A 3D cartilage tissue and its preparation method and application

Abstract

The present invention belongs to the field of tissue engineering and regenerative medicine, and specifically relates to a 3D cartilage tissue, a preparation method thereof, and an application thereof. In response to the problems in the prior art such as the limited source of mesenchymal stem cells and the easy fibrosis of induced cartilage, the present invention reprograms skin fibroblasts into cartilage precursor cells through transcription factor transfection combined with multi-stage small molecule regulation, and utilizes a dynamic 3D culture system to optimize the formation and functional maintenance of cartilage particles. Experiments have confirmed that the expression of cartilage tissue marker genes induced by this method is normal, COL10 is negative, Alcian blue staining and immunohistochemistry verify its hyaline cartilage characteristics, and in vivo repair experiments show significant therapeutic effects. The raw materials of this solution are easily available and the cost is controllable, providing an efficient and stable technical platform 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 in particular to a 3D cartilage tissue and a preparation method and application thereof. Background Art

[0002] Cartilage is a translucent, elastic tissue that forms the skeleton of vertebrate embryos and very small larvae. In higher vertebrates, most of this tissue is converted to bone, but in primitive species (such as sturgeons and elasmobranchs), it remains throughout life and is the primary component of the skeleton. Cartilage is composed of chondrocytes, fibers, and a matrix. The matrix is ​​70% water, and its organic components are primarily various proteins, such as cartilaginous mucin, collagen, and chondrosclerosis.

[0003] Cartilage tissue engineering is a crucial area of ​​regenerative medicine, but current mainstream chondrogenic methods rely heavily on mesenchymal stem cells (MSCs), such as bone marrow mesenchymal stem cells (BMSCs), umbilical cord mesenchymal stem cells (UMSCs), or adipose-derived mesenchymal stem cells (ADSCs). These cell sources present significant limitations: BMSCs require invasive bone marrow aspiration, UMSCs are limited to neonatal umbilical cord samples, and ADSCs require liposuction. All of these methods present ethical challenges, high donor dependency, and limited clinical accessibility. Furthermore, existing chondrogenic techniques generally rely on high concentrations of TGF-β and BMP family factors, making the induced cartilage tissue susceptible to fibrosis (as evidenced by high expression of type X collagen) and unable to maintain the physiological functions of hyaline cartilage (such as joint cushioning and lubrication).

[0004] Although serum-free culture medium and 3D culture technology have partially improved the culture environment, limited cell sources and fibrosis problems are still the core bottlenecks restricting the clinical transformation of cartilage repair.

[0005] Therefore, there is an urgent need for an alternative solution that is widely available, cost-effective, and can stably produce 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 provides an efficient and stable solution for cartilage defect repair and organoid research.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing 3D cartilage tissue, comprising the following steps:

[0009] The skin fibroblasts were digested to obtain cell pellets;

[0010] transfecting the cell pellet with a plasmid containing or capable of activating OCT4, SOX2, and KIF4 to obtain an electroporated cell suspension;

[0011] The electroporated cell suspension was cultured using medium B to obtain high-density cells;

[0012] separating cells with a high nuclear-to-cytoplasmic ratio from the high-density cells, culturing and passage-generating them to obtain passage cells;

[0013] The passaged cells were cultured using culture medium C, and then transferred into a 3D culture container and cultured in culture medium D;

[0014] Replace with medium E and continue 3D culture;

[0015] Replace with culture medium D to obtain 3D cartilage tissue;

[0016] The culture medium B is used to regulate cell gene expression and signaling pathways, thereby assisting cell reprogramming to acquire the potential for chondrocyte differentiation, and contains TGF-β3, FGF2, Chir99021, PD0325901, A-83-01, a pH regulator, LDN-193189, FSK, VPA, BMP4, DZNeP, EPZ5676, RepSox, AM580, and Brdu;

[0017] The culture medium C is used to guide the reprogrammed cells to differentiate into chondrocytes, so that they gradually acquire chondrocyte characteristics, and contains FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A, and GluMax;

[0018] The culture medium D is used to promote cell differentiation into cartilage tissue and form cartilage tissue in a 3D environment;

[0019] The medium E is used to support the maturation and maintenance of cartilage tissue.

