid containing or capable of activating OCT4, SOX2, and KIF4 into the cell pellets to obtain an electrotransfected cell suspension; The cell suspension after electroporation is cultured with medium B to obtain high-density cells; The high karyoplasmic ratio cells in the high-density cells are isolated, cultured and passaged to obtain passaged cells; The passaged cells are cultured with medium C, and then transferred to a 3D culture container after culturing, and medium D is added for culturing; It is replaced with medium E and 3D culture is continued; It is replaced with medium D, and 3D cartilage tissue is cultured and obtained; The medium B is used to regulate cell gene expression and signal pathways, and assist cell reprogramming to obtain the potential to differentiate into chondrocytes, and contains TGF-β3, FGF2, Chir99021, PD0325901, A-83-01, pH regulator, LDN-193189, FSK, VPA, BMP4, DZNeP, EPZ5676, RepSox, AM580 and Brdu; The medium C is used to guide the reprogrammed cells to differentiate in the direction of chondrocytes, so that they gradually possess the characteristics of chondrocytes, and contains FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A and GluMax; The medium D is used to promote the differentiation of cells into cartilage tissue in a 3D environment and form cartilage tissue; The medium E is used to support the maturation and maintenance of cartilage tissue.

[0008] Preferably, the 3D culture container is a CERO culture tube.

[0009] Preferably, the number of days of culture is 1-30 d.

[0010] Preferably, the medium B contains Insulin, ascorbic acid diphosphate, transferrin, selenium salt, TGF-β3, FGF2, Chir99021, PD0325901, A-83-01, pH regulator, LDN-193189, FSK, VPA, BMP4, DZNeP, EPZ5676, RepSox, AM580 and Brdu; The final concentration of the Insulin is 18-22 μg / mL; The final concentration of the ascorbic acid diphosphate is 180-220 μg / mL; The final concentration of the transferrin is 18-22 μg / mL; The final concentration of the selenium salt is 18-22 ng / mL; The final concentration of the TGF-β3 is 1.8-2.2 ng / mL; The final concentration of the FGF2 is 36-44 ng / mL; The final concentration of Chir99021 is 45 - 55 nM; The final concentration of PD0325901 is 9 - 11 nM; The final concentration of A - 83 - 01 is 4.5 - 5.5 nM; The final concentration of LDN - 193189 is 4.5 - 5.5 nM; The final concentration of FSK is 9 - 11 μM; The final concentration of VPA is 0.09 - 0.11 mM; The final concentration of BMP4 is 9 - 11 ng / mL; The final concentration of DZNeP is 0.045 - 0.055 μM; The final concentration of EPZ5676 is 4.5 - 5.5 μM; The final concentration of RepSox is 4.5 - 5.5 μM; The final concentration of AM580 is 0.045 - 0.055 μM; The final concentration of Brdu is 9 - 11 μM.

[0011] Preferably, the medium B is based on DMEM / F12.

[0012] Preferably, the medium C is based on DMEMKO and contains FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A, and GluMax; The final volume percentage concentration of FBS is 0.83 - 1.67%; The final volume percentage concentration of KSR is 1.67 - 3.33%; The final concentration of BMP4 is 10 - 20 ng / mL; The final concentration of Chir99021 is 10 - 30 nM; The final concentration of FGF2 is 5 - 20 ng / mL; The final concentration of Wnt3A is 5 - 20 ng / mL; The final volume percentage concentration of GluMax is 0.5 - 2%.

[0013] Preferably, the medium D is based on DMEM and contains KSR, GluMax, TGFβ - 3, BMP - 4, dexamethasone, ascorbic acid, proline, and iTS - plus; The final volume percentage concentration of KSR is 3 - 5%; The final volume percentage concentration of GluMax is 0.5 - 1.5%; The final concentration of the TGFβ-3 is 5-10 ng / mL; The final concentration of the BMP-4 is 10-20 ng / mL; The final concentration of the dexamethasone is 80-120 nM; The final concentration of the ascorbic acid is 30-80 μg / mL; The final concentration of the proline is 20-60 μg / mL; The final volume percentage concentration of the iTS-plus is 0.8-1.2%; And / or, the medium E is based on DMEM and contains KSR, GluMax, NEAA, pyruvate, dexamethasone, GDF5, FGF2, iTS-plus, ascorbic acid and proline; The final volume percentage concentration of the KSR is 3-5%; The final volume percentage concentration of the GluMax is 0.5-1.5%; The final volume percentage concentration of the NEAA is 0.5-1.5%; The final volume percentage concentration of the pyruvate is 0.5-1.5%; The final concentration of the dexamethasone is 80-120 nM; The final concentration of the GDF5 is 20-30 ng / mL; The final concentration of the FGF2 is 1-5 ng / mL; The final volume percentage concentration of the iTS-plus is 0.5-1.5%; The final concentration of the ascorbic acid is 30-80 μg / mL; The final concentration of the proline is 20-60 μg / mL.

