Method for preparing shaped cartilage tissue specimen

Abstract

To provide a method for obtaining a cartilage body in any shape.SOLUTION: A method for preparing a shaped cartilage body includes inoculating cartilaginous progenitors in a cell culture space formed by a frame provided on a substrate, culturing them to make a cell aggregate, and further culturing it in a cartilage differentiation induced medium.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a shaped cartilage tissue structure. As used herein, "chondroprogenitor cells" is used to mean chondroprogenitor cells or a population of chondroprogenitor cells, and "LBM" is used to mean limb bud mesenchymal cells or a population of limb bud mesenchymal cells. [Background technology]

[0002] Foreign materials such as silicone and autologous costal cartilage are used to treat malignant tumors on the face, trauma from traffic accidents, cleft lip and palate accompanied by nasal deformity, microtia, etc. The implantation of foreign materials such as silicone carries the risk of infection and inflammation, and while the use of autologous costal cartilage reduces the risk of infection and inflammation, it is difficult to obtain a sufficient amount of cartilage. Patent Document 1 discloses a method for obtaining cartilage from chondroprogenitor cells. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2021 / 054449 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a method for obtaining a cartilage tissue mass in any desired shape. [Means for solving the problem]

[0005] The present invention provides the following method for preparing a shaped cartilage tissue structure. [1] A method for preparing a shaped cartilage tissue body, comprising seeding chondroprogenitor cells into a cell culture space formed by a formwork provided on a substrate, culturing them to produce a cell aggregate, and further culturing them in a cartilage differentiation induction medium. [2] The seeding density of chondroprogenitor cells was 1.4 × 10 6 ~2.8×10 6 cells / cm 2 The method for preparing a shaped cartilage tissue structure according to [1], [3] A method for preparing a shaped cartilage tissue structure according to [1] or [2], wherein the substrate portion in the cell culture space is coated with a material that promotes cell adhesion, and then chondroprogenitor cells are cultured. [4] A method for preparing a shaped cartilage tissue according to any one of [1] to [3], which comprises culturing chondroprogenitor cells in a cartilage differentiation-inducing medium to produce aggregates, and then stacking and further culturing multiple aggregates to obtain a cartilage tissue. [5] A method for preparing a shaped cartilage tissue specimen according to any one of [1] to [4], in which the chondrocyte precursor cells are cultured at a reduced BMP4 concentration for a certain period at the end of the culture period, thereby suppressing ossification when transplanted into a living body. [6] A method for preparing a shaped cartilage tissue according to any one of [1] to [5], which comprises culturing chondroprogenitor cells in a cartilage differentiation-inducing medium, extracting the resulting shaped cartilage tissue, and treating it with glutaraldehyde and / or ethanol. [7] A shaped cartilage tissue derived from human iPS cells that undergoes a volume change of 25% or less when transplanted into a mammalian body. [Effects of the Invention]

[0006] According to the present invention, nasal cartilage, auricular cartilage, etc. can be prepared in any shape. Unlike foreign substances such as silicone, the shaped cartilage tissue obtained by the present invention can reduce problems such as infection and inflammation. The cartilage obtained by the present invention can be used to repair cartilage deformation or defect, bone or soft tissue defect, etc., and can also be used in cosmetic surgery, including cartilage deformation, defect, or injury due to trauma, combat, fighting, sports, exercise, etc., osteoarthritis, rheumatoid arthritis, osteochondritis dissecans, achondroplasia, relapsing polychondritis, articular cartilage damage, intervertebral disc herniation, ear dysplasia, cartilage defects in anotia and microtia, nasal dysplasia, eyelid defects, and tissue defects following malignant tumor resection. [Brief explanation of the drawings]