[0020] Preferably, the 3D culture container is a CERO culture tube.

[0021] Preferably, the culture period is 1 to 30 days.

[0022] Preferably, the culture 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;

[0023] The final concentration of the insulin is 18-22 μg / mL;

[0024] The final concentration of ascorbyl diphosphate is 180-220 μg / mL;

[0025] The final concentration of transferrin is 18-22 μg / mL;

[0026] The final concentration of the selenium salt is 18-22 ng / mL;

[0027] The final concentration of TGF-β3 is 1.8-2.2 ng / mL;

[0028] The final concentration of FGF2 is 36-44 ng / mL;

[0029] The final concentration of Chir99021 is 45-55 nM;

[0030] The final concentration of PD0325901 is 9-11 nM;

[0031] The final concentration of A-83-01 is 4.5~5.5nM;

[0032] The final concentration of LDN-193189 is 4.5-5.5 nM;

[0033] The final concentration of FSK is 9-11 μM;

[0034] The final concentration of VPA is 0.09-0.11 mM;

[0035] The final concentration of BMP4 is 9-11 ng / mL;

[0036] The final concentration of DZNeP is 0.045-0.055 μM;

[0037] The final concentration of EPZ5676 is 4.5-5.5 μM;

[0038] The final concentration of RepSox is 4.5-5.5 μM;

[0039] The final concentration of AM580 is 0.045-0.055 μM;

[0040] The final concentration of BrdU is 9-11 μM.

[0041] Preferably, the culture medium B uses DMEM / F12 as the basal medium.

[0042] Preferably, the culture medium C is based on DMEMKO and contains FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A and GluMax;

[0043] The final volume percentage concentration of the FBS is 0.83-1.67%;

[0044] The final volume percentage concentration of the KSR is 1.67-3.33%;

[0045] The final concentration of BMP4 is 10-20 ng / mL;

[0046] The final concentration of Chir99021 is 10-30 nM;

[0047] The final concentration of FGF2 is 5-20 ng / mL;

[0048] The final concentration of Wnt3A is 5-20 ng / mL;

[0049] The final volume percentage concentration of the GluMax is 0.5-2%.

[0050] Preferably, the culture medium D is based on DMEM and contains KSR, GluMax, TGFβ-3, BMP-4, dexamethasone, ascorbic acid, proline and iTS-plus;

[0051] The final volume percentage concentration of the KSR is 3-5%;

[0052] The final volume percentage concentration of the GluMax is 0.5-1.5%;

[0053] The final concentration of TGFβ-3 is 5-10 ng / mL;

[0054] The final concentration of BMP-4 is 10-20 ng / mL;

[0055] The final concentration of dexamethasone is 80-120 nM;

[0056] The final concentration of ascorbic acid is 30-80 μg / mL;

[0057] The final concentration of proline is 20-60 μg / mL;

[0058] The final volume percentage concentration of the iTS-plus is 0.8-1.2%;

[0059] and / or, the culture medium E is based on DMEM and contains KSR, GluMax, NEAA, pyruvate, dexamethasone, GDF5, FGF2, iTS-plus, ascorbic acid and proline;

[0060] The final volume percentage concentration of the KSR is 3-5%;

[0061] The final volume percentage concentration of the GluMax is 0.5-1.5%;

[0062] The final volume percentage concentration of the NEAA is 0.5-1.5%;

[0063] The final volume percentage concentration of the pyruvate is 0.5-1.5%;

[0064] The final concentration of dexamethasone is 80-120 nM;

[0065] The final concentration of GDF5 is 100 ng / mL;

[0066] The final concentration of FGF2 is 1-5 ng / mL;

[0067] The final volume percentage concentration of the iTS-plus is 0.5-1.5%;

[0068] The final concentration of ascorbic acid is 30-80 μg / mL;

[0069] The final concentration of proline is 20-60 μg / mL.

[0070] Preferably, the skin fibroblasts are obtained by a preparation method comprising the following steps:

[0071] digesting and culturing the skin tissue to obtain a culture fluid containing skin fibroblasts;

[0072] Add proliferation medium A to culture skin fibroblasts;

[0073] The proliferation medium A is based on DMEM and contains penicillin-streptomycin, FBS, GluMax and NEAA;

[0074] The final volume percentage concentration of the penicillin-streptomycin is 0.8-1.2%;

[0075] The final volume percentage concentration of the FBS is 3-8%;

[0076] The final volume percentage concentration of the GluMax is 0.3-0.8%;

[0077] The final volume percentage concentration of the NEAA is 0.3-0.8%.