[0014] Preferably, the skin fibroblasts are obtained by a preparation method including the following steps: Digest and culture the skin tissue to obtain a culture solution containing skin fibroblasts; Add the proliferation medium A and culture to obtain skin fibroblasts; The proliferation medium A is based on DMEM and contains penicillin-streptomycin, FBS, GluMax and NEAA; The final volume percentage concentration of the penicillin-streptomycin is 0.8-1.2%; The final volume percentage concentration of the FBS is 3-8%; The final volume percentage concentration of the GluMax is 0.3-0.8%; The final volume percentage concentration of the NEAA is 0.3-0.8%.

[0015] The present invention also provides a 3D cartilage tissue prepared by the above preparation method.

[0016] The present invention also provides the application of the above 3D cartilage tissue in the preparation of clinical cartilage repair materials, drug research and development and screening, or cartilage biological research.

[0017] Beneficial effects of the present invention: The present invention uses skin fibroblasts (FCs) as raw materials. Abundant cells can be obtained through simple separation and amplification, without invasive sampling, significantly reducing donor dependence and ethical risks; by optimizing the combination of small molecules, the induced cartilage tissue shows negative expression of COL10, and alcian blue staining and COL2 immunohistochemistry confirm its hyaline cartilage characteristics, effectively avoiding the formation of fibrous cartilage; the present invention can also combine a dynamic rotating culture instrument (CERO system) with a multi-stage culture medium to achieve efficient amplification and morphology maintenance of cartilage particles, shortening the culture period to 14 days, and the cell density and functional indicators are highly consistent with those of natural cartilage. Animal experiments show that CLFC cartilage particles combined with GelMA hydrogel can effectively repair rat articular cartilage defects, providing a scalable production solution for traumatic cartilage injuries or degenerative joint diseases. Description of the drawings

[0018] Figure 1 It is a physical photograph of the cartilage tissue induced by the present invention; Figure 2 It is a comparison chart of marker expressions between the cartilage tissue derived from skin cells of the present invention and normal cartilage tissue; Figure 3 It is a result chart of alcian blue staining of the induced cartilage tissue of the present invention; Figure 4 It is a result chart of COL2 immunohistochemistry of paraffin sections of the induced cartilage tissue of the present invention; Figure 5 It is a result chart of COL10 immunohistochemistry of paraffin sections of the induced cartilage tissue of the present invention; Figure 6 It is an experimental effect chart of using the cartilage particles of the present invention for cartilage repair in rats in vivo (experimental group); Figure 7 It is an experimental effect chart of using the cartilage particles of the present invention for cartilage repair in rats in vivo (sham operation control group). Detailed implementation manners

