A method for preparing high-regeneration fibrochondrocytes, products and applications

Abstract

The present invention discloses a method for preparing fibrochondrocytes with high regeneration and passage, a product and application thereof, and develops a maintenance culture medium formula that can stably expand a large number of cells in vitro, belonging to the field of cell biology and regenerative medicine. The method aims at the problems of limited proliferation of autologous fibrochondrocytes in current clinical applications, poor repair effect, trauma caused by sampling, and relatively poor safety. A method for efficiently deriving fibrochondrocytes with high regeneration ability from hiPSCs is invented by manipulating the transforming factor TGFβ3, Shh (Sonic hedgehog) and retinoic acid RA signaling pathways. Since the experimental operation of the method is relatively simple, the fibrochondrocytes produced can be expanded in large quantities and maintain cartilage characteristics for a long time under the conditions of the maintenance culture medium, and can be used as a potential therapy for the regeneration and repair of dense connective tissues (including tendons, ligaments, menisci and annuli) in the future.

Description

Technical Field

[0001] The invention belongs to the field of cell biology and regenerative medicine, and relates to a preparation method, product and application of high-regeneration passage fibrochondrocytes. Background Art

[0002] Fibrocartilage is a transitional form of tissue between hyaline cartilage and dense connective tissue, mainly distributed in intervertebral discs, articular cartilage discs, pubic symphysis, and tendon joint capsule ligament attachment parts. Fibrocartilage is composed of chondrocytes, fibroblasts, and cartilage matrix (proteoglycans and collagen fibers), and its main function in the body is to resist pressure and stress. However, due to the lack of blood vessels in fibrocartilage tissue, it is difficult to repair the tissue after damage.

[0003] Fibrochondrocytes have become candidate cell types for repairing knee meniscus because of their ability to form cartilage tissue and synthesize and secrete cartilage matrix components (such as collagen and chondroitin sulfate). Autologous fibrochondrocyte tissue (or cell) transplantation and surgical repair are currently the main methods for treating adult dense connective tissue (including tendons, ligaments, menisci and annulus fibrosus) injuries. A large number of clinical studies have confirmed that the healing effect of these dense connective tissue injuries is very unsatisfactory, mainly because the proliferation and regeneration ability of autologous fibrochondrocytes in vitro is weak, and ECM synthesis and secretion are low, making it difficult to achieve rapid and effective repair. In addition, autologous materials can also cause trauma and infection to the body itself, while the transplantation of allogeneic cartilage cannot effectively resist pressure and stress. Therefore, it is necessary to develop new methods to obtain a large number of fibrochondrocytes with high regeneration ability for the development of potential therapies for the regeneration and repair of these dense connective tissues. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides the following embodiments:

[0005] The present invention provides a product, which is used for preparing fibrochondrocytes with high regeneration and passage ability; the product comprises culture medium 1, culture medium 2, culture medium 3 and / or culture medium 4; wherein, the culture medium 1 is an E8 complete culture medium containing Rocki, the culture medium 2 is an E8 complete culture medium containing LDN193189, the culture medium 3 is an E8 complete culture medium containing LDN193189, CHIR-99021 and AGN193109, and the culture medium 4 is a DMEM / F12 nutrient solution containing ITS-G, PUNO1-TGF-β3, SAG, FGF-2 and AGN193109.

[0006] Furthermore, the concentration of the Rocki component in the culture medium 1 is 10 μM.

[0007] Furthermore, the concentration of the component LDN193189 in the culture medium 2 is 250 nM.

[0008] Furthermore, the concentration of the component LDN193189 in the culture medium 3 is 250 nM.

[0009] Furthermore, the concentration of the component CHIR-99021 in the culture medium 3 is 5 μM.

[0010] Furthermore, the concentration of component AGN193109 in the culture medium 3 is 100 nM.

[0011] Furthermore, the mass percentage of the component ITS-G in the culture medium 4 is 1%.

[0012] Furthermore, the concentration of the component PUNO1-TGF-β3 in the culture medium 4 is 10 ng / ml.

[0013] Furthermore, the concentration of the component SAG in the culture medium 4 is 300 nM.

[0014] Furthermore, the concentration of the component FGF-2 in the culture medium 4 is 20 ng / ml.

[0015] Furthermore, the concentration of component AGN193109 in the culture medium 4 is 100 nM.