[0007] [Figure 1] Examination of self-aggregation conditions [Figure 2] Examination of self-aggregation conditions [Figure 3] Development of small ring-shaped cartilage tissue structures [Figure 4] Development of small ring-shaped cartilage tissue structures (using FBS-containing medium) [Figure 5] Consideration of differentiation protocol (first 3 weeks of STEP 3) [Figure 6] Consideration of differentiation protocol (the last 3 weeks of STEP 3) [Figure 7] Differentiation protocol considerations [Figure 8] Application of ring-shaped cartilage tissue structures (schematic diagram) [Figure 9] Application of ring-shaped cartilage tissue (actual example) [Figure 10] Example of large ring-shaped cartilage [Figure 11] Various culture protocols and post-transplant changes [Figure 12] Subcutaneous implantation of glutaraldehyde-treated cultured cartilage [Figure 13] Example of ear frame formation using glutaraldehyde-cultured cartilage DETAILED DESCRIPTION OF THE INVENTION

[0008] The substrate used in the present invention is provided with a mold, and a cell culture space is formed by the mold and the substrate. The mold is preferably detachable. The mold is preferably made of a biocompatible material such as silicone, or its surface is preferably coated with a biocompatible material. The cell culture space may have any shape, such as a rod, ring (doughnut), cylinder, ellipsoid, sheet, disk, or plate. The width of the coated area of ​​the cell culture space is preferably approximately 3 to 15 mm, more preferably approximately 5 to 10 mm. The size of the cell culture space affects the formation of aggregates of chondroprogenitor cells. The cell culture space may be shaped to suit the transplant site of cartilage, such as nasal cartilage, auricular cartilage, or articular cartilage. Alternatively, the shaped cartilage tissue may be formed by the present invention and then shaped into the desired shape.

[0009] Culturing chondroprogenitor cells in a cell culture space results in the formation of aggregates of chondroprogenitor cells. When these aggregates are removed and multiple aggregates are stacked, they can easily combine to form a single larger aggregate. By further culturing these stacked aggregates and inducing them into cartilage, larger, more shaped cartilage tissue structures can be obtained.

[0010] The surface of the substrate exposed to the cell culture space may be coated with a material that promotes cell adhesion, after which chondroprogenitor cells may be cultured. The exposed "surface" of the substrate refers to the surface that comes into contact with chondroprogenitor cells or culture medium, etc., and particularly when the substrate is part of a cell culture vessel, refers to the bottom surface that comes into contact with chondroprogenitor cells or culture medium, etc.

[0011] In the preparation method of the present invention, chondroprogenitor cells are seeded into a cell culture space. Examples of chondroprogenitor cells include cells derived from mammals such as humans, mice, rats, rabbits, goats, guinea pigs, dogs, cats, monkeys, and chimpanzees, with human chondroprogenitor cells being preferred. The chondroprogenitor cells are preferably human chondroprogenitor cells that are positive for PRRX1 protein and derived from pluripotent stem cells. A method for preparing PRRX1 protein-positive human chondroprogenitor cells derived from pluripotent stem cells is disclosed in detail in WO2021 / 054449, and the entirety of WO2021 / 054449 is incorporated herein by reference.

[0012] "Pluripotent stem cells" refer to cells that have both the ability to self-renew and the ability to differentiate into cells of multiple lineages (multipotency), and "progenitor cells" refer to cells that are in the process of differentiating from stem cells into functional cells, which are their final differentiation destination. Therefore, human chondroprogenitor cells derived from pluripotent stem cells refer to cells that have been induced to differentiate from pluripotent stem cells as source cells and are in the process of differentiating into chondrocytes, which are their final differentiation destination. Examples of pluripotent stem cells include multipotent cells (cells that have the ability to differentiate into all somatic cells and germline cells), such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

[0013] Furthermore, in a preferred embodiment of the present invention, the "chondroprogenitor cells" are positive for the PRRX1 protein. The PRRX1 (Paired related homeobox 1) protein is a transcription factor with a homeodomain, and is known to be specifically expressed in limb buds derived from the lateral plate mesoderm and in head mesoderm derived from the paraxial mesoderm during development.