[0019] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0020] Sources of reagents and materials in the embodiments of the present invention: Insulin (Company: MedChemExpress, Catalog Number: HY-P73243) Ascorbic acid diphosphate (Company: Sigma-Aldrich, CAS Number: 113170-55-1) Transferrin (Company: Sigma-Aldrich, Catalog Number: T8158) Selenium salt (Company: Sigma-Aldrich, Catalog Number: S5261) TGF-β3 (Company: STEMCELL Technologies, Catalog Number: # 78156) FGF2 (Company: Thermo Fisher Scientific, Catalog Number: 100-18BHS-50UG) Chir99021 (Company: Selleck, Catalog Number: S1263) PD0325901 (Company: Selleck, Catalog Number: S1036) A-83-01 (Company: Selleck, Catalog Number: S7692) LDN-193189 (Company: Selleck, Catalog Number: S2618) FSK (Company: MedChemExpress, Catalog Number: HY-15371) VPA (Company: Selleck, Catalog Number: S3944) BMP4 (Company: R&D System, Catalog Number: 314E-GMP) DZNeP (Company: MedChemExpress, Catalog Number: HY-10442) EPZ5676 (Company: Selleck, Catalog Number: S7062) RepSox (Company: Selleck, Catalog Number: S7223) AM580 (Company: Selleck, Catalog Number: S2933) Brdu (Company: Selleck, Catalog Number: S7918) FBS (Company: Thermo Fisher Scientific, Catalog Number: 10099141C) KSR (Company: Thermo Fisher Scientific, Catalog Number: 10828028) Wnt3A (Company: R&D System, Catalog Number: 5036-GMP) GluMax (Company: Thermo Fisher Scientific, Catalog Number: 35050061) Dexamethasone (Company: Selleck, Catalog Number: S1322) Ascorbic acid (Company: SparkJade, Catalog Number: SJ-MN0658) Proline (Company: Sigma-Aldrich, Catalog Number: 1074340100) iTS-plus (Company: Sigma-Aldrich, Catalog Number: I2521-5ML) NEAA (Company: Thermo Fisher Scientific, Catalog Number: 11140050) Pyruvic acid (Company: Thermo Fisher Scientific, Catalog Number: 11360070) GDF5 (Company: R&D System, Catalog Number: 8340-G5-050) Penicillin-Streptomycin (Company: Thermo Fisher Scientific, Catalog Number: 15070063) The plasmid was purchased from Addgene Addgene: pCXLE-hOCT3 / 4-shp53-F Addgene: pCXLE-hSK Two plasmids, where the function of pCXLE-hOCT3 / 4-shp53-F is to activate the OCT4 transcription factor, and pCXLE-hSK provides SOX2 and KLF4.

[0021] Examples I. Obtaining of skin fibroblasts 1. Preparation in advance: 1 hour before taking the sample, add 0.1% gelatin to each well of a 6-well plate in a laminar flow hood. Place the plate coated with gelatin in the cell culture incubator for 30 minutes. Take a 15 ml centrifuge tube filled with culture medium, preheat it to 37 degrees, and then take the sample at the hospital. Record the age of the sample donor and the sampling site.

[0022] 2. Rinsing and separation: Add a sterilized PBS solution containing double antibiotics to the culture dish. Wipe the 15 ml centrifuge tube for sampling with 75% alcohol, place the sample in the culture dish, and use sterilized forceps to rinse the skin tissue at least 3 times. Then, in the culture dish, use ophthalmic scissors and a scalpel to remove the white subcutaneous fat and slightly remove the epidermis, separate the epidermis and subcutaneous tissue, and leave the dermis. Place the epidermis side up and the dermis adheres to the wall.

[0023] 3. Prepare a petri dish. Place the sample skin pieces in a new petri dish. Use forceps to lift the epidermis and remove it. Cut the remaining dermal tissue into small pieces with scissors and a scalpel.

[0024] 4. Transfer it to a 50 ml centrifuge tube, add type I collagenase, digest it by constant shaking at 37 °C, and harvest fibroblasts in stages (the intervals are 2 h and 4 h respectively).

[0025] 5. After each stage of time has passed, operate in a laminar flow hood, filter with a cell sieve to remove undigested tissue, and collect the cell suspension.

[0026] 6. Centrifuge at 200 g for 8 min, discard the supernatant, resuspend the cells with the culture medium, and inoculate them into a culture plate.

[0027] 7. Check for contamination the next day. Change the medium or passage the cells according to the cell growth conditions.

[0028] 8. Discard the supernatant, retain less than 1 mL of liquid, transfer the liquid to a gelatin-coated cell culture plate, and add 5 mL of medium A (formula of medium A: 465 mL of DMEM, 25 mL of FBS (South America), 2.5 mL of GluMax, 2.5 mL of NEAA, 5 mL of penicillin-streptomycin). Observe cell attachment within 5 - 14 days. When the cells proliferate to 2×10 7 cells, proceed to the next step.

[0029] II. Transfection of skin fibroblasts 1. Digest the proliferated fibroblasts with trypsin. After terminating the digestion, centrifuge at 200 g for 5 minutes to collect the precipitate.