[0078] The present invention also provides 3D cartilage tissue prepared by the above preparation method.

[0079] The present invention also provides the use of the above-mentioned 3D cartilage tissue in the preparation of clinical cartilage repair materials, drug development and screening, or cartilage biology research.

[0080] Beneficial effects of the present invention:

[0081] This method uses dermal fibroblasts (FCs) as raw material. Sufficient cells can be obtained through simple isolation and expansion, eliminating the need for invasive sampling and significantly reducing donor dependency and ethical risks. By optimizing the combination of small molecules, the resulting cartilage tissue is negative for COL10 expression, as confirmed by Alcian blue staining and COL2 immunohistochemistry, effectively preventing the formation of fibrocartilage. Furthermore, the method combines a dynamic rotary culture apparatus (CERO system) with a multi-stage culture medium to achieve efficient expansion and morphological maintenance of cartilage particles, shortening the culture period to 14 days. Cell density and functional indicators are highly consistent with those of native cartilage. Animal experiments have demonstrated that CLFC cartilage particles combined with GelMA hydrogel can effectively repair articular cartilage defects in rats, providing a scalable solution for traumatic cartilage injury or degenerative joint disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 This is a photo of the cartilage tissue induced by the present invention;

[0083] Figure 2 This is a comparison chart of marker expression between the skin cell-derived cartilage tissue of the present invention and normal cartilage tissue;

[0084] Figure 3 This is the result of Alcian blue staining of the induced cartilage tissue of the present invention;

[0085] Figure 4 This is the result of COL2 immunohistochemistry in paraffin sections of cartilage tissue induced by the present invention;

[0086] Figure 5 This is the result of COL10 immunohistochemistry in paraffin sections of cartilage tissue induced by the present invention;

[0087] Figure 6 This is a diagram showing the effect of the cartilage particles of the present invention on cartilage repair in rats (experimental group);

[0088] Figure 7 This is a diagram showing the effect of the cartilage particles of the present invention on cartilage repair in rats (sham-operated control group). DETAILED DESCRIPTION

[0089] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0090] Sources of reagents and materials in the embodiments of the present invention:

[0091] Insulin (Company: MedChemExpress, Product Number: HY-P73243)

[0092] Ascorbyl diphosphate (Company: Sigma-Aldrich CAS No.: 113170-55-1)

[0093] Transferrin (Sigma-Aldrich, Catalog No. T8158)

[0094] Selenium salt (Sigma-Aldrich, Catalog No. S5261)

[0095] TGF-β3 (Company: STEMCELL Technologies, Catalog No.: 78156)

[0096] FGF2 (Company: Thermo Fisher Scientific, Catalog Number: 100-18BHS-50UG)

[0097] Chir99021 (Company: Selleck Item No.: S1263)

[0098] PD0325901 (Company: Selleck, Product No.: S1036)

[0099] A-83-01 (Company: Selleck Part No.: S7692)

[0100] LDN-193189 (Company: Selleck, Part No.: S2618)

[0101] FSK (Company: MedChemExpress, Product No.: HY-15371)

[0102] VPA (Company: Selleck, Item No.: S3944)

[0103] BMP4 (Company: R&D System, Catalog Number: 314E-GMP)

[0104] DZNeP (Company: MedChemExpress, Product No.: HY-10442)

[0105] EPZ5676 (Company: Selleck, Product No.: S7062)

[0106] RepSox (Company: Selleck, Product No.: S7223)

[0107] AM580 (Company: Selleck Part No.: S2933)

[0108] Brdu (Company: Selleck, Item No.: S7918)

[0109] FBS (Company: Thermo Fisher Scientific, Catalog Number: 10099141C)

[0110] KSR (Company: Thermo Fisher Scientific Catalog No.: 10828028)

[0111] Wnt3A (Company: R&D System, Catalog Number: 5036-GMP)

[0112] GluMax (Company: Thermo Fisher Scientific, Catalog Number: 35050061)