[0016] The term "culture" refers to the growth of cells, organisms, multicellular entities or tissues in a culture medium. The term "culture" refers to any method for achieving such growth, and may include multiple steps. The term "further culture" refers to culturing cells, organisms, multicellular entities or tissues to a certain growth stage, and then using another culture method to make the cells, organisms, multicellular entities or tissues reach another growth stage. "Cell culture" refers to the growth of cells in vitro. In such a culture, cells proliferate, but they themselves are not organized into tissues. "Tissue culture" refers to the maintenance or growth of tissues, such as in vitro explants of organ primordia or adult organs, in order to maintain their structure and function. "Monolayer culture" refers to a culture in which cells are propagated in a suitable culture medium, and most of them are connected to each other and adhere to a substrate. In addition, "suspension culture" refers to a culture in which cells are propagated in a suitable culture medium and suspended in the suitable culture medium.

[0017] The term "culture medium" or "culture fluid" is recognized in the art and generally refers to any substance or preparation used to culture living cells. The term "culture medium" as used in reference to cell culture includes components of the environment surrounding the cells. The culture medium can be a solid, liquid, gas, or a mixture of phases and materials. Culture medium includes liquid growth medium and liquid culture medium that does not maintain cell growth. Culture medium also includes gel-like culture media, such as agar, agarose, gelatin, and collagen matrices. The term "culture medium" also refers to materials intended for cell culture, even if it has not yet been contacted with cells. In other words, a nutrient-rich liquid prepared for bacterial culture is a culture medium.

[0018] In certain specific embodiments, the product for preparing fibrochondrocytes with high regeneration ability comprises culture medium 1, whose components are 10 μM Rocki's E8 complete medium. In certain specific embodiments, the product for preparing fibrochondrocytes with high regeneration ability comprises culture medium 2, whose components are 250nM LDN193189's E8 complete medium. In certain specific embodiments, the product for preparing fibrochondrocytes with high regeneration ability comprises culture medium 3, whose components are 250nM LDN193189, 5μM CHIR-99021, 100nMAGN193109's E8 complete medium. In certain specific embodiments, the product for preparing fibrochondrocytes with high regeneration ability comprises culture medium 4, whose components are 1% ITS-G, 10ng / ml PUNO1-TGF-β3, 300nM SAG, 20ng / ml FGF-2, 100nMAGN193109's DMEM / F12 nutrient solution.

[0019] The present invention provides a method for differentiating fibrochondrocytes with high regeneration and passage ability, the method comprising the following steps:

[0020] 1) iPS cells were digested into single cells, centrifuged and resuspended in medium 1 as described above, and inoculated into low-adhesion culture dishes overnight to form EB spheres;

[0021] 2) replacing the aforementioned medium 2 and inducing the culture for 2 days;

[0022] 3) Inoculate in Matrigel-coated plates and replace the aforementioned medium 3 for 4 days;

[0023] 4) Replace the culture medium 4 described above and culture for 6 days to complete the differentiation.

[0024] Furthermore, the iPS cells in step 1) are hiPSCs.

[0025] Furthermore, the reagent used for iPS cell digestion in step 1) of the differentiation method is Tryp.LE.

[0026] Furthermore, the concentration of the single cells digested in step 1) is 1.5×10 6 cells / mL.

[0027] Furthermore, the culture dish inoculated in step 1) is a low-adsorption well plate.

[0028] Furthermore, the matrix gel-coated plate inoculated in step 3) is a Matrigel-coated 12-well plate.

[0029] As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent that keeps the fibrochondrocytes used in the present invention alive. For a fibrochondrocyte population, the term "substantially pure" refers to a cell population in which at least about 75%, at least about 85%, at least about 90%, or at least about 95% of the cells are MSCs. In other words, for an MSC population, the term "substantially pure" refers to a cell population that contains less than about 20%, less than about 10%, or less than about 5% lineage-committed cells by number.

[0030] In certain specific embodiments, the method for differentiating fibrochondrocytes with high regeneration ability comprises the following steps: 1) iPS cells are digested with Tryp.LE into single cells (1.5×10 6 cells / mL, centrifuged and resuspended in the aforementioned culture medium 1, inoculated in a low-adsorption 6-well plate and cultured overnight to form EB spheres; 2) replaced the aforementioned culture medium 2 for 2 days of induction culture; 3) EB spheres were inoculated in a Matrigel-coated 12-well plate and replaced with the aforementioned culture medium 3 for 4 days; 4) replaced with the aforementioned culture medium 4, cultured for 5 days, differentiation was completed, and fibrochondrocytes with high regeneration and passage ability were obtained.