[0030] 2. Use the Lonza electroporation kit (VPI - 1005) to transfect the plasmid containing OCT4, SOX2, and KIF4 according to the instructions. Select electroporation program C - 017 or X - 001.

[0031] 3. Transfer the electroporated cell suspension to a cell culture plate pre-coated with MG. Place it in an incubator at 37 °C and 5% CO2. After continuous culture for 5 - 7 days, proceed to the next step.

[0032] 4. Change the medium of the cells with medium B every day and culture for 21 days, and high-density cells can be observed.

[0033] Formula of medium B: 500 mL of DMEM / F12; 20 μg / mL of Insulin; 200 μg / mL of ascorbic acid diphosphate; 20 μg / mL of transferrin; Selenium salt 20 ng / mL; TGF-β3 2 ng / mL; FGF2 40 ng / mL; Chir99021 50 nM; PD0325901 10 nM; A-83-01 5 nM; Bicarbonate 2438 μg / mL; LDN-193189 5 nM; FSK 10 μM; VPA 0.1 mM; BMP4 10 ng / mL; DZNeP 0.05 μM; EPZ5676 5 μM; RepSox 5 μM; AM580 0.05 μM; Brdu 10 μM.

[0034] 5. Use the physical passage method to separate cells with a high nuclear-cytoplasmic ratio, and continuously culture them using mTeSR 1 medium (Stemcell Technologies). When the cell density reaches 70%-90%, passage the cells.

[0035] III. Induction of cells 1. After single-cell passage of the 10th-generation cells, the next day, culture the cells using medium C. After 5 days, treat the cells with 0.5% mM EDTA, collect the cells, and passage them at a ratio of 1:3 to 1:6 (the specific ratio is determined according to the density before passage, cell growth rate, and actual situation).

[0036] Formula of medium C: DMEM KO 480 mL; FBS (South America) 5 mL; KSR 10 mL; BMP4 10 ng / mL; Chir99021 20 nM; FGF2 10 ng / mL; Wnt3A 10 ng / mL; GluMax 5 mL.

[0037] 2. Transfer to a 3D culture centrifuge tube and culture using medium D.

[0038] Formulation of Medium D: 470 mL of DMEM; 20 mL of KSR; 5 mL of GluMax; 10 ng / mL of TGFβ-3; 10 ng / mL of BMP-4; 100 nM of dexamethasone; 50 μg / mL of ascorbic acid; 40 μg / mL of proline; 5 mL of iTS-plus.

[0039] 3. Place the CERO culture tube in the slot of the CERO automatic 3D cell culture instrument, set the program to dynamic mode, set the rotation speed of the rotation function to 100 revolutions per minute, the running time to 5 seconds, and the pause time to 2 seconds. Set the running duration to continuous operation.

[0040] 4. Replace the medium every 3 days. After culturing for 6 to 9 days, take out the CERO culture tube, suck out the medium in the tube, and discard the waste liquid.

[0041] 5. Add 20 mL of Medium E (the same volume as Medium B) to the CERO culture tube. Put the culture tube back into the CERO automatic 3D cell culture instrument. Set the program to dynamic mode, set the rotation speed of the rotation function to 100 revolutions per minute, the running time to 1 second, and the pause time to 2 seconds. Set the running duration to continuous operation.

[0042] Formulation of Medium E: 460 mL of DMEM; 20 mL of KSR; 5 mL of GluMax; 5 mL of NEAA; 5 mL of sodium pyruvate; 100 nM of dexamethasone; 100 ng / mL of GDF5; 5 ng / mL of FGF2; 5 mL of iTS-plus; 50 μg / mL of ascorbic acid; 40 μg / mL of proline.

[0043] 6. Take out the CERO culture tube, suck out and discard half of the original volume of the medium. Re-supplement Medium D to the original volume. Repeat the steps every 2 days and continue culturing for 7 - 14 days to obtain cartilage particles that are positive for Alcian blue staining and COL2.

[0044] Figure 1 Photograph of cartilage induced by the present invention.

[0045] Detection method for cartilage marker (1). Transfer the cartilage particles from the CERO culture tube to a 15 mL centrifuge tube, centrifuge at 100 g for 5 min using a centrifuge. Discard the supernatant, add 1 mL of PBS to each tube to wash the surface of the pellets, and repeat three times.

