A method for isolating and culturing temporomandibular joint disc stem cells

Abstract

The present invention belongs to the field of cell culture technology, and provides a method for isolating and culturing temporomandibular joint disc stem cells. The method of the present invention includes the steps of sampling, sample processing, enzymatic digestion, obtaining a single cell suspension, sorting of joint disc stem cells, primary cell seeding and subculture. The method of the present invention has the advantages of short extraction time, accelerated experimental cycle, wide application range, and can be applied to rare samples or small samples. The present invention obtains joint disc stem cells by magnetic bead sorting of CD90-positive cells, and meets the standards of mesenchymal stem cells, providing support for future joint disc stem cell therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a method for isolating and culturing temporomandibular joint disc stem cells. Background Art

[0002] The temporomandibular joint (TMJ) is a synovial joint consisting of the mandibular condylar head, articular disc, temporal bone fossa, and articular tubercle. The articular disc is attached to the joint capsule and condyle, dividing the joint cavity into upper and lower parts. The articular disc bears stress, provides cushioning, and increases stability during complex joint movements, similar to the meniscus of the knee joint. The articular disc is a fibrocartilaginous tissue, of which chondrocytes account for approximately 30% and fibroblasts account for approximately 70% (Detamore MS, Orfanos JG, Almarza AJ, et al. Quantitative analysis and comparative regional investigation of the extracellular matrix of the porcine temporomandibular joint disc [J]. Matrix Biol, 2005, 24(1): 45-57.). The main extracellular matrix includes collagen I, a small amount of collagen II, a trace amount of type III collagen, and non-fibrous collagens (VI, IX, XII) (Landesberg R, Takeuchi E, Puzas JE. Cellular, biochemical and molecular characterization of the bovine temporomandibular joint disc [J]. Arch Oral Biol, 1996, 41 (8-9): 761-7.). Collagen fibers are arranged in a ring around the periphery of the articular disc and extend medially and laterally from the center of the articular disc. These arrangements affect the tensile strength of different regions. The middle part of the articular disc is the thinnest, but contains the highest density of collagen fibers, enabling it to bear high stress (Detamore MS, Athanasiou KA. Structure and function of the temporomandibular joint disc: implications for tissue engineering [J]. J Oral Maxillofac Surg, 2003, 61 (4): 494-506.). The extracellular matrix of the articular disc also includes glycosaminoglycans and elastic fibers.Glycosaminoglycans are primarily located in the central region of the articular disc and are thought to enhance the disc's compressive strength (Detamore MS, Orfanos JG, Almarza AJ, et al. Quantitative analysis and comparative regional investigation of the extracellular matrix of the porcine temporomandibular joint disc [J]. Matrix Biol, 2005, 24(1): 45-57.). Elastic fibers are typically oriented parallel to collagen and are thought to help the disc recover its shape after stress (Detamore MS, Athanasiou K A. Structure and function of the temporomandibular joint disc: implications for tissue engineering [J]. J Oral Maxillofac Surg, 2003, 61(4): 494-506.). There are no blood vessels in the articular disc, so the synovial fluid and blood vessels around the articular disc are needed to provide the necessary cellular nutrients for the articular disc (Detamore MS, Orfanos JG, Almarza AJ, et al. Quantitative analysis and comparative regional investigation of the extracellular matrix of the porcinetemporomandibular joint disc [J]. Matrix Biol, 2005, 24 (1): 45-57.).Compared with other fibrocartilage tissues, the temporomandibular joint disc has a higher cell density and nutrient consumption rate (Kuo J, Shi C, Cisewski S, et al. Regional cell density distribution and oxygen consumption rates inporcine TMJ discs: an explant study[J]. Osteoarthritis Cartilage, 2011, 19(7): 911-8.), and a lower solute diffusion rate (Shi C, Wright GJ, Ex-Lubeskie CL, et al. Relationship between anisotropic diffusion properties and tissue morphology in porcine TMJ disc[J]. Osteoarthritis Cartilage, 2013, 21(4): 625-33.). Therefore, temporomandibular joint disc cells are more sensitive to factors that hinder nutrient supply. The main theory of articular disc disease is that the cellular composition of the articular disc changes, thereby reducing the level of extracellular matrix deposition and water accumulation, further affecting the homeostasis and load-bearing capacity of the articular disc, and ultimately leading to degenerative deterioration of the tissue (Lavi A, Pelled G, Tawackoli W, et al. Isolation and characterization of mesenchymal stromal progenitors from the temporomandibular joint disc [J]. J Tissue Eng Regen Med, 2017, 11 (5): 1553-1561.).

[0003] Temporomandibular joint disorder is considered to be the second most common musculoskeletal disease affecting the general population. The main symptoms of temporomandibular joint disorder are pain, clicking, and noise in the temporomandibular joint area. These problems may lead to impaired joint function, develop into joint structure disorder, and even organic damage. According to epidemiological surveys, 60-70% of the population will experience symptoms related to temporomandibular joint disorder (Sharma S, Gupta DS, Pal US, et al. Etiological factors of temporomandibular joint disorders [J]. Natl J Maxillofac Surg, 2011, 2 (2): 116-9.). Among them, lesions of the articular disc include internal articular derangement, articular disc thinning, and articular disc perforation. Approximately 5-15% of patients with internal articular derangement will eventually develop articular disc perforation ( MF, Rodríguez-Campo FJ, Escorial Hernández V, et al. Temporomandibular joint disc perforation: long-term results after operative arthroscopy[J]. J Oral Maxillofac Surg, 2013, 71(4): 667-76.)(Kuribayashi A, Okochi K, Kobayashi K, et al. MRI findings of temporomandibular joints with disk perforation[J]. Oral Surg Oral Med OralPathol Oral Radiol Endod, 2008, 106(3): 419-25.). Currently, repair of articular disc injury remains a major difficulty in clinical practice, and the main treatment option for severe temporomandibular joint lesions is joint replacement.