[0031] The terms "comprising" and "including" are used in an inclusive, open sense, meaning that additional elements may be included.

[0032] The present invention provides a fibrochondrocyte, which is prepared by the above-mentioned differentiation method.

[0033] The invention provides a 10% TY expansion culture medium, which can expand a large number of fibrochondrocytes and maintain the characteristics of fibrochondrocytes; the 10% TY expansion culture medium comprises a DMEM / F12 culture medium whose components are ITS-G, FBS, PUNO1-TGFβ3, SAG, FGF-2, PDGF-BB, Dexamethasone, GlutaMAX, NEAA and Penicilin-Streptomycin.

[0034] Furthermore, the mass percentage of the ITS-G is 1%.

[0035] Furthermore, the mass percentage of the FBS is 10%.

[0036] Furthermore, the concentration of PUNO1-TGFβ3 is 10 ng / ml.

[0037] Furthermore, the concentration of SAG is 100 nM.

[0038] Furthermore, the concentration of FGF-2 is 20 ng / ml.

[0039] Furthermore, the concentration of the PDGF-BB is 2 ng / ml.

[0040] Furthermore, the concentration of Dexamethasone is 100 nM.

[0041] Furthermore, the mass percentage of the GlutaMAX is 1%.

[0042] Furthermore, the mass percentage of the NEAA is 1%.

[0043] Furthermore, the mass percentage of the Penicilin-Streptomycin is 1%.

[0044] In certain specific embodiments, the 10% TY expansion medium for culturing and expanding fibrochondrocytes and maintaining the characteristics of fibrochondrocytes is based on DMEM / F12 medium, and the following components are added thereto: 1% ITS-G, 10% FBS, 10ng / ml PUNO1-TGFβ3, 100nM SAG, 20ng / ml FGF-2, 2ng / ml PDGF-BB, 100nM Dexamethasone, 1% GlutaMAX, 1% NEAA, and 1% Penicilin-Streptomycin. The unit of the component in the medium is % means mass percentage.

[0045] The term "basal medium" refers to a medium that promotes the growth of many types of microorganisms that do not require any special nutritional supplements. Most basal media usually contain four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. Basal media are usually used as the basis of more complex culture media, and supplements such as serum, buffer, growth factors, lipids, etc. are added to the basal medium. Examples of basal media include but are not limited to Eagle's basal medium, minimal essential medium, Dulbecco's modified Eagle's medium, culture medium 199, nutrient mixture Ham's F-10 and Ham's F 12, McCoy's 5A, Dulbecco's MEM / FI 2, RPMI 1640 and Iscov's modified Dulbecco's medium (IMDM).

[0046] The present invention provides a method for high-expansion and passage of fibrochondrocytes. The method comprises the following steps: converging the fibrochondrocytes described above, washing, digesting with Trypsin-EDTA, terminating the digestion, collecting cell precipitates by centrifugation, passage in proportion, resuspending the cells in the 10% TY expansion medium described above, and inoculating the cells in a culture dish pre-coated with gelatin.

[0047] Furthermore, the confluence of the fibrochondrocytes is 90%-95%.

[0048] Furthermore, the fibrochondrocytes were washed with DPBS after confluence.

[0049] Furthermore, the mass percentage of Trypsin-EDTA used in the digestion is 0.25%.

[0050] Furthermore, the digestion time is 3 minutes.

[0051] Furthermore, the digestion environment is an incubator at 37° C. and 5% CO 2 .

[0052] Furthermore, the culture medium used for terminating the digestion is a culture medium containing FBS.

[0053] Furthermore, the ratio of proportional subculture is 1:6 or 1:12.

[0054] Furthermore, the mass percentage of the pre-coated gelatin is 0.2%.

[0055] In certain specific embodiments, the method for high expansion and high passaging of fibrochondrocytes comprises the following steps: converging fibrochondrocytes having fibrochondrocyte proliferation characteristics, and when the confluence of the fibrochondrocytes reaches 90-95%, washing with DPBS, then adding 0.25% Trypsin-EDTA and digesting for 3 minutes in a 37°C, 5% CO2 incubator, then terminating the digestion with a culture medium containing FBS, then collecting the cell precipitate by centrifugation at 1200rpm for 3 minutes, and then passaging at a ratio of 1:6 or 1:12, resuspending the cells in the 10% TY expansion medium described above, and inoculating them in a culture dish pre-coated with 0.2% gelatin.