[0046] (2). Take out the cartilage particles from PBS and transfer them into liquid nitrogen to quickly freeze the cartilage tissue.

[0047] (3). Put 10 - 12 pieces of cartilage tissue into a mortar, crush it, and make it as uniform as possible.

[0048] (4). Add 1 mL of Trizol reagent to the mortar to completely cover the fragmented cartilage tissue, grind it again, let it stand for 5 min to fully lyse, pipette up and down, and transfer it to a 1.5 mL enzyme-free EP tube.

[0049] (5). Add 200 μL of chloroform to every 1 mL of Trizol, vortex for 30 seconds to mix well, let it stand for 5 minutes. Centrifuge at 4℃, 12,000 g for 15 min.

[0050] (6). After centrifugation, the EP tube is divided into three phases. Carefully aspirate the upper clear liquid phase. The volume of the upper liquid phase is about 60% of the volume of Trizol.

[0051] (7). Add isopropanol with a volume of 50% of Trizol, shake vigorously to mix well. Isopropanol can precipitate RNA. Let it stand for 10 min after mixing well, centrifuge at 4℃, 12,000 g for 10 min.

[0052] (8). Discard the supernatant, avoid sucking the visible white precipitate at the bottom of the EP tube, sometimes it may also be a colorless precipitate. Add 1 mL of 75% ethanol, mix well to wash the RNA precipitate, centrifuge at 4℃, 7500 g for 10 min.

[0053] (9). Discard the supernatant, invert and dry for 10 min, add 100 μL of RNase-Free water to dissolve the RNA.

[0054] (10). Use NanoDrop to detect the RNA concentration.

[0055] (11). The obtained RNA is placed in a water bath at 65℃ for 5 min and then cooled on ice.

[0056] (12). Configure the reverse transcription system according to the reverse transcription kit instructions, use a PCR instrument for reaction, 37 °C for 15 min for transcription reaction; 98 °C for 5 min to inactivate the enzyme; store at 4 °C for later use.

[0057] (13). Add SYBGreen, water, upstream primer, downstream primer and cDNA template in a ratio of 5:2:1:1:1 respectively, mix well, transfer to an eight-strip enzyme gel strip, briefly centrifuge to make the liquid fall to the bottom, and seal.

[0058] (14). Use Light Cycler 480 II for RT-PCR reaction. The program selects an initial denaturation at 95 °C for 1 minute, and then performs 45 amplification cycles (95 °C for 10 s, 60 °C for 15 s, 72 °C for 20 s).

[0059] (15). Finally, perform relative quantification on the CT value by the 2^-ΔΔCt calculation method, and the results are shown in Figure 2 .

[0060] It can be seen from Figure 2 that compared with normal cartilage tissue, markers and others are normally expressed in the cartilage tissue derived from skin cells prepared by the present invention.

[0061] Alcian blue staining of sections: (1). Discard the medium in the centrifuge tube, and rinse the cartilage tissue with PBS, mainly being careful not to use too much force to cause the chondrocytes on the surface of the cartilage tissue to be lost.

[0062] (2). Add tissue fixative (4% paraformaldehyde solution) for paraffin embedding and sectioning with a microtome.

[0063] (3). Use xylene to dewax.

[0064] (4). Add the sections to gradient ethanol in sequence, and finally hydrate the sections with distilled water.

[0065] (5). Immerse the sections in the acidified working solution (acidifying solution: distilled water = 1:2) for 5 min.

[0066] (6) After removing the sections, re-add Alcian staining solution and stain for 30 min.

[0067] (7). After rinsing the sections, re-put them into nuclear fast red staining solution and stain for 5 min.

[0068] (8). After rinsing, dehydrate, clear and mount the sections, and the results are shown in Figure 3 , proving the existence of cartilage tissue.

[0069] Immunohistochemistry of sections: (1). Discard the culture medium in the centrifuge tube, and rinse the cartilage tissue with PBS. Be careful not to apply excessive force to prevent the loss of chondrocytes on the surface of the cartilage tissue.

[0070] (2). Add tissue fixative (4% paraformaldehyde solution), perform paraffin embedding, and section with a microtome.