[0113] Dexamethasone (Company: Selleck, Product No.: S1322)

[0114] Ascorbic acid (Company: SparkJade, Product No.: SJ-MN0658)

[0115] Proline (Sigma-Aldrich, Catalog Number: 1074340100)

[0116] iTS-plus (Company: Sigma-Aldrich, Catalog Number: I2521-5ML)

[0117] NEAA (Company: Thermo Fisher Scientific, Catalog Number: 11140050)

[0118] Pyruvate (Company: Thermo Fisher Scientific Catalog No.: 11360070)

[0119] GDF5 (Company: R&D System, Catalog Number: 8340-G5-050)

[0120] Penicillin-streptomycin (Company: Thermo Fisher Scientific Catalog No.: 15070063)

[0121] Plasmids were purchased from Addgene

[0122] Addgene: pCXLE-hOCT3 / 4-shp53-F

[0123] Addgene: pCXLE-hSK

[0124] Two plasmids, pCXLE-hOCT3 / 4-shp53-F activates the OCT4 transcription factor, and pCXLE-hSK provides SOX2 and KLF4.

[0125] Example

[0126] 1. Obtaining Skin Fibroblasts

[0127] 1. Pre-preparation: One hour before sampling, add 0.1% gelatin to each well of a 6-well plate in a clean bench. Place the plate in a cell culture incubator for 30 minutes. Preheat the culture medium to 37°C in a 15ml centrifuge tube before sampling at the hospital. Record the donor's age and the sampling site.

[0128] 2. Rinse and Separate: Add a sterile PBS solution containing double-antibody to a culture dish. Wipe the 15ml centrifuge tube containing the sample with 75% alcohol, place the sample in the culture dish, and rinse the skin tissue at least three times using autoclaved forceps. 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, leaving the dermis. Place the sample with the epidermis facing up, with the dermis adherent to the wall.

[0129] 3. Prepare a culture dish, place the sample skin piece in a new culture dish, lift the epidermis with tweezers and remove it, and use scissors and a scalpel to cut the remaining dermis into small pieces.

[0130] 4. Transfer to a 50ml centrifuge tube, add type 1 collagenase, and digest at 37°C with shaking. Harvest fibroblasts in stages (at intervals of 2 hours and 4 hours respectively).

[0131] 5. After each stage, operate in a clean bench, filter with a cell sieve to remove undigested tissue, and collect the cell suspension.

[0132] 6. Centrifuge at 200g for 8 minutes, discard the supernatant, resuspend the cells in culture medium, and inoculate the culture plate.

[0133] 7. Check for contamination the next day. Change the medium or subculture the cells depending on the cell growth.

[0134] 8. Discard the supernatant, retaining less than 1 mL of liquid, transfer the liquid to a gelatin-coated cell culture plate, and add 5 mL of medium A.

[0135] Medium A formulation:

[0136] DMEM 465 mL

[0137] FBS (South American) 25 mL

[0138] GluMax 2.5 mL

[0139] NEAA 2.5 mL

[0140] Penicillin-streptomycin 5 mL.

[0141] 8. Observe cell attachment within 5-14 days. When the cell proliferation reaches 2×10 7 , go to the next step.

[0142] 2. Transfection of Skin Fibroblasts

[0143] 1. Digest the proliferating fibroblasts with trypsin. After terminating the digestion, centrifuge at 200g for 5 minutes and collect the precipitate.

[0144] 2. Use the Lonza electroporation kit (VPI-1005) to transfect the plasmids containing OCT4, SOX2, and KIF4 according to the instructions. Select the electroporation program C-017 or X-001.

[0145] 3. Transfer the electroporated cell suspension to a pre-coated MG-coated cell culture plate. Place in an incubator at 37°C and 5% CO2. Culture for 5-7 days before proceeding to the next step.

[0146] 4. Use medium B to change the cell culture medium every day. After 21 days of culture, high-density cells can be observed.