[0056] The present invention provides an application of a combination of ITS-G, FBS, PUNO1-TGFβ3, SAG, FGF-2, PDGF-BB, Dexamethasone, GlutaMAX, NEAA, Penicilin-Streptomycin, and DMEM / F12 culture medium in preparing a product of fibrochondrocytes capable of continuously proliferating and maintaining fibrochondrocyte characteristics.

[0057] Advantages and beneficial effects of the present invention:

[0058] The present invention solves the problem of poor proliferation ability and inability to pass down differentiated fibrochondrocytes. The present invention regulates the PUNO1-TGFβ3, SHH, and retinoic acid RA signaling pathways, undergoes the transition of paraxial mesoderm PM-TSPCs tendon progenitor cells, and establishes and optimizes a method for efficiently deriving fibrochondrocytes with high regeneration ability from hiPSCs. At the same time, we have also optimized a culture medium 10% TY that can amplify and maintain the characteristics of fibrochondrocytes in vitro. In the future, the mechanical functions of these fibrochondrocytes will be further tested, and potential therapies for the regeneration and repair of dense connective tissue will be developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the Alcian blue staining of differentiated fibrochondrocytes induced by different TGF-β factors and the cell growth after passage;

[0060] Figure 2 This is the live and dead cell staining of fibrochondrocytes on day 12 of differentiation;

[0061] Figure 3 is the toluidine blue and alcian blue staining of AGN1 fibrochondrocytes;

[0062] Figure 4 This is a graph showing the statistical results of the proliferation curve and doubling time of fibrochondrocytes;

[0063] Figure 5It is a graph of gene expression levels of fibrocartilage markers detected by qPCR during passage;

[0064] Figure 6 This is a picture of fibrocartilage tissue formed by fibrochondrocytes under the skin of nude mice. DETAILED DESCRIPTION

[0065] Example 1 Method for differentiating fibrochondrocytes from hiPSCs

[0066] 1. Experimental Materials

[0067] E8 complate medium(Stem Cell, #05990); Y27632 / Rocki (abcam, ab120129); CHIR-99021 (Selleck, S1263); LDN1931 89 (Selleck, S2618); TrypLE (Gibco, 12604-021); AGN193109 (MCE, HY-U00449); PUNO1-TG F-β3 (invivoGen); TGFβ3 (peprotech, 100-36E); TGF-β1 (Cellapybio, CA32002); SAG (Selleck, S7779); Matrigel (Corning, 354277); 0.25% Trypsin-EDTA (Gibco, 2520072); Fetal Bovine Serum(Cellma,SA211.02);DMEM / F12 withhepes (Gibco, 11320-033); Penicilin-Streptomycin (HyClone, SV30010); Beta-Mercaptoethanol (1000×) (Gibco, 21985-023); Dexamethasone (MK-1 25) (Selleck, S1322); Insulin-Transferrin-Selenium (ITS-G) (100×) (Gibco, 41400045); FGF-2 (peprotech, AF-100-18B); PDGF-BB (MCE, HY-P7055); MEM NEAA (Gibco, 11140050); GlutaMax (Gibco, 35050061); Gelatin (Sigma, 48722)

[0068] 2. Experimental methods

[0069] A. Differentiation Method

[0070] 1) Digest hiPSCs cells into single cells using Tryp.LE and adjust the cell concentration to 1.5×10 6 cells / mL, centrifuged and resuspended in E8 complete medium containing 10 μM Rocki, inoculated into low-adhesion 6-well plates and cultured overnight in suspension to form EB spheres;

[0071] 2) Change to E8 complete medium containing 250 nM LDN193189 for 2 days of induction culture;

[0072] 3) EB spheres were seeded in Matrigel-coated 12-well plates and replaced with E8 complete medium containing 250 nM LDN193189, 5 μM CHIR-99021, and 100 nM AGN193109 for 4 days;

[0073] 4) The culture medium was replaced with DMEM / F12 medium containing 1% ITS-G, 10 ng / ml PUNO1-TGF-β3, 300 nM SAG, 20 ng / ml FGF-2, and 100 nM AGN193109. The cells were cultured for 6 days to terminate differentiation.