[0014] The cDNA nucleotide sequence of the human (Homo sapiens) PRRX1 gene and the amino acid sequence of the PRRX1 protein have been registered in GenBank, provided by the National Center for Biotechnology Information (NCBI), under the following accession numbers. Note that if multiple revisions are registered, the most recent revision is understood to be the one being referred to. - Human PRRX1 gene: NM_006902 (NM_006902.5), NM_022716 (NM_022716.4) - Human PRRX1 protein: NP_008833 (NP_008833.1), NP_073207 (NP_073207.1)

[0015] Whether a cell is positive for the PRRX1 protein can be detected by known techniques, such as detection using a reporter gene whose expression is controlled by the transcription promoter sequence of the PRRX1 gene, or detection by immunostaining using an antibody specific to the PRRX1 protein.

[0016] PRRX1 protein-positive chondroprogenitor cells can be produced, for example, by a method comprising the following steps: - inducing differentiation of mammalian pluripotent stem cells into lateral plate mesoderm cells; - culturing the cells induced to differentiate by the above step under an environment that activates Wnt signaling to prepare limb bud mesenchymal cells; - A step of culturing the limb bud mesenchymal cells prepared in the above step in an environment that activates Wnt signaling to prepare chondrocyte precursor cells.

[0017] In the above-mentioned method, pluripotent stem cells used as starting cells can be, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. ES cells and iPS cells can be newly prepared or already established.

[0018] In the above-described method, first, pluripotent stem cells are induced to differentiate into lateral plate mesoderm cells. Known methods can be used to induce differentiation of pluripotent stem cells into lateral plate mesoderm cells. For example, a method similar to the method described in the literature (Loh et al., 2016, Cell, 451-467) can be employed. Specifically, pluripotent stem cells are first induced to differentiate into a primitive streak (mid-primitive streak), and then the primitive streak is induced to differentiate into lateral plate mesoderm cells.

[0019] The induction of differentiation of lateral plate mesoderm cells can be confirmed, for example, by detecting the expression of HAND1 protein, a specific marker for lateral plate mesoderm cells. The differentiation-induced lateral plate mesoderm cells are then cultured in an environment that activates Wnt signaling, and are induced to differentiate into lateral plate mesoderm-derived PRRX1-positive cells that are positive for PRRX1 protein. To reduce the influence of the previous culture environment, it is preferable to wash the cells appropriately with PBS buffer or the like before culturing them in an environment that activates Wnt signaling.

[0020] The differentiation induction of lateral plate mesoderm cells into limb bud mesenchymal cells is also preferably carried out in the absence of an FGF signal activator such as FGF2, more preferably in an environment that activates Wnt signaling and in the presence of one, two, or three inhibitors selected from the group consisting of a TGFβ signal inhibitor, a BMP signal inhibitor, and a hedgehog signal inhibitor, and even more preferably in the presence of a TGFβ signal inhibitor, a BMP signal inhibitor, a TGFβ signal inhibitor, and a hedgehog signal inhibitor.

[0021] Culturing under an environment that activates Wnt signaling can be achieved, for example, by culturing in the presence of an effective amount of a Wnt signaling activator. Wnt signaling activators enhance signal transduction mediated by Wnt (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), XAV939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), and LiCl. When CHIR99021 is used as the Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

[0022] BMP signal inhibitors suppress (inhibit) signal transduction mediated by BMP. Examples of BMP signal inhibitors include LDN193189 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline) or a salt thereof (e.g., hydrochloride), DMH-1, etc., and the amount of the inhibitor added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM. TGFβ signal inhibitors suppress (inhibit) signal transduction mediated by TGFβ. Examples of TGFβ signal activators include A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) and SB431542, and the amount of these activators added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM. Hedgehog signal inhibitors suppress (inhibit) signal transduction mediated by hedgehog. Examples of hedgehog signal activators include vismordegib, cyclopamine, and sonidegib, and the amount of each agent added can be, for example, about 10 nM to 1 μM, preferably about 50 nM to 500 nM.