[0147] Medium B formulation:

[0148] DMEM / F12 500 mL

[0149] Iusulin 20 μg / mL

[0150] Ascorbyl diphosphate 200 μg / mL

[0151] Transferrin 20 μg / mL

[0152] Selenium salt 20 ng / mL

[0153] TGF-β3 2 ng / mL

[0154] FGF2 40 ng / mL

[0155] Chir99021 50 nM

[0156] PD0325901 10 nM

[0157] A-83-01 5 nM

[0158] Bicarbonate 2438 μg / mL

[0159] LDN-193189 5 nM

[0160] FSK 10 μM

[0161] VPA 0.1 mM

[0162] BMP4 10 ng / mL

[0163] DZNeP 0.05 μM

[0164] EPZ5676 5 μM

[0165] RepSox 5 μM

[0166] AM580 0.05 μM

[0167] Brdu 10 μM.

[0168] 5. Use physical passaging to separate cells with a high nuclear-to-cytoplasmic ratio and culture them continuously in mTeSR 1 medium (Stemcell Technologies). When the cell density reaches 70%-90%, the cells are passaged.

[0169] 3. Cell Induction

[0170] 1. After single-cell passage of the 10th generation cells, culture the cells in Medium C the next day. After about 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 by the density before passage and the cell growth rate, and the actual situation).

[0171] Medium C formulation:

[0172] DMEM KO 480 mL

[0173] FBS (South American) 5 mL

[0174] KSR 10 mL

[0175] BMP4 10 ng / mL

[0176] Chir99021 20 nM

[0177] FGF2 10 ng / mL

[0178] Wnt3A 10 ng / mL

[0179] GluMax 5 mL.

[0180] 2. Transfer the cells to a 3D culture centrifuge tube and culture in Medium D.

[0181] Medium D formulation:

[0182] DMEM 470 mL

[0183] KSR 20 mL

[0184] GluMax 5 mL

[0185] TGFβ-3 10 ng / mL

[0186] BMP-4 10 ng / mL

[0187] Dexamethasone 100 nM

[0188] Ascorbic acid 50 μg / mL

[0189] Proline 40 μg / mL

[0190] iTS-plus 5 mL

[0191] 3. Place the CERO culture tube into the CERO fully automated 3D cell culture apparatus. Set the program to dynamic mode, with a rotation speed of 100 rpm, a time of 5 seconds, and a pause of 2 seconds. Set the duration to forever.

[0192] 4. Replace the culture medium every 3 days. After 6-9 days, remove the centrifuge tube, aspirate the culture medium and discard it.

[0193] 5. Add 20 mL of Medium E (the same volume as Medium B). Return the cells to the CERO Automated 3D Cell Culture Instrument. Set the instrument to dynamic mode, with a rotation speed of 100 rpm, a time of 1 second, and a pause of 2 seconds. Set the duration to forever.

[0194] Medium E formulation:

[0195] DMEM 460 mL

[0196] KSR 20 mL

[0197] GluMax 5 mL

[0198] NEAA 5 mL

[0199] 5 mL of pyruvate

[0200] Dexamethasone 100 nM

[0201] GDF5 100 ng / mL

[0202] FGF2 5 ng / mL

[0203] iTS-plus 5 mL

[0204] Ascorbic acid 50 μg / mL

[0205] Proline 40 μg / mL

[0206] 6. Remove the CERO culture tube, aspirate half of the original culture medium, and discard. Refill the tube with Culture Medium D to the original volume. Repeat this process every two days for 7-14 days to obtain cartilage particles that are positive for Alcian blue staining and COL2.

[0207] Figure 1 This is a photo of the cartilage induced by the present invention.

[0208] Detection method of cartilage markers:

[0209] (1) Transfer the cartilage particles from the CERO culture tube to a 15 mL centrifuge tube and centrifuge at 100g for 5 minutes. Discard the supernatant and add 1 mL of PBS to each tube to wash the surface of the pellet. Repeat three times.

[0210] (2) Remove the cartilage particles from PBS, transfer them into liquid nitrogen, and quickly freeze the cartilage tissue.

[0211] (3) Take 10-12 cartilage tissues and put them into a mortar and crush them as evenly as possible.

[0212] (4) Add 1 mL of Trizol reagent to the mortar and pestle to completely cover the cartilage tissue fragments. Grind again, let it stand for 5 minutes to fully lyse, blow and transfer to a 1.5 mL enzyme-free EP tube.