[0004] The development of stem cell tissue engineering offers hope for the repair and regeneration of temporomandibular joint (TMJ) disc lesions. Stem cells possess the potential for self-renewal and differentiation, secrete a wide range of trophic factors, and possess potent regenerative and immunomodulatory properties (Meirelles Lda S, Fontes AM, Covas DT, et al. Mechanisms involved in the therapeutic properties of mesenchymal stem cells [J]. Cytokine Growth Factor Rev, 2009, 20(5-6):419-27). These properties provide a foundation for the treatment of TMJ disorders. According to the minimum criteria for defining mesenchymal stem cells by the International Society for Cellular Therapy, mesenchymal stem cells should be plastic-adherent, express CD73, CD90, and CD105, but not express CD34, CD45, CD14 (or CD11b), CD19 (or CD79a), and HLA-DR, and these cells should have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes (Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement [J]. Cytotherapy, 2006, 8 (4): 315-7.). Mesenchymal stem cells are a potential cell source for fibrocartilage repair. Extracting and studying the function of these cell populations in articular disc development and repair will help explore stem cell-based repair therapy methods in fibrocartilage. Currently, multipotent stem cells have been discovered in the human meniscus, which are phenotypically similar to mesenchymal stem cells (Segawa Y, Muneta T, Makino H, et al. Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles [J]. JOrthop Res, 2009, 27 (4): 435-41.).Mesenchymal stem cells isolated from rabbit meniscus exhibit common stem cell characteristics, including clonogenicity, multipotency, self-renewal capacity, and expression of stem cell markers, and exhibit a clear tendency to differentiate into chondrogenic cartilage both in vitro and in vivo (Ding Z, Huang H. Mesenchymal stem cells in rabbit meniscus and bone marrow exhibit a similar feature but a heterogeneous multi-differentiation potential: superiority of meniscus as a cell source for meniscus repair [J]. BMC Musculoskelet Disord, 2015, 16: 65.). Stem / progenitor cells have also been isolated from mouse meniscus, and endogenous progenitor cells may exist on the surface and external regions of the meniscus in vivo (Gamer LW, Shi RR, Gendelman A, et al. Identification and characterization of adult mouse meniscus stem / progenitor cells [J]. Connect Tissue Res, 2017, 58(3-4): 238-245.).Fibrocartilage stem cells have also been found in the fibrous surface layer of the condyle of the temporomandibular joint. Currently, fibrocartilage stem cells in rats, mice, rabbits and humans have been identified and located using mesenchymal cell markers (Embree MC, Chen M, Pylawka S, et al. Exploiting endogenous fibrocartilage stem cells to regenerate cartilage and repair joint injury[J]. Nat Commun, 2016, 7: 13073.)(Nathan J, Ruscitto A, Pylawka S, et al. Fibrocartilage Stem Cells Engraft and Self-Organize into Vascularized Bone[J]. J Dent Res, 2018, 97(3): 329-337.)(Bi R, Yin Q, Mei J, et al. Identification of human temporomandibular joint fibrocartilage stem cells with distinct chondrogenic capacity[J]. Osteoarthritis Cartilage, 2020, 28(6): 842-852.)(Ruscitto A, Scarpa V, Morel M, et al. Notch Regulates Fibrocartilage Stem Cell Fate and Is Upregulated in Inflammatory TMJ Arthritis[J]. J Dent Res, 2020, 99(10): 1174-1181.). Currently, there are no patented technology reports on the extraction and research of stem cells in the articular disc. The present invention obtains articular disc stem cells by magnetic bead sorting of CD90-positive cells, which meet the standards of mesenchymal stem cells, providing support for future articular disc stem cell therapy. Summary of the Invention

[0005] To overcome the above-mentioned defects in the prior art, the present invention provides a method for isolating and culturing temporomandibular joint disc stem cells. The stem cells extracted by this method have a high survival rate, can be successfully cultured and passaged, and have basic stem cell characteristics such as multidirectional differentiation potential and colony-forming ability.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for isolating and culturing temporomandibular joint disc stem cells, comprising the following steps:

[0008] (1) Sampling: After the temporomandibular joint disc is washed in a buffer solution, the joint capsule attached to the disc is removed to obtain the disc sample;

[0009] (2) Sample processing: The articular disc sample is placed in a buffer solution and washed 2 to 4 times to remove blood clots, tissue fragments, and impurities on the surface of the articular disc, and the buffer solution is discarded;

[0010] (3) Enzyme digestion: Add α-MEM solution containing pronase storage solution and digest for 0.8-1.2 h. Centrifuge and discard the supernatant. Then add α-MEM solution containing collagenase P storage solution and digest for 40-60 min to obtain digestion solution.

[0011] (4) Obtaining a single-cell suspension: adding a complete cell culture medium twice the volume of the digestion solution to terminate the enzymatic digestion reaction, filtering, and obtaining a single-cell suspension;

[0012] (5) Sorting of articular disc stem cells: The single cell suspension was centrifuged and the supernatant was discarded. The cells were labeled with 85-95 μL magnetic bead sorting buffer and 8-12 μL CD90.2 magnetic beads. After incubation in the dark, 0.8-1.2 mL magnetic bead sorting buffer was added to collect CD90 + The cells are articular disc stem cells;

[0013] (6) Primary cell seeding plate: The obtained articular disc stem cells are seeded into a plate and cultured in complete cell culture medium;

[0014] (7) Subculture: Change the medium every 2 to 4 days until the cell confluence is ≥80%. Remove the old medium, digest with trypsin for 1 to 2 minutes, and then subculture.

[0015] Preferably, the volume ratio of the temporomandibular joint disc to the buffer solution in step (1) is 1:5-10, and the temperature during the cleaning process is 3-5°C.

[0016] Preferably, the volume ratio of the articular disc sample to the buffer solution in step (2) is 1:2-4.

[0017] Preferably, the buffers in step (1) and step (2) are independently based on PBS, containing 1-3% FBS and 0.8-1.2% penicillin-streptomycin.