[0023] The culturing method can be exemplified by, but not limited to, culturing at about 37°C and with a carbon dioxide concentration of about 5%. Culturing under the above conditions can be performed, for example, by controlling the temperature and CO2 concentration using a known CO2 incubator. The culture can be performed by two-dimensional cell culture (plate culture). Two-dimensional cell culture can be performed by coating the culture equipment as needed to promote cell adhesion.

[0024] The period of culturing under an environment that activates Wnt signaling is not particularly limited, but can be, for example, about 6 hours to 4 days, preferably about 1 to 3 days, and more preferably about 2 days (about 48 hours). The medium can be changed as necessary. Culture conditions are preferably in accordance with conventional methods. During culture, passage can be performed as necessary. When passage is performed, the cells are collected before or immediately after reaching a confluent state and seeded in a new medium. The medium can also be replaced as appropriate.

[0025] It is preferable to use a serum-free medium such as IMDM medium, F12 medium, or a mixture thereof. Differentiation induction and maintenance culture can be performed without containing animal-derived components. The serum-free medium can also be supplemented with various medium additives, such as antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), ROCK inhibitors (e.g., Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl)), hormones such as insulin, proteins such as transferrin and albumin, lipids, polyvinyl alcohol, and monothioglycerol.

[0026] The prepared limb bud mesenchymal stem cells are then cultured under an environment that activates Wnt signaling, and induced to differentiate into PRRX1 protein-positive chondroprogenitor cells. The "environment that activates Wnt signaling" has the same meaning as above. Furthermore, it is preferable that the differentiation of limb bud mesenchymal cells into chondroprogenitor cells be further induced in the presence of an FGF signal activator such as FGF2. The chondroprogenitor cells thus obtained can be expanded in the same lot and stored under certain quality control conditions.

[0027] The seeding of chondroprogenitor cells into the cell culture space is carried out by a method known per se, such as suspending the cells in a medium to prepare a cell suspension and adding this to the cell culture space. The seeding density of chondroprogenitor cells is 1.4 × 10 6 ~2.8×10 6 cells / cm 2 is preferred, and 1.8 × 10 6 ~2.2×10 6 cells / cm 2 If the seeding density is too low, the formation of cell aggregates will be slow, and holes may be formed in the cell aggregates, or the strength of the cartilage tissue may be insufficient to maintain its shape. The chondroprogenitor cells seeded in the cell culture space are cultured to obtain a cell aggregate. The culturing method is not particularly limited as long as it is a method capable of producing a cell aggregate from chondroprogenitor cells, and is appropriately selected depending on the properties of the chondroprogenitor cells, etc.

[0028] For example, PRRX1 protein-positive chondroprogenitor cells derived from pluripotent stem cells are preferably cultured in an environment that activates Wnt signaling and / or an environment that suppresses TGFβ signaling. Culturing under an environment that activates Wnt signaling can be achieved, for example, by culturing in the presence of an effective amount of a Wnt signaling activator. Wnt signaling activators enhance signal transduction mediated by Wnt (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), XAV939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), and LiCl. When CHIR99021 is used as the Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably about 1 to 10 μM.

[0029] Culturing under an environment that suppresses TGFβ signaling can be achieved, for example, by culturing in the presence of an effective amount of a TGFβ signaling inhibitor. A TGFβ signaling inhibitor suppresses (inhibits) signal transduction mediated by TGFβ. Examples of TGFβ signaling inhibitors include A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazol-1-carbothioamide) and SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide). When A-83-01 is used as the TGFβ signaling inhibitor, the amount added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM.

[0030] The culturing of chondroprogenitor cells can be performed, for example, at about 37°C and with a carbon dioxide concentration of about 5%. The culturing under the above conditions can be performed, for example, by controlling the temperature and CO2 concentration using a known CO2 incubator. The culturing period is not particularly limited, but can be, for example, about 6 hours to 4 days, preferably about 1 to 3 days, and more preferably about 2 days (about 48 hours). The medium can be changed as necessary. The culturing conditions are preferably in accordance with conventional methods.