[0074] B. Amplification and Passaging Methods

[0075] The differentiated fibrochondrocytes were collected, and when the confluence of the fibrochondrocytes reached 90-95%, they were washed with DPBS, digested with 0.25% Trypsin-EDTA in a 37°C 5% CO2 incubator for 3 minutes, and digested with a medium containing FBS. The cell pellets were collected by centrifugation at 1200 rpm for 3 minutes, passaged at a ratio of 1:6 or 1:12, resuspended in 10% TY expansion medium, and inoculated in a culture dish pre-coated with 0.2% gelatin.

[0076] The components of the expansion medium 10% TY include 1% ITS-G, 10% FBS, 10 ng / ml PUNO1-TGFβ3, 100 nM SAG, 20 ng / ml FGF-2, 2 ng / ml PDGF-BB, 100 nM Dexamethasone, 1% GlutaMAX, 1% NEAA, and 1% Penicilin-Streptomycin.

[0077] Example 2 Identification of fibrochondrocytes

[0078] 1. Experimental Materials

[0079] PBS buffer (Solebol, P1003); Toluidine blue staining solution (Solebol, G2543); Alcian blue staining kit PH2.5 (Solebol, G2542); Sterile deionized water.

[0080] 2. Experimental methods

[0081] According to the method of hiPSCs differentiation into fibrochondrocytes described in Example 1, the fibrochondrocytes were passaged 4 times (P4) and the cell concentration was adjusted to 2×10 4 Cells / well were inoculated into 12-well plates pre-coated with 0.2% gelatin, and when the cell confluence reached 80-90%, the cells were cultured and washed once with DPBS, and fixed with 4% PFA at room temperature for 30 min. Toluidine blue and Alcian blue staining were performed according to the instructions provided by the reagent supplier, and fibriblasts served as the control group.

[0082] Toluidine blue staining: The fixed fibrochondrocytes were washed with sterile deionized water for 2 minutes; then, the toluidine blue staining solution was used for 30 minutes, and the residual staining solution was removed by washing with sterile deionized water for 2 minutes. The fibrochondrocytes showed a clear blue-purple color. The bright field electron microscope (CKX53PH, Olympus) was used to collect the photos.

[0083] Alcian blue staining: The fixed fibrochondrocytes were washed with sterile deionized water for 2 minutes; immersed in Alcian acidified solution for 3 minutes; then immersed in Alcian staining solution (PH2.5) for 30 minutes; washed with sterile deionized water for 2 minutes to remove the residual staining solution, and the fibrochondrocytes appeared blue. The bright field electron microscope (CKX53PH, Olympus) was used to collect photos.

[0084] 3. Experimental results

[0085] The present differentiation method is compared with the differentiation method of the prior art, and the results are as follows Figure 1 As shown, the results showed that PUNO1-TGFβ3 in this differentiation method promoted the proliferation and differentiation of chondrocytes, and differentiated into fibrochondrocytes with high regenerative ability. The cell growth morphology of the obtained fibrochondrocytes after the first passage and cultured for 72 hours showed that the cells differentiated by TGF-β1 did not proliferate and gradually apoptosis. Figure 1 The results showed that compared with the TGF-β1 group, TGF-β3 induced a higher proportion of hiPSCs differentiated into fibrochondrocytes, as manifested by a higher Alcian blue-positive area and faster cell proliferation rate. The fibrochondrocyte growth activity of the PUNO1-TGFβ3 experimental case was higher.

[0086] In the control experiment, a large number of cells showed apoptosis on day 12. Figure 2 As shown, Figure 2The results show that the differentiation method used in the present invention does not cause cell death and has excellent effects. Taking fibroblasts as a control, the fibrochondrocytes AGN1 differentiated from hiPSCs can still be stained with toluidine blue and alcian blue after 4 subcultures, proving that these cells are fibrochondrocytes. Figure 3 Example 3 Test of fibrochondrocyte proliferation ability and maintenance of cartilage properties

[0087] 1. Experimental Materials

[0088] Hoechest33342 (Beyotime, C1028); DPBS (Hyclone, SH30028.02); cck8 (YEASEN, 40203ES76).

[0089] 2. Experimental methods

[0090] According to the method of differentiating hiPSCs into fibrochondrocytes as described in Example 1, the fibrochondrocytes were passaged to P5 (5th generation) and P11 (11th generation) to prepare for testing the proliferation curve and doubling time.