[0018] Preferably, the working conditions of the enzymatic digestion in step (3) are 35-40° C., 80-120 rpm horizontal rotary oscillation, the pronase storage solution uses ddH2O as a solvent and contains 18-22 mg / ml of pronase, the final concentration of pronase in the α-MEM solution containing the pronase storage solution is 3-5 mg / ml, the collagenase P storage solution uses PBS as a solvent and contains 18-22 mg / ml of collagenase P, the final concentration of collagenase P in the α-MEM solution containing the collagenase P storage solution is 1-3 mg / ml, the centrifugal speed is 250×g~350×g, and the centrifugal time is 4-6 min.

[0019] Preferably, the pore size of the filtration in step (4) is 35 to 45 μm.

[0020] Preferably, the number of cells required for the sorting in step (5) is ≤ 1×10 7 The centrifugal speed is 250×g~350×g, the centrifugal time is 4~6min, the magnetic bead sorting buffer is based on PBS, contains 0.4~0.6% BSA, 1~3mM EDTA and 0.8~1.2% penicillin-streptomycin, the light-proof incubation temperature is 3~5°C, and the light-proof incubation time is 8~12min.

[0021] Preferably, the density of the articular disc stem cell seed plate in step (6) is 2 to 4×10 4 pieces / cm 2 The amount of complete cell culture medium added is 1.5-2 ml / cm 2 The culture conditions are 35-40° C. and 4-6% CO 2 .

[0022] Preferably, the complete cell culture medium in step (4) and step (6) is based on α-MEM, containing 18-22% FBS and 0.8-1.2% penicillin-streptomycin.

[0023] Preferably, the seed plate density during the subculture process in step (7) is 1.5 to 2×10 4 pieces / cm 2 The conditions for the subculture are 35-40° C. and 4-6% CO 2 .

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. Short extraction time: The extraction step in the present invention includes two steps: enzyme digestion and sorting, which takes a total of about 3 to 4 hours to complete. Traditional articular discs are digested with type I collagenase, but the digestion time is as long as 6 hours, and it is not easy to fully digest. Articular discs contain not only collagen I, but also a small amount of collagen II and a trace amount of type III collagen and non-fibrous collagen (VI, IX, XII), which are difficult to completely digest with a single enzyme. The present invention uses two enzymes (pronase and collagenase P) for digestion in succession, and the total digestion time is only 1.5h to 2h, and no obvious tissue fragments are seen under the microscope (see Table 1 and Table 1 for specific differences). Figure 1 ). The present invention adopts CD90 + Magnetic bead separation is used to obtain articular disc stem cells. The operation is simple and only takes 0.5 hours. The invention also shortens the extraction time, helps ensure cell activity, increases stem cell preservation rate, and reduces the possibility of cell contamination.

[0026] Table 1

[0027] Pronase + collagenase P (the present invention) Traditional collagenase type I Digestion time 1.5-2h 6h Digestive activity 80-95％ 30-50％ Is digestion sufficient? Almost no tissue fragments Some tissue fragments are visible

[0028] 2. Speed up the experimental cycle: Multiple passages will lead to changes in stem cell morphology, phenotype and genetics, so subsequent experiments should be carried out as early as possible. + Subsequent stem cell experiments can be carried out after magnetic bead sorting, while traditional monoclonal culture requires waiting for the monoclonal cells to form beads before experiments can be carried out (see Table 2 for specific differences).

[0029] Table 2

[0030] CD90 magnetic bead sorting (present invention) Traditional monoclonal culture Cell mass <![CDATA[CD90 + About 10% of total disc cells Monoclonal cell beads account for approximately 1% of the total disc cells Experimental cycle After sorting, you can start the experiment Monoclonal cell beads take at least 2 weeks to form

[0031] 3. Wide range of applications, applicable to rare or tiny samples: In traditional monoclonal culture, only 1% of 96-well plates can obtain target stem cells, while the method based on the present invention can instantly obtain a large number of stem cells, which can be used to extract stem cells from rare or tiny samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0033] Figure 1 The microscopic examination results after treatment with different enzyme digestion methods (Note: the left picture shows the digestion of mouse articular disc by pronase and collagenase P; the right picture shows the digestion of mouse articular disc by collagenase I);

[0034] Figure 2 These are the articular discs of 3-day-old and 16-month-old mice (Note: the left picture is the articular disc of a 3-day-old mouse; the right picture is the articular disc of a 16-month-old mouse);

[0035] Figure 3 The changes of mouse articular disc stem cells from Day 1 to Day 7 after seeding in Example 1 of the present invention;

[0036] Figure 4 This is a three-way differentiation experiment of mouse articular disc stem cells (adipogenic induction in young mice);

[0037] Figure 5 This is a three-way differentiation experiment of mouse articular disc stem cells (adipogenic induction in aged mice);

[0038] Figure 6 This is a three-way differentiation experiment of mouse articular disc stem cells (osteogenesis induction in young mice);

[0039] Figure 7 This is a three-way differentiation experiment of mouse articular disc stem cells (osteogenesis induction in aged mice);

[0040] Figure 8 This is a three-way differentiation experiment of mouse articular disc stem cells (chondrogenic induction in young mice);

[0041] Figure 9 This is a three-way differentiation experiment of mouse articular disc stem cells (chondrogenic induction in aged mice);

[0042] Figure 10 This is an experiment to detect the proliferation capacity of mouse articular disc stem cells (young mice) (Note: **** indicates that the proliferation capacity of articular disc stem cells after screening and culture is significantly enhanced compared with the control cells);

[0043] Figure 11 This is an experiment to detect the proliferation capacity of mouse articular disc stem cells (aged mice);

[0044] Figure 12 This is a mouse articular disc stem cell colony formation experiment. DETAILED DESCRIPTION

[0045] The present invention provides a method for isolating and culturing temporomandibular joint disc stem cells, comprising the following steps:

[0046] (1) Sampling: After the temporomandibular joint disc is washed in a buffer solution, the joint capsule attached to the disc is removed to obtain the disc sample;

[0047] (2) Sample processing: The articular disc sample is placed in a buffer solution and washed 2 to 4 times to remove blood clots, tissue fragments, and impurities on the surface of the articular disc, and the buffer solution is discarded;

[0048] (3) Enzyme digestion: Add α-MEM solution containing pronase storage solution and digest for 0.8-1.2 h. Centrifuge and discard the supernatant. Then add α-MEM solution containing collagenase P storage solution and digest for 40-60 min to obtain digestion solution.