[0213] (5) Add 200 μL of chloroform to each mL of Trizol, vortex for 30 seconds to mix thoroughly, and let stand for 5 minutes. Centrifuge at 4°C, 12,000 g, for 15 minutes.

[0214] (6) After centrifugation, the Eppendorf tube is separated into three phases. Carefully aspirate the upper transparent liquid phase. The volume of the upper liquid phase is approximately 60% of the volume of Trizol.

[0215] (7) Add 50% volume of isopropanol to Trizol and shake vigorously to mix. Isopropanol can precipitate RNA. After mixing, let it stand for 10 minutes and centrifuge at 4℃, 12,000g, for 10 minutes.

[0216] (8) Discard the supernatant and avoid aspirating the white precipitate visible at the bottom of the EP tube. Sometimes it may be a colorless precipitate. Add 1 mL of 75% ethanol and mix thoroughly. Wash the RNA precipitate and centrifuge at 4°C, 7500 g, for 10 min.

[0217] (9) Discard the supernatant, invert and dry for 10 min, add 100 μL RNase-Free water to dissolve the RNA.

[0218] (10) Use NanoDrop to detect RNA concentration.

[0219] (11) The obtained RNA was placed on ice after being heated in a constant temperature water bath at 65°C for 5 min.

[0220] (12) Prepare the reverse transcription system according to the instructions of the reverse transcription kit. Use a PCR instrument to perform the reaction at 37°C for 15 min; inactivate the enzyme at 98°C for 5 min; and store at 4°C for later use.

[0221] (13) Add SYBGreen, water, upstream primer, downstream primer and cDNA template in a ratio of 5:2:1:1:1, mix thoroughly, transfer to eight consecutive enzyme strips, centrifuge briefly to let the liquid fall to the bottom, and seal.

[0222] (14) RT-PCR reaction was performed using Light Cycler 480 II. The program selected was an initial denaturation at 95°C for 1 minute, followed by 45 amplification cycles (95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 20 seconds).

[0223] (15). Finally, the CT value was relatively quantified by the 2^-ΔΔCt calculation method. The results are shown in Figure 2 .

[0224] Depend on Figure 2 It can be seen that compared with normal cartilage tissue, the markers and the like in the skin cell-derived cartilage tissue prepared in the present invention are normally expressed.

[0225] Alcian blue staining of sections:

[0226] (1) Discard the culture medium in the centrifuge tube and rinse the cartilage tissue with PBS. Be careful not to use too much force to cause the chondrocytes on the surface of the cartilage tissue to be lost.

[0227] (2) Add tissue fixative (4% paraformaldehyde solution) for paraffin embedding and slice using a microtome.

[0228] (3) Dewax using xylene.

[0229] (4) Add gradient ethanol to the slices in sequence, and finally use distilled water to hydrate the slices.

[0230] (5) Soak the sections in acidified working solution (acidified solution: distilled water = 1:2) for 5 minutes.

[0231] (6) After removing the slices, re-add Alcian staining solution and stain for 30 minutes.

[0232] (7) After washing the sections, re-place them in the Nuclear Fast Red staining solution and stain for 5 minutes.

[0233] (8) After washing, dehydration, transparency, and sealing, the results are shown in Figure 3 , proving the existence of cartilage tissue.

[0234] Immunohistochemistry of sections:

[0235] (1) Discard the culture medium in the centrifuge tube and rinse the cartilage tissue with PBS. Be careful not to use too much force to cause the chondrocytes on the surface of the cartilage tissue to be lost.

[0236] (2) Add tissue fixative (4% paraformaldehyde solution), embed in paraffin, and slice using a microtome.

[0237] (3) Place the slices in a 65℃ oven and bake for 2 hours to melt the paraffin oil.

[0238] (4) After the paraffin oil melts, wash with PBS, and then transfer to xylene and gradient alcohol.

[0239] (5) Perform antigen retrieval on the sections using boiling citric acid buffer.

[0240] (6) Cool to room temperature, rinse with PBS, and place in diluted primary antibody at 4°C overnight.

[0241] (7) Remove the primary antibody, wash with PBS, add the secondary antibody and incubate at room temperature for 30 min in the dark.