[0071] (3). Place the sections in an oven at 65 °C and bake for 2 hours to melt the paraffin oil.

[0072] (4). After the paraffin oil melts, wash with PBS, and then transfer to xylene and gradient ethanol.

[0073] (5). Repair the antigens of the sections with boiling citrate buffer solution.

[0074] (6). Cool to room temperature, rinse with PBS, and then place in the diluted primary antibody at 4 °C overnight.

[0075] (7). Remove the primary antibody, wash with PBS, add the secondary antibody in the dark, and incubate at room temperature for 30 min.

[0076] The immunohistochemical results of COL2 in paraffin sections are shown in Figure 4 , its expression is normal, and it is cartilage tissue; type X collagen (COL10) is a marker of cartilage fibrosis, and COL10 is not expressed in hyaline cartilage. The immunohistochemical results of COL10 after paraffin section show that its expression is negative, as shown in Figure 5 , proving that it is hyaline cartilage and has not undergone fibrosis.

[0077] The above results show that the present invention has successfully achieved the induction of 3D cartilage.

[0078] Use the CLFC cartilage tissue and hydrogel to conduct in-vivo cartilage repair experiments on rats. The specific steps are as follows: (1) Anesthetize the rats with isoflurane and shave the hair around the knee joint.

[0079] (2) Use surgical scissors, forceps and other instruments to cut open the skin on the inner side of the rat knee joint.

[0080] (3) Peel back the ligament (without damaging the ligament), and use a 2-mm drill bit to grind and damage the cartilage between the femoral condyles distally.

[0081] (4) Add the CLFC cartilage tissue.

[0082] (5) Add GelMA pretreated at 37 °C, and use a blue light flashlight to fix the CLFC cartilage tissue with GelMA.

[0083] (6) Suture the wound surgically.

[0084] (7) Penicillin injection was used after the operation to prevent infection.

[0085] (8) After 12 weeks, an overdose of sodium pentobarbital was injected to sacrifice the rats. The cartilage repair was observed.

[0086] On the basis of the above experimental groups, the sham operation group did not add CLFC cartilage and only had hydrogel.

[0087] The results showed that compared with the sham operation, the cartilage particles provided by the present invention could well repair the cartilage tissue ( Figure 6 ), while the sham operation group could not repair the cartilage ( Figure 7 ).

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing 3D cartilage tissue, characterized in that, It includes the following steps: Digest skin fibroblasts to obtain cell pellets; Transfect the cell pellets with plasmids containing or capable of activating OCT4, SOX2, and KIF4 to obtain an electrotransfected cell suspension; Culture the electrotransfected cell suspension with Medium B to obtain high-density cells; Isolate the high nuclear-cytoplasmic ratio cells from the high-density cells, culture and passage them to obtain passage cells; Culture the passage cells with Medium C, and then transfer them to a 3D culture container and add Medium D for culture; Replace it with Medium E and continue 3D culture; Replace it with Medium D and culture to obtain 3D cartilage tissue; The Medium B is used to regulate cell gene expression and signal pathways, facilitate cell reprogramming to obtain the potential to differentiate into chondrocytes, and contains TGF-β3, FGF2, Chir99021, PD0325901, A-83-01, pH regulator, LDN-193189, FSK, VPA, BMP4, DZNeP, EPZ5676, RepSox, AM580, and Brdu; The Medium C is used to guide the reprogrammed cells to differentiate in the direction of chondrocytes and gradually endow them with chondrocyte characteristics, and contains FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A, and GluMax; The Medium D is used to promote the differentiation of cells into cartilage tissue in a 3D environment and form cartilage tissue; The Medium E is used to support the maturation and maintenance of cartilage tissue.