[0049] (4) Obtaining a single-cell suspension: adding a complete cell culture medium twice the volume of the digestion solution to terminate the enzymatic digestion reaction, filtering, and obtaining a single-cell suspension;

[0050] (5) Sorting of articular disc stem cells: The single cell suspension was centrifuged and the supernatant was discarded. The cells were labeled with 85-95 μL magnetic bead sorting buffer and 8-12 μL CD90.2 magnetic beads. After incubation in the dark, 0.8-1.2 mL magnetic bead sorting buffer was added to collect CD90 + The cells are articular disc stem cells;

[0051] (6) Primary cell seeding plate: The obtained articular disc stem cells are seeded into a plate and cultured in complete cell culture medium;

[0052] (7) Subculture: Change the medium every 2 to 4 days until the cell confluence is ≥80%. Remove the old medium, digest with trypsin for 1 to 2 minutes, and then subculture.

[0053] In the present invention, the volume ratio of the temporomandibular joint disc to the buffer solution in step (1) is preferably 1:5-10, more preferably 1:9; the temperature during the cleaning process is preferably 3-5°C, more preferably 4°C.

[0054] In the present invention, in step (2), the articular disc sample is placed in a buffer solution and washed 2 to 4 times to wash away blood clots, tissue fragments and impurities on the surface of the articular disc, and the buffer solution is discarded. It is further preferred that the articular disc sample is placed in a buffer solution and washed 3 times to wash away blood clots, tissue fragments and impurities on the surface of the articular disc, and the buffer solution is discarded.

[0055] In the present invention, the volume ratio of the articular disc sample to the buffer solution in step (2) is preferably 1:2-4, more preferably 1:3.

[0056] In the present invention, the buffer solutions in step (1) and step (2) are preferably based on PBS, containing 1-3% FBS and 0.8-1.2% penicillin-streptomycin, and more preferably based on PBS, containing 2% FBS and 1% penicillin-streptomycin.

[0057] In the present invention, the step (3) is to add an α-MEM solution containing a pronase storage solution to digest for 0.8 to 1.2 hours, centrifuge and discard the supernatant, then add an α-MEM solution containing a collagenase P storage solution to digest for 40 to 60 minutes to obtain a digestion solution. More preferably, the step is to add an α-MEM solution containing a pronase storage solution to digest for 1 hour, centrifuge and discard the supernatant, then add an α-MEM solution containing a collagenase P storage solution to digest for 40 minutes to obtain a digestion solution. During the enzymatic digestion process, if only pronase is used without collagenase P, although obvious rough edges can be observed on the tissue edge, further digestion and separation of cells cannot be achieved; if only collagenase P is used without pronase, cells tend to clump and become entangled in a flocculent state.

[0058] In the present invention, the working conditions of the enzymatic digestion in step (3) are preferably 35-40°C, 80-120 rpm horizontal rotary oscillation, more preferably 37°C, 100 rpm horizontal rotary oscillation; the pronase storage solution preferably uses ddH2O as a solvent and contains 18-22 mg / ml of pronase, more preferably uses ddH2O as a solvent and contains 20 mg / ml of pronase; the final concentration of pronase in the α-MEM solution containing the pronase storage solution is preferably 3-5 mg / ml, more preferably The collagenase P storage solution preferably uses PBS as a solvent and contains 18 to 22 mg / ml of collagenase P, and more preferably uses PBS as a solvent and contains 20 mg / ml of collagenase P; the final concentration of collagenase P in the α-MEM solution containing the collagenase P storage solution is preferably 1 to 3 mg / ml, and more preferably 2 mg / ml; the centrifugal speed is preferably 250×g to 350×g, and more preferably 300×g; the centrifugal time is preferably 4 to 6 min, and more preferably 5 min.

[0059] In the present invention, the pore size of the filtration in step (4) is preferably 35 to 45 μm, more preferably 38 to 42 μm, and even more preferably 40 μm.

[0060] In the present invention, the single cell suspension is centrifuged and the supernatant is discarded in step (5), and the cells are labeled with 85-95 μL magnetic bead sorting buffer and 8-12 μL CD90.2 magnetic beads. After incubation in the dark, 0.8-1.2 mL magnetic bead sorting buffer is added to collect CD90 + The cells are articular disc stem cells. It is further preferred that the single cell suspension is centrifuged and the supernatant is discarded. The cells are labeled with 90 μL of magnetic bead sorting buffer and 10 μL of CD90.2 magnetic beads. After incubation in the dark, 1 mL of magnetic bead sorting buffer is added to collect CD90 + The cells are called articular disc stem cells.

[0061] In the present invention, the number of cells required for the sorting in step (5) is preferably ≤1×10 7 , more preferably 1×10 7 ; The centrifugal speed is preferably 250×g~350×g, more preferably 300×g; the centrifugal time is preferably 4~6min, more preferably 5min; the magnetic bead sorting buffer is preferably based on PBS, containing 0.4~0.6% BSA, 1~3mM EDTA and 0.8~1.2% penicillin-streptomycin, more preferably based on PBS, containing 0.5% BSA, 2mM EDTA and 1% penicillin-streptomycin; the temperature of the light-proof incubation is preferably 3~5°C, more preferably 4°C; the light-proof incubation time is preferably 8~12min, more preferably 10min.