[0242] The results of COL2 immunohistochemistry in paraffin sections are shown in Figure 4 , its expression is normal, it is cartilage tissue; type X collagen (COL10) is a marker of cartilage fibrosis, COL10 is not expressed in hyaline cartilage, and the COL10 immunohistochemistry results after paraffin section show that its expression is negative, see Figure 5 , proving that it is hyaline cartilage without fibrosis.

[0243] The above results show that the present invention successfully achieved the induction of 3D cartilage.

[0244] In vivo cartilage repair experiments in rats were conducted using the CLFC cartilage tissue and hydrogel:

[0245] Specific steps:

[0246] (1) Rats were anesthetized with isoflurane and the hair around the knee joint was shaved.

[0247] (2) Use surgical scissors, surgical forceps and other instruments to cut the skin on the inside of the rat knee joint.

[0248] (3) Peel off the ligament (without destroying it) and use a 2mm drill to grind and destroy the cartilage between the condyles of the distal femur.

[0249] (4) Adding CLFC cartilage tissue

[0250] (5) Add GelMA pretreated at 37°C and use a blue light flashlight to fix the CLFC cartilage tissue with GelMA.

[0251] (6) Surgical suture of wounds

[0252] (7) Penicillin injection is used after surgery to prevent infection.

[0253] (8) After 12 weeks, the rats were killed by an overdose of sodium pentobarbital injection and the cartilage repair was observed.

[0254] On the basis of the above experimental groups, the sham operation group did not add CLFC cartilage but only hydrogel.

[0255] The results showed that compared with sham surgery, the cartilage particles provided by the present invention can repair cartilage tissue well ( Figure 6 ), while the sham-operated group was unable to repair cartilage ( Figure 7 ).

[0256] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing 3D cartilage tissue, characterized in that: The following steps are involved: The skin fibroblasts were digested to obtain cell pellets; transfecting the cell pellet with a plasmid containing or capable of activating OCT4, SOX2, and KIF4 to obtain an electroporated cell suspension; The electroporated cell suspension was cultured using medium B to obtain high-density cells; separating cells with a high nuclear-to-cytoplasmic ratio from the high-density cells, culturing and passage-generating them to obtain passage cells; The passaged cells were cultured using culture medium C, and then transferred into a 3D culture container and cultured in culture medium D; Replace with medium E and continue 3D culture; Supplement medium D to obtain 3D cartilage tissue; The culture medium B is used to regulate cell gene expression and signaling pathways, thereby assisting cell reprogramming to obtain the potential for chondrocyte differentiation; The culture medium C is used to guide the reprogrammed cells to differentiate into chondrocytes, so that they gradually acquire chondrocyte characteristics; The culture medium D is used to promote cell differentiation into cartilage tissue and form cartilage tissue in a 3D environment; The culture medium E is used to support the maturation and maintenance of cartilage tissue; The culture medium B consists of the following components: basal medium DMEM / F12, 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 ascorbyl diphosphate is 180-220 μg / mL; The final concentration of transferrin is 18-22 μg / mL; The final concentration of the selenium salt is 18-22 ng / mL; The final concentration of TGF-β3 is 1.8-2.2 ng / mL; The final concentration of 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 4.5~5.5nM; 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; The culture medium C consists of the following components: basal medium DMEMKO, FBS, KSR, BMP4, Chir99021, FGF2, Wnt3A and GluMax; The final volume percentage concentration of the FBS is 0.83-1.67%; The final volume percentage concentration of the 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 the GluMax is 0.5-2%; The culture medium D consists of the following components: basal medium DMEM, KSR, GluMax, TGFβ-3, BMP-4, dexamethasone, ascorbic acid, proline and iTS-plus; 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 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 the iTS-plus is 0.8-1.2%; The culture medium E consists of the following components: basal medium DMEM, 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 dexamethasone is 80-120 nM; The final concentration of GDF5 is 100 ng / mL; The final concentration of FGF2 is 1-5 ng / mL; The final volume percentage concentration of the 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.

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 culture period is 1 to 30 days.

4. The preparation method according to claim 1, characterized in that The skin fibroblasts are obtained by a preparation method comprising the following steps: digesting and culturing the skin tissue to obtain a culture fluid containing skin fibroblasts; Add proliferation medium A to culture skin fibroblasts; The proliferation medium A consists of the following components: basal medium DMEM, 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%.

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