[0091] CCK8 test for cell proliferation: On day 0, 2000 AGN1 cells were inoculated per well of a 96-well plate, and 10 parallel wells were made, for a total of 5 plates; on the next day, the culture medium was discarded, and a volume ratio of culture medium: CCK8 was added at 10:1 and mixed, 100 μL was added to each well and incubated in a 37°C 5% CO2 incubator for 2 h, leaving 3 CCK8 cell-free wells (background), and the absorbance OD value was measured at 450 nm by a thermo microplate reader.

[0092] Doubling time statistics (P6 and P12): The number of cells inoculated (24-well plate, 20,000 cells / well), 3-4 parallel wells and timing, when the cell confluence reaches 90-95%, 0.25% Trypsin-EDTA digestion cells for counting. Doubling time was calculated according to the formula: PDT = t × [log2 / (logNt-logN0)], where t is the total cell culture time (h), Nt is the number of cells after culturing for t time, and N0 is the number of cells initially inoculated.

[0093] qPCR detection of gene expression levels of fibrocartilage markers during the passage process: Fibrocartilage cells of different generations during the passage process (P3, P6 and P12) were taken, lysed with trizol, and RNA was extracted and reverse transcribed. Real-time fluorescence quantitative PCR instrument (LightCycler 480II, Roche) was used for qPCR detection of gene expression levels of Sox9, ACAN, Col 1A1, Col 2A1, Col 1A2, FSP1, a-SMA, and Oct4.

[0094] 3. Experimental results

[0095] The statistical results of the proliferation curve and doubling time of CCK8 are as follows Figure 4 As shown in the figure, the cck8 proliferation curve results show that fibrochondrocyte AGN1 can proliferate in large quantities in vitro, and the higher the generation (P12), the slower the proliferation. The doubling time statistical results also show that the population doubling time of AGN1 cells increases with the increase of cell generation.

[0096] Using hiPSCs as a control, the expression levels of gene markers during the passage of fibrochondrocytes (P3, P6 and P12) were detected. Figure 5 The results showed that AGN1 fibrochondrocytes highly expressed chondrocyte (Sox9 / ACAN / COL2A1) and fibroblast (FSP1 and a-SMA) markers, indicating that AGN1 fibrochondrocytes can be expanded in vitro and maintain fibrocartilage characteristics.

[0097] Example 4 Formation of immortalized fibrochondrocytes in vivo

[0098] 1. Experimental Materials

[0099] 6-8 week old NOD SCID female mice; 1 mL syringe (KDL); Matrigel (Corning, 354277); collagen type I rat tail (Corning, 354236).

[0100] 2. Experimental methods

[0101] According to the hiPSCs differentiation method described in Example 1, the fibrochondrocytes were passaged to P6. When the cell confluence reached 90%, they were digested with 0.25% Typsin-EDTA in a 37°C, 5% CO2 incubator for 3 min. Then, the digestion was terminated with a medium containing FBS, and the cell pellet was collected by centrifugation at 1200 rpm for 3 min. The cells were counted and 1.5×10 7 AGN1 fibrochondrocytes were mixed with 50% volume of Matrigel+collagen type I+DMEM / F12 (100uL Matrigel+0.4mg collagen type I+50uL DMEM / F12) and injected subcutaneously at a single point in the groin of nude mice. The samples were collected after 6-8 weeks.

[0102] 3. Experimental results

[0103] Fibrocartilage cells AGN1 P6 (generation 6) were injected subcutaneously into nude mice. Six weeks later, samples were collected for observation to form 0.5×0.4×0.4 cm non-transparent fibrocartilage tissue. The results of the formed fibrocartilage tissue are shown in the figure. Figure 6 As shown, the results indicate that AGN1 fibrochondrocytes injected subcutaneously into nude mice formed immortalized fibrocartilage tissue without losing the characteristics of fibrochondrocytes, showing excellent performance.