[0062] In the present invention, the density of the articular disc stem cell seed plate in step (6) is preferably 2 to 4×10 4 pieces / cm 2 , more preferably 3×10 4 pieces / cm 2 The amount of complete cell culture medium added is preferably 1.5 to 2 ml / cm 2 , more preferably 1.8 ml / cm 2 The culture conditions are preferably 35-40°C, 4-6% CO2, and more preferably 37°C, 5% CO2.

[0063] In the present invention, the complete cell culture medium in step (4) and step (6) is preferably based on α-MEM, containing 18-22% FBS and 0.8-1.2% penicillin-streptomycin, and further preferably based on α-MEM, containing 20% FBS and 1% penicillin-streptomycin.

[0064] In the present invention, the medium is changed every 2 to 4 days in step (7) until the cell confluence is ≥80%, the old medium is removed, the cells are digested with trypsin for 1 to 2 minutes, and subculture is performed. It is further preferred that the medium is changed every 3 days until the cell confluence is 85%, the old medium is removed, the cells are digested with trypsin for 1.5 minutes, and subculture is performed.

[0065] In the present invention, the seed plate density in the subculture process in step (7) is preferably 1.5 to 2×10 4 pieces / cm 2 , more preferably 1.8×10 4 pieces / cm 2 The conditions for the subculture are preferably 35-40°C and 4-6% CO2, and more preferably 37°C and 5% CO2.

[0066] 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.

[0067] Phosphate buffered saline (PBS), α-MEM medium, fetal bovine serum, penicillin-streptomycin mixture, and trypsin in the following examples were purchased from Gibco; pronase and collagenase P were purchased from Roche; the magnetic bead separation system was purchased from Miltenyi Biotec; EDTA in the magnetic bead separation buffer was purchased from J&K; and BSA was purchased from BioFroxx. Cell counts were performed using a Bio-Rad cell counter.

[0068] The article numbers of the reagents in the following examples are:

[0069] PBS:Gibco,C200112500BT,US

[0070] FBS:Gibco,10091148,US

[0071] Pronase: Roche 10165921001, Switzerland

[0072] Collagenase P: Roche 11213865001, Switzerland

[0073] α-MEM culture medium: Gibco, C12571500BT, US

[0074] Penicillin-streptomycin mixture: Gibco, 15140122, US

[0075] BSA: BioFroxx, 4240GR500, Germany

[0076] EDTA: J&K, 932641, China

[0077] CD90.2 / THY1 magnetic beads: 130-121-278, Miltenyi Biotec, Germany

[0078] Example 1

[0079] Taking 10 3-day-old to 1-week-old mouse articular discs as an example, the temporomandibular joint disc stem cells were isolated and cultured as follows:

[0080] (1) After the sudden death of mice, samples were processed in a biosafety cabinet, and all instruments were sterilized in advance.

[0081] (2) The mouse zygomatic arch was cut with microscissors to expose the temporomandibular joint area. The joint capsule was opened with microtweezers. The articular disc was separated and removed under a stereomicroscope or under the naked eye. The disc was placed in a 3 cm culture dish containing 3 ml of buffer (PBS + 2% FBS + 1% penicillin-streptomycin mixture) and washed (4°C). After washing, the disc was trimmed under a stereomicroscope and the joint capsule attached to the disc was removed to obtain the disc sample. The disc size of the young mouse articular disc is approximately 700-1000 μm.

[0082] (3) Place the articular disc sample into a 1.5 ml EP tube, add 1 ml of buffer, centrifuge briefly (300 × g) to allow the articular disc to sink to the bottom, discard the supernatant, repeat washing three times, and discard the buffer.

[0083] (4) Enzyme digestion: Enzyme digestion was performed at 37°C with a horizontal rotary shaker at 100 rpm. The enzymatic digestion was divided into two steps. First, 4 mg / mL pronase was added for digestion for 1 hour. After pronase digestion, obvious rough edges were observed at the edge of the articular disc. Centrifuge at 300 × g for 5 minutes, discard the supernatant, and add 2 mg / mL collagenase P for digestion for 40 minutes. After collagenase P digestion, there were basically no large tissue fragments.

[0084] (5) Obtaining single-cell suspension: Add twice the volume of complete cell culture medium (α-MEM + 20% FBS + 1% penicillin-streptomycin mixture) to terminate the enzyme reaction. Pass the cell suspension through a 40 μm cell strainer to obtain a single-cell suspension, count it, and aliquot it so that the number of cells per tube is less than 1 × 10 7 indivual.

[0085] (6) Sorting of articular disc stem cells: centrifuge at 300×g for 5 min and discard the supernatant. Protect from light and sort according to the requirements. 7 Within 90 μL magnetic bead sorting buffer and 10 μL CD90.2 magnetic beads can fully label CD90 + Cells. Incubate at 4°C in the dark for 10 minutes. After incubation, no further operations are required to protect cells from light. Add 1 mL of magnetic bead separation buffer (0.5% BSA + 2mM EDTA + 1% penicillin-streptomycin mixture + PBS). Place the separation column on the magnetic separation rack, wet the separation column, add the cell suspension, and collect all CD90 - Remove the separation column from the magnetic separation rack, add 1 mL of magnetic bead separation buffer, and collect all CD90 + Cells were counted.

[0086] (7) P0 seed plate: cells were plated at 3×10 4 pieces / cm 2 Seed plate, add 1.8ml / cm 2Complete cell culture medium. Culture in a 37°C, 5% CO2, saturated humidity incubator.

[0087] (8) Passaging: Change the medium every 3 days. After 5-6 days, when the cell confluence reaches more than 80%, remove the old medium, wash twice with PBS, and trypsinize for 1.5 minutes. When the P0 cells attached to the wall fall off from the bottom of the culture dish, add double volume of complete cell culture medium to stop the digestion. Centrifuge at 300×g for 5 minutes, discard the supernatant, add complete culture medium, resuspend, count and passage. The density of the P1 and P2 seed plates is 1.8×10 4 pieces / cm 2 The subculture conditions are culturing in an incubator at 37°C, 5% CO2, and saturated humidity, and the cells will grow to full size in about 3 days.