[0031] It is preferable to use a serum-free medium such as IMDM medium, F12 medium, or a mixture thereof. Differentiation induction and maintenance culture are possible without containing animal-derived components. Furthermore, various medium additives can be added to the serum-free medium, such as antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), ROCK inhibitors (e.g., Y-27632), cell growth factors such as EGF and FGF, hormones such as insulin, proteins such as transferrin and albumin, lipids, polyvinyl alcohol, and monothioglycerol.

[0032] In this way, a self-aggregated cell aggregate is obtained from human chondroprogenitor cells. The obtained cell aggregate is then induced to differentiate into a cartilage tissue. The production of the cell aggregate and its induction into a cartilage tissue may be carried out successively by changing the medium, or the cell aggregate may be removed from the cell culture space at the cell aggregate stage, and multiple cell aggregates may be used to form the desired shape and then induced to differentiate into a cartilage tissue. In the differentiation induction step, the cell aggregate is cultured to produce a cartilage tissue construct. The culturing method is not particularly limited as long as it is a method that can produce a cartilage tissue from the cell aggregate, and is appropriately selected depending on the properties of the cell aggregate obtained from the human chondroprogenitor cells.

[0033] For example, it is preferable to culture a cell aggregate obtained from PRRX1 protein-positive human chondroprogenitor cells derived from pluripotent stem cells using a method including a step of culturing the cells in an environment that activates Wnt signaling.

[0034] Culturing under an environment that activates Wnt signaling can be achieved, for example, by culturing in the presence of an effective amount of a Wnt signaling activator. Wnt signaling activators enhance signal transduction mediated by Wnt (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), XAV939 (3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), and LiC1. When CHIR99021 is used as the Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

[0035] It is also preferable to culture in multiple stages. (i) culturing in the presence of a Wnt signal activator and an FGF signal activator; (ii) culturing in the presence of an FGF signal activator (and preferably in the absence of a Wnt signal activator); (iii) preferably culturing the cells in the absence of a Wnt signal activator and an FGF signal activator; It is preferable to carry out the above three steps.

[0036] Culturing under an environment that activates FGF signaling can be carried out, for example, by culturing in the presence of an effective amount of an FGF signal activator. FGF signal activators enhance fibroblast growth factor (FGF) signaling. Examples of FGF signal activators include FGF1 / aFGF and FGF2 / bFGF. When FGF2 is used as the FGF signal activator, the amount added can be, for example, about 0.1 to 100 ng / mL, preferably 1 to 50 ng / mL.

[0037] When culturing in the three stages, steps (i) and (ii) can be performed by two-dimensional cell culture (plate culture) or three-dimensional culture. Two-dimensional cell culture can be performed by coating the culture equipment as needed to promote cell adhesion. Step (iii) is preferably performed by three-dimensional culture. Between each stage of culture, it is preferable to appropriately wash the cells with PBS buffer or the like before culturing in order to reduce the influence of the previous culture environment.

[0038] The culture can be carried out in a suitable container for storing the cells and medium. Suitable culture methods include, but are not limited to, culturing at approximately 37°C and a carbon dioxide concentration of approximately 5%. Culture under the above conditions can be carried out, for example, using a known CO2 incubator while controlling the temperature and CO2 concentration.

[0039] The period of culturing under an environment that activates Wnt signaling is not particularly limited, as long as it does not impair the effects of the present invention. For example, it can be about 2 hours to 12 days, about 2 days to 8 days, or about 3 days to 6 days. Furthermore, when culturing is performed in multiple stages, for example, when culturing in the three stages described above, step (i) and step (ii) can be about 2 hours to 12 days, about 2 days to 8 days, or about 3 days to 6 days, respectively, and step (iii) can be about 2 hours to 60 days, about 8 days to 54 days, or about 21 days to 50 days, particularly about 35 days to 45 days. If necessary, the medium can be changed during the culturing period. Culture conditions are preferably in accordance with conventional methods. The culture can be passaged as necessary. When passaged, the cells are harvested before or immediately after reaching a confluent state and seeded in a new medium.