[0104] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A product, characterized in that The product is used to prepare fibrochondrocytes with high regeneration and passage ability; the product includes culture medium 1, culture medium 2, culture medium 3 and culture medium 4; wherein, culture medium 1 is E8 complete culture medium containing Rocki, culture medium 2 is E8 complete culture medium containing LDN193189, culture medium 3 is E8 complete culture medium containing LDN193189, CHIR-99021 and AGN193109, culture medium 4 is DMEM / F12 nutrient solution containing ITS-G, PUNO1-TGF-β3, SAG, FGF-2 and AGN193109, and PUNO1-TGF-β3 is from invivoGen; The concentration of the Rocki component in the culture medium 1 is 10 μM; The concentration of component LDN193189 in the culture medium 2 is 250 nM; The concentration of component LDN193189 in the culture medium 3 is 250 nM; The concentration of component CHIR-99021 in the culture medium 3 is 5 μM; The concentration of component AGN193109 in the culture medium 3 is 100 nM; The mass percentage of the component ITS-G in the culture medium 4 is 1%; The concentration of the component PUNO1-TGF-β3 in the culture medium 4 is 10 ng / ml; The concentration of the component SAG in the culture medium 4 is 300 nM; The concentration of the component FGF-2 in the culture medium 4 is 20 ng / ml; The concentration of the component AGN193109 in the culture medium 4 is 100 nM.

2. A method for differentiating fibrochondrocytes with high regeneration ability, characterized in that: The preparation method comprises the following steps: 1) digesting iPS cells into single cells, centrifuging and resuspending them in the medium 1 of claim 1, and seeding them on a low-adhesion culture dish overnight to form EB spheres; 2) replacing the culture medium 2 described in claim 1 and inducing the culture for 2 days; 3) inoculating in a matrigel-coated plate, replacing the medium 3 described in claim 1 and culturing for 4 days; 4) replacing the culture medium 4 described in claim 1, culturing for 6 days, and then the differentiation is completed.

3. The differentiation method according to claim 2, characterized in that The reagent used for iPS cell digestion in step 1) of the differentiation method is TrypLE. The differentiation method according to claim 2 , wherein the iPS cells are hiPSCs.

5. The differentiation method according to claim 2, wherein the concentration of the single cells digested in step 1) is 1.5×10 6 cells / mL. 6 . The differentiation method according to claim 2 , wherein the culture dish inoculated in step 1) is a low-adsorption well plate. 7 . The differentiation method according to claim 2 , wherein the matrix gel-coated plate in step 3) is a Matrigel-coated 12-well plate.

8. A 10% TY expansion medium, characterized in that The 10% TY expansion medium can expand in large quantities and maintain the characteristics of fibrochondrocytes; the 10% TY expansion medium is a DMEM / F12 medium comprising ITS-G, FBS, PUNO1-TGF-β3, SAG, FGF-2, PDGF-BB, Dexamethasone, GlutaMAX, NEAA, and Penicilin-Streptomycin, and the PUNO1-TGF-β3 is from invivoGen; The mass percentage of the ITS-G is 1%; The mass percentage of the FBS is 10%; The concentration of PUNO1-TGF-β3 is 10 ng / ml; The concentration of SAG is 100 nM; The concentration of FGF-2 is 20 ng / ml; The concentration of the PDGF-BB is 2 ng / ml; The concentration of Dexamethasone is 100 nM; The mass percentage of the GlutaMAX is 1%; The mass percentage of the NEAA is 1%; The mass percentage of the Penicilin-Streptomycin is 1%.

9. A method for high proliferation and passage of fibrochondrocytes, characterized in that: The method comprises converging the fibrochondrocytes prepared by the method according to any one of claims 2 to 7, washing, digesting with Trypsin-EDTA, terminating the digestion, collecting the cell precipitate by centrifugation, passage in proportion, resuspending the cells in the 10% TY expansion medium according to claim 8, and inoculating the cells in a culture dish pre-coated with gelatin.

10. The method according to claim 9, characterized in that The confluence of the fibrochondrocytes is 90%-95%.

11. The method according to claim 9, wherein the fibrochondrocytes are washed with DPBS after confluence.

12. The method according to claim 9, wherein the mass percentage of Trypsin-EDTA used in the digestion is 0.25%.

13. The method according to claim 9, wherein the digestion time is 3 min.

14. The method according to claim 9, wherein the digestion environment is an incubator at 37°C and 5% CO2.

15. The method according to claim 9, wherein the culture medium used to terminate the digestion is a culture medium containing FBS. The method according to claim 9 , wherein the ratio of proportional subculturing is 1:6 or 1:

12.

17. The method according to claim 9, wherein the mass percentage of the pre-coated gelatin is 0.2%.

18. Use of the 10% TY expansion medium according to claim 8 in preparing a product of fibrochondrocytes capable of continuously expanding and maintaining fibrochondrocyte characteristics.

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