[0088] Example 2

[0089] Using six 16-month-old mouse articular discs as an example, temporomandibular joint disc stem cells were isolated and cultured using the following steps:

[0090] (1) After the sudden death of mice, samples were processed in a biosafety cabinet, and all instruments were sterilized in advance.

[0091] (2) Use microscissors to cut the mouse zygomatic arch to expose the temporomandibular joint area. Use microtweezers to open the joint capsule. Separate and remove the articular disc under a stereomicroscope or under the naked eye. Place it in a 3 cm culture dish containing 3 ml of buffer (PBS + 2% FBS + 1% penicillin-streptomycin mixture) and wash it (4°C). After washing, trim the articular disc under a stereomicroscope and remove the part of the joint capsule attached to the articular disc to obtain the articular disc sample. The articular disc size of old mice is approximately 1600-2500 μm.

[0092] (3) Place the articular disc sample in a 1.5ml EP tube, add 1ml of buffer, centrifuge briefly (300×g) to allow the articular disc to settle to the bottom, discard the supernatant, and repeat the washing three times. The articular disc volume of old mice is significantly larger than that of young mice. To ensure more complete tissue digestion, use sterile microscissors to cut the articular disc of old mice into 1.5mm pieces. 2 Centrifuge briefly and discard the buffer.

[0093] (4) Enzyme digestion: Enzyme digestion was performed at 37°C with a horizontal rotary shaker at 100 rpm. The enzymatic digestion was divided into two steps. First, 4 mg / mL pronase was added for digestion for 1 hour. After pronase digestion, obvious rough edges were observed at the edge of the articular disc. Centrifuge at 300 × g for 5 minutes and discard the supernatant. Add 2 mg / mL collagenase P and digest for 60 minutes. After collagenase P digestion, there were basically no large tissue fragments.

[0094] (5) Obtaining single-cell suspension: Add twice the volume of complete cell culture medium (α-MEM + 20% FBS + 1% penicillin-streptomycin mixture) to terminate the enzyme reaction. Pass the cell suspension through a 40 μm cell strainer to obtain a single-cell suspension, count it, and aliquot it so that the number of cells per tube is less than 1 × 10 7 indivual.

[0095] (6) Sorting of articular disc stem cells: centrifuge at 300×g for 5 min and discard the supernatant. Protect from light and sort according to the requirements. 7 Within 90 μL magnetic bead sorting buffer and 10 μL CD90.2 magnetic beads can fully label CD90 + Cells. Incubate at 4°C in the dark for 10 minutes. After incubation, no further operations are required to protect cells from light. Add 1 mL of magnetic bead separation buffer (0.5% BSA + 2mM EDTA + 1% penicillin-streptomycin mixture + PBS). Place the separation column on the magnetic separation rack, wet the separation column, add the cell suspension, and collect all CD90 - Remove the separation column from the magnetic separation rack, add 1 mL of magnetic bead separation buffer, and collect all CD90 + Cells were counted.

[0096] (7) P0 seed plate: cells were plated at 3×10 4 pieces / cm 2 Seed plate, add 1.8ml / cm 2 Complete cell culture medium. Culture in a 37°C, 5% CO2, saturated humidity incubator.

[0097] (8) Passaging: Change the medium every 3 days. After 5-6 days, when the cell confluence reaches more than 80%, remove the old medium, wash twice with PBS, and trypsinize for 1.5 minutes. When the P0 cells attached to the wall fall off from the bottom of the culture dish, add double volume of complete cell culture medium to stop the digestion. Centrifuge at 300×g for 5 minutes, discard the supernatant, add complete culture medium, resuspend, count and pass the cells. The density of the passage plate is 1.8×10 4 pieces / cm 2 The subculture conditions were cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0098] Experimental Example 1

[0099] Taking the mouse articular disc in Example 1 as an example, the optimal enzyme concentration was studied.

[0100] 1. Exploration of the optimal working solution concentration of pronase:

[0101] After the 3-day to 1-week mouse articular discs were harvested, trimmed, and cleaned, 1 ml of freshly prepared α-MEM digestion working solution containing pronase (stock solution: 20 mg / ml) was added to every 10 mouse articular discs.

[0102] The preparation methods of different concentrations of pronase working solutions are shown in Table 3:

[0103] Table 3

[0104]

[0105] The results of 1 h digestion with different concentrations of pronase are shown in Table 4:

[0106] Table 4

[0107]

[0108] As shown in Table 4, when the pronase digestion concentration was 4 mg / ml, the tissue fragments already had obvious rough edges. Considering the comprehensive cost, the optimal pronase digestion concentration was 4 mg / ml.

[0109] 2. Exploration of the optimal working solution concentration of collagenase P:

[0110] The sample digested with 4 mg / ml pronase for 1 hour was centrifuged at 300×g for 5 minutes, the supernatant was discarded, and 1 ml of freshly prepared α-MEM digestion working solution containing collagenase P (stock solution: 20 mg / ml) was added.

[0111] The preparation methods of different collagenase P working solutions are shown in Table 5:

[0112] Table 5

[0113]

[0114] The results of 40 min digestion with different concentrations of collagenase P are shown in Table 6:

[0115] Table 6

[0116] Collagenase P 1mg / ml 2mg / ml 3mg / ml 4mg / ml Cell viability 67％ 92％ 83％ 75％ Number of living cells <![CDATA[1.7x10 5 ]]> <![CDATA[3.7x10 5 ]]> <![CDATA[3.5x10 5 ]]> <![CDATA[3.3x10 5 ]]> Observation under light microscope Cell clumping No obvious clumping No obvious clumping No obvious clumping

[0117] As shown in Table 6, when the optimal digestion concentration of collagenase P was 2 mg / ml, the cells had no obvious clumping. Considering the comprehensive cost, the optimal digestion concentration of collagenase P was 2 mg / ml.