[0040] The medium used in the differentiation induction step is not particularly limited. Serum-free media such as IMDM medium, F12 medium, or a mixture thereof can be used. Furthermore, known cartilage induction media supplemented with L-ascorbic acid, ITS (insulin-transferrin-sodium selenite medium supplement), GDF5, and / or BMP4 can also be used. If necessary, various known medium additives can also be added, such as antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), ROCK inhibitors (e.g., Y-27632), cell growth factors such as EGF and TGFβ, hormones such as insulin, proteins such as transferrin and albumin, lipids, polyvinyl alcohol, and monothioglycerol.

[0041] In a particularly preferred embodiment of the present invention, differentiation of a cell aggregate into a cartilage tissue can be performed, for example, by the following steps 1 to 3. (i) Step 1 The culture period is 3 to 6 days, and the culture temperature is around 37°C. The medium components include CHIR99021, FGF2, ascorbic acid, ITS (insulin, transferrin, selenium), and FBS (fetal bovine serum). The medium is replaced as necessary. In step 1 and process (i), it is preferable to use FBS at a concentration of about 1 to 10% (w / v) in order to maintain the shape of the aggregates.

[0042] (ii) Step 2 The culture period is 3 to 6 days, and the culture temperature is around 37°C. Medium components include BMP4, TGFb1, GDF5, FGF2, ascorbic acid, ITS, and FBS. The medium is replaced as needed. In step 2 and process (ii), using FBS at a concentration of approximately 1 to 10% (w / v) prevents deformation of the cartilage tissue and allows the formation of smooth, uniform cartilage tissue of the desired thickness. Histologically, the production of cartilage matrix, which stains red with safranin O and blue with alcian blue, can be confirmed.

[0043] (iii) Step 3 The culture period is 6 to 8 weeks, and the culture temperature is around 37°C. Medium components include BMP4, TGFb1, GDF5, ascorbic acid, ITS, and FBS. The medium is replaced as needed. In step 3 and step (iii), FBS is preferably used at a concentration of approximately 1 to 10% (w / v) to obtain a cartilage tissue with the desired shape and uniformity and smoothness. A differentiation-inducing medium containing 10% (w / v) FBS is sometimes referred to below as "STEP 3 + 10% FBS." The resulting cartilage tissue stains red with Safranin O and blue with Alcian blue, confirming the production of cartilage matrix.

[0044] The present inventors prepared a medium that did not contain TGFb1 or BMP4 and induced differentiation. The culture period for STEP3 was divided into a first 3-week period and a second 3-week period, and culture was performed using STEP3 + 10% FBS, STEP3 + 10% FBS-TGFb1, or STEP3 + 10% FBS-BMP4. In the first three weeks, when STEP3 + 10% FBS-TGFb1 was used, cartilage tissue expansion occurred, which became more pronounced as the cartilage tissue became larger. On the other hand, no such deformation occurred during the first three weeks when STEP3 + 10% FBS or STEP3 + 10% FBS-BMP4 was used. These findings suggest that TGFb1 is essential for maintaining the ring morphology during the first three weeks. Furthermore, gene expression analysis by qPCR revealed that hypertrophic cartilage markers such as RUNX2, Col10, and IHH were elevated during the first three weeks when STEP3 + 10% FBS-TGFb1 was used, but not when STEP3 + 10% FBS or STEP3 + 10% FBS-BMP4 were used. These findings suggest that BMP4 promotes cartilage hypertrophy, while TGFb1 inhibits it. From the above, a preferred medium for the first three weeks is a medium composed of BMP4, TGFb1, GDF5, ascorbic acid, ITS, and FBS, or a medium composed of TGFb1, GDF5, ascorbic acid, ITS, and FBS.