2. The preparation method according to claim 1, characterized in that, The 3D culture container is a CERO culture tube.

3. The preparation method according to claim 2, characterized in that, The number of days of culture is 1 - 30 d.

4. The preparation method according to claim 2, wherein The Medium B contains Insulin, ascorbic acid diphosphate, transferrin, selenium salt, TGF-β3, FGF2, Chir99021, PD0325901, A-83-01, pH regulator, LDN-193189, FSK, VPA, BMP4, DZNeP, EPZ5676, RepSox, AM580, and Brdu; The final concentration of the Insulin is 18 - 22 μg / mL; The final concentration of the ascorbic acid diphosphate is 180 - 220 μg / mL; The final concentration of the transferrin is 18 - 22 μg / mL; The final concentration of the selenium salt is 18 - 22 ng / mL; The final concentration of the TGF-β3 is 1.8 - 2.2 ng / mL; The final concentration of the FGF2 is 36 - 44 ng / mL; The final concentration of the Chir99021 is 45 - 55 nM; The final concentration of the PD0325901 is 9 - 11 nM; The final concentration of the A-83-01 is 4.5 - 5.5 nM; The final concentration of the LDN-193189 is 4.5 - 5.5 nM; The final concentration of the FSK is 9 - 11 μM; The final concentration of the VPA is 0.09 - 0.11 mM; The final concentration of the BMP4 is 9 - 11 ng / mL; The final concentration of the DZNeP is 0.045 - 0.055 μM; The final concentration of EPZ5676 is 4.5 - 5.5 μM; The final concentration of RepSox is 4.5 - 5.5 μM; The final concentration of AM580 is 0.045 - 0.055 μM; The final concentration of Brdu is 9 - 11 μM.

5. The preparation method according to claim 4, characterized in that, The medium B is based on DMEM / F12 as the basal medium.

6. The preparation method according to claim 1, characterized in that, The medium C is based on DMEMKO as the basal medium, containing FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A and GluMax; The final volume percentage concentration of FBS is 0.83 - 1.67%; The final volume percentage concentration of KSR is 1.67 - 3.33%; The final concentration of BMP4 is 10 - 20 ng / mL; The final concentration of Chir99021 is 10 - 30 nM; The final concentration of FGF2 is 5 - 20 ng / mL; The final concentration of Wnt3A is 5 - 20 ng / mL; The final volume percentage concentration of GluMax is 0.5 - 2%.

7. The preparation method according to claim 1, characterized in that, The medium D is based on DMEM as the basal medium, containing KSR, GluMax, TGFβ-3, BMP-4, dexamethasone, ascorbic acid, proline and iTS-plus; The final volume percentage concentration of KSR is 3 - 5%; The final volume percentage concentration of GluMax is 0.5 - 1.5%; The final concentration of TGFβ-3 is 5 - 10 ng / mL; The final concentration of BMP-4 is 10 - 20 ng / mL; The final concentration of dexamethasone is 80 - 120 nM; The final concentration of ascorbic acid is 30 - 80 μg / mL; The final concentration of proline is 20 - 60 μg / mL; The final volume percentage concentration of iTS-plus is 0.8 - 1.2%; And / or, the medium E is based on DMEM as the basal medium, containing KSR, GluMax, NEAA, pyruvate, dexamethasone, GDF5, FGF2, iTS-plus, ascorbic acid and proline; The final volume percentage concentration of KSR is 3 - 5%; The final volume percentage concentration of GluMax is 0.5 - 1.5%; The final volume percentage concentration of NEAA is 0.5 - 1.5%; The final volume percentage concentration of pyruvate is 0.5 - 1.5%; The final concentration of dexamethasone is 80 - 120 nM; The final concentration of GDF5 is 20 - 30 ng / mL; The final concentration of FGF2 is 1 - 5 ng / mL; The final volume percentage concentration of iTS-plus is 0.5 - 1.5%; The final concentration of ascorbic acid is 30 - 80 μg / mL; The final concentration of proline is 20 - 60 μg / mL.

8. The preparation method according to claim 1, wherein The skin fibroblasts are obtained by a preparation method including the following steps: Digest and culture the skin tissue to obtain a culture solution containing skin fibroblasts; Add the proliferation medium A and culture to obtain skin fibroblasts; The proliferation medium A uses DMEM as the basal medium and contains penicillin-streptomycin, FBS, GluMax, and NEAA; The final volume percentage concentration of the penicillin-streptomycin is 0.8 - 1.2%; The final volume percentage concentration of the FBS is 3 - 8%; The final volume percentage concentration of the GluMax is 0.3 - 0.8%; The final volume percentage concentration of the NEAA is 0.3 - 0.8%.

9. A 3D cartilage tissue prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the 3D cartilage tissue according to claim 9 in the preparation of a clinical cartilage repair material, drug research and development and screening, or cartilage biological research.

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