[0118] Experimental Example 2

[0119] Taking the mouse articular disc in Example 1 as an example, the optimal digestion time was studied.

[0120] 1. After sampling, trimming, and cleaning the articular discs of 3-day-1-week-old mice, add 1 ml of freshly prepared α-MEM digestion working solution containing 4 mg / ml pronase (stock solution: 20 mg / ml) for every 10 mouse articular discs.

[0121] The results of 4 mg / ml pronase digestion at different times are shown in Table 7:

[0122] Table 7

[0123]

[0124] As shown in Table 7, the optimal digestion time of pronase is 1 h.

[0125] 2. Take the sample digested with 4 mg / ml pronase for 1 hour, centrifuge at 300×g for 5 minutes, discard the supernatant, and add 1 ml of freshly prepared α-MEM digestion working solution containing 2 mg / ml collagenase P (stock solution: 20 mg / ml).

[0126] The results of 1 h digestion with 4 mg / ml pronase and different digestion times with 2 mg / ml collagenase P are shown in Table 8:

[0127] Table 8

[0128]

[0129]

[0130] As shown in Table 8, the optimal digestion time of collagenase P is 40 min.

[0131] Experimental Example 3

[0132] Taking the mouse articular disc in Example 1 as an example, the optimal inoculation density (ie, seed plate density) was studied.

[0133] After the 3-day to 1-week-old mouse articular discs were harvested, trimmed, and cleaned, 10 mouse articular discs were digested with 4 mg / ml pronase for 1 hour and 2 mg / ml collagenase P for 40 minutes. The discs were plated in 24-well plates with a bottom area of 2 cm. 2 .

[0134] The results of different primary inoculation densities after 7 days of culture are shown in Table 9:

[0135] Table 9

[0136] <![CDATA[1x10 4 pieces / cm 2 ]]> <![CDATA[2x10 4 pieces / cm 2 ]]> <![CDATA[3x10 4 pieces / cm 2 ]]> <![CDATA[4x10 4 pieces / cm 2 ]]> Observation under light microscope Unable to survive 30-40% full grown Full Overgrown, multi-layered

[0137] As shown in Table 9, the optimal seeding rate of primary cells is 3×10 4 pieces / cm 2 .

[0138] Experimental Example 4

[0139] Mouse articular disc stem cell three-way differentiation experiment:

[0140] Osteogenic induction: P2 cells were cultured at 2×10 4 pieces / cm 2 Inoculate in 24-well plates and culture in complete medium. When the cell confluence reaches 50-70% (about 2-2.5 days), replace with fresh medium containing 10% FBS, 10 -8 α-MEM osteogenic induction medium containing 100 μM dexamethasone (D8040, Solarbio), 50 μg / ml ascorbic acid (231406, J&K), and 10 mM β-glycerophosphate (G8100, Solarbio) was replaced every 3 days. After 3 weeks of culture, cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 1% Alizarin Red (G1452, Solarbio).

[0141] Adipogenic induction: P2 cells were plated at 2×10 4 pieces / cm 2 Cells were seeded in 24-well plates and cultured in complete medium until confluent (approximately 3-4 days). Adipogenic medium A was prepared: 20% FBS, 500 μM IBMX (I7018, Sigma), 1 μM dexamethasone (D8040, Solarbio), 1 μM rosiglitazone (R8470, Solarbio), 10 μg / ml insulin (I8040, Solarbio), and α-MEM medium. After 48 hours of culture in medium A, cells were washed twice with 1 ml of PBS and replaced with medium B. Adipogenic medium B was prepared: 20% FBS, 10 μg / ml insulin, 1 μM rosiglitazone, and α-MEM medium. Medium B was completely replaced every 2 days. After 6 days of induction, cells were fixed with 4% paraformaldehyde for 15 minutes and stained with Oil Red O.

[0142] Chondrogenic induction: P2 cells were cultured at 2.5×10 5 10 cells / well were seeded in a V-bottom 96-well plate. -7The cells were centrifuged at 0.3 × g for 5 minutes to form a pellet. The culture medium was changed every 3 days, and the medium was freshly prepared each time. After three weeks, the pellet was pelleted with 4% paraformaldehyde, and frozen and paraffin sections were prepared. The cells were stained with Movat, SOX9, Aggrecan, and Safranin O-Fast Green.

[0143] The results are as follows Figures 4 to 9 As shown. Figures 4 to 9 It can be seen that mouse articular disc CD90 + The cells have the ability to form bones, adipocytes, and cartilage. + The osteogenic, adipogenic and chondrogenic abilities of the cells were significantly stronger than those of the CD90 cells in the articular disc of young mice. - Cells. CD90 in articular disc of aged mice + The osteogenic, adipogenic and chondrogenic abilities of cells were relative to those of CD90 in the articular disc of young mice. + Cells decreased.

[0144] Experimental Example 5

[0145] Mouse articular disc stem cell proliferation ability detection experiment:

[0146] The proliferation capacity of cells was measured using the C0071 BeyoClickTM EdU-488 kit. In this experiment, P2 cells were plated at 2×10 4 pieces / cm 2 Inoculate in 24-well plates (juvenile mouse articular disc CD90 + and CD90 - cells, and CD90 in articular discs of aged mice + cell).

[0147] When cell confluency reached 50%, EdU was added to the culture medium at a final concentration of 10 μM, and the cells were incubated for 4 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes at room temperature. The fixative was removed, and the cells were washed three times with 1 ml of PBS per well for 5 minutes each. The PBS was removed, and the cells were permeabilized with 1 ml of 0.3% Triton X-100 in PBS per well for 13 minutes at room temperature. The permeabilization solution was removed, and the cells were washed twice with 1 ml of PBS per well for 4 minutes each. Fresh Click reaction solution was prepared and used within 15 minutes of preparation. Incubate in the dark for 30 minutes. The Click reaction solution was aspirated, and the cells were washed three times with 1 ml of PBS per well for 4 minutes each. The cells were stained with DAPI containing the anti-fluorescence quencher for 10 minutes. Positive cells were imaged and counted using a Lecia fluorescence microscope. The percentage of EdU-positive cells was calculated by dividing the number of green fluorescent cells by the number of blue fluorescent DAPI-stained cells.