[0045] On the other hand, in the last three weeks, even when TGFb1 was removed, there was almost no expansion of the cartilage tissue. The thickness of the cartilage tissue increased in the following order: STEP3 + 10% FBS-TGFb1, STEP3 + 10% FBS, and STEP3 + 10% FBS-BMP4. Here again, the results suggest that BMP4 promotes cartilage hypertrophy, while TGFb1 inhibits it. Based on the above, preferred culture media for the last three weeks are a medium composed of BMP4, GDF5, ascorbic acid, ITS, and FBS, a medium composed of BMP4, TGFb1, GDF5, ascorbic acid, ITS, and FBS, or a medium composed of TGFb1, GDF5, ascorbic acid, ITS, and FBS. The volume change rate of the shaped cartilage tissue when transplanted into the living body of a mammal, including a human, is preferably 25% or less, more preferably 20% or less, even more preferably 15% or less, and particularly preferably 10% or less.

[0046] The shape of the substrate surface may be flat or uneven, but a flat shape is preferred. The substrate may also be a so-called cell culture vessel or a part thereof. Examples of cell culture vessels include Petri dishes or dishes such as tissue culture dishes and multi-dishes commonly used for cell culture, flasks such as cell culture flasks and spinner flasks, bags such as plastic bags, Teflon (registered trademark) bags and culture bags, plates such as microplates, microwell plates, multi-plates and multi-well plates, chamber slides, tubes, trays, bottles such as roller bottles, and the like.

[0047] Examples of materials for the substrate include glass, metals, metal-containing compounds or metalloid-containing compounds, activated carbon, and resins. Examples of metals include typical metals (aluminum group elements: Al, Ga, In; iron group elements: Fe, Co, Ni; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; and noble metals: Cu, Ag, Au). Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds such as metal oxides or metalloid oxides (silicon oxide, alumina, etc.); metal carbides or metalloid carbides; metal nitrides or metalloid nitrides (silicon nitride, etc.); and metal borides or metalloid borides; aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0048] The resin constituting the substrate may be either a natural resin or its derivative, or a synthetic resin. Examples of natural resins or their derivatives include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose immobilized with dextran sulfate. Examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), and polyvinyl alcohol (PVA). ), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), or Teflon (registered trademark) is preferably used. The substrate for producing a shaped cartilage tissue used in the present invention does not require high-temperature treatment, and therefore resins with low heat resistance can be used. The substrate material may be one type or a combination of two or more types.

[0049] The substrate may have a surface that suppresses cell adhesion. In this case, the substrate surface exposed to the cell culture space is preferably coated with a material that promotes cell adhesion. Examples of such coating materials include those represented by the following formula (I): [ka] [In the formula, U a1 and U a2 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms], and a repeating unit derived from a monomer represented by the following formula (II): [ka]

[0050] [In the formula, R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. The molar ratio of the repeating units derived from the monomer represented by formula (I) to the total of the repeating units derived from the monomer represented by formula (I) and the repeating units derived from the monomer represented by formula (II) is preferably 99 mol % to 51 mol %. The copolymer may further comprise a compound represented by the following formula (III): [ka]

[0051] [In the formula, R c and R d each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R e represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.

[0052] In this specification, the above-described coating of the substrate may be referred to as CAT (Cell self-aggregation technology) coating. The substrate of the present invention comprises a copolymer (P) containing a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [ka]

[0053] [In the formula, U a11 , U a12 , U b11 , U b12 and U b13 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. It is preferable that the coating film contains the following on at least a part of the surface.

[0054] The substrate coating can be formed by applying the copolymer, preferably a primer containing the copolymer. The primer can be prepared by mixing the copolymer with a water-containing solution by a method known per se. Such a primer is useful for promoting cell aggregate formation