[0148] The results are as follows Figure 10 and Figure 11 As shown. Figure 10 and Figure 11 It is known that CD90 + The proliferation ability of the cells was significantly stronger than that of CD90- cells in the articular disc of young mice. + CD90 cell proliferation ability relative to that of young mouse articular disc + Cells decreased.

[0149] Experimental Example 6

[0150] Mouse articular disc stem cell colony formation experiment:

[0151] The cells at P2 stage were harvested and seeded into 6-well plates at 1000 cells / well. The complete culture medium was replaced every 3 days (CD90 + and CD90 - cells, and CD90 in articular discs of aged mice + After 12 days of culture, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, stained with 0.1% crystal violet dye (G1072, Solarbio) for 2 minutes, washed three times with ddH2O, and the culture dishes were air-dried and photographed.

[0152] The results are as follows Figure 12 As shown. Figure 12 It is known that CD90 + The colony-forming ability of cells was significantly stronger than that of CD90- cells in the articular disc of young mice. + The colony-forming ability of CD90 cells was relative to that of CD90 cells in the articular disc of young mice + Cells decreased.

[0153] 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 isolating and culturing temporomandibular joint disc stem cells, characterized in that: The steps include: (1) Sampling: After the temporomandibular joint disc is washed in a buffer solution, the joint capsule attached to the disc is removed to obtain the disc sample; (2) Sample processing: The articular disc sample is placed in a buffer solution and washed 2 to 4 times to remove blood clots, tissue fragments, and impurities on the surface of the articular disc, and the buffer solution is discarded; (3) Enzyme digestion: Add α-MEM solution containing pronase storage solution to digest for 0.8-1.2 hours, centrifuge and discard the supernatant, then add α-MEM solution containing collagenase P storage solution to digest for 40-60 minutes to obtain the digestion solution; The final concentration of pronase in the α-MEM solution containing the pronase storage solution is 3-5 mg / ml, and the final concentration of collagenase P in the α-MEM solution containing the collagenase P storage solution is 2-3 mg / ml; (4) Obtaining a single-cell suspension: Adding twice the volume of complete cell culture medium of the digestion solution to terminate the enzymatic digestion reaction, filtering, and obtaining a single-cell suspension; (5) Sorting of articular disc stem cells: centrifuge the single cell suspension and discard the supernatant, label the cells with 85-95 μL magnetic bead sorting buffer and 8-12 μL CD90.2 magnetic beads, incubate in the dark, add 0.8-1.2 mL magnetic bead sorting buffer, and collect CD90 + The cells are articular disc stem cells; (6) Primary cell seeding plate: The obtained articular disc stem cells are seeded into a plate and cultured in complete cell culture medium; (7) Subculture: Change the medium every 2 to 4 days until the cell confluence is ≥80%. Remove the old culture medium, digest with trypsin for 1 to 2 minutes, and then subculture.

2. The method for isolating and culturing temporomandibular joint stem cells according to claim 1, wherein: The volume ratio of the temporomandibular joint disc to the buffer solution in step (1) is 1:5-10, and the temperature during the cleaning process is 3-5°C.

3. The method for isolating and culturing temporomandibular joint disc stem cells according to claim 1 or 2, characterized in that: The volume ratio of the articular disc sample to the buffer solution in step (2) is 1:2-4.

4. The method for isolating and culturing temporomandibular joint stem cells according to claim 3, wherein: The buffer solutions in step (1) and step (2) are independently based on PBS and contain 1-3% FBS and 0.8-1.2% penicillin-streptomycin.

5. The method for isolating and culturing temporomandibular joint stem cells according to claim 4, characterized in that: The working conditions of the enzymatic digestion in step (3) are 35-40° C. and 80-120 rpm horizontal rotation oscillation. The pronase storage solution uses ddH2O as a solvent and contains 18-22 mg / ml of pronase. The collagenase P storage solution uses PBS as a solvent and contains 18-22 mg / ml of collagenase P. The centrifugal speed is 250×g-350×g, and the centrifugal time is 4-6 min.

6. The method for isolating and culturing temporomandibular joint stem cells according to claim 5, characterized in that: The pore size of the filtration in step (4) is 35-45 μm.

7. The method for isolating and culturing temporomandibular joint stem cells according to claim 6, characterized in that: The number of cells required for sorting in step (5) is ≤ 1 × 10 7 The centrifugal speed is 250×g~350×g, the centrifugal time is 4~6 min, the magnetic bead sorting buffer is based on PBS, contains 0.4~0.6% BSA, 1~3mM EDTA and 0.8~1.2% penicillin-streptomycin, the light-proof incubation temperature is 3~5°C, and the light-proof incubation time is 8~12 min.

8. The method for isolating and culturing temporomandibular joint stem cells according to claim 7, characterized in that: The density of the articular disc stem cell seed plate in step (6) is 2~4×10 4 pieces / cm 2 The amount of complete cell culture medium added is 1.5~2ml / cm 2 The culture conditions are 35-40° C. and 4-6% CO 2 .

9. The method for isolating and culturing temporomandibular joint disc stem cells according to claim 8, characterized in that: The complete cell culture medium in step (4) and step (6) is based on α-MEM, which contains 18-22% FBS and 0.8-1.2% penicillin-streptomycin.

10. The method for isolating and culturing temporomandibular joint stem cells according to claim 9, characterized in that: The seed plate density during the subculture process in step (7) is 1.5~2×10 4 pieces / cm 2 The conditions for the subculture are 35-40°C and 4-6% CO2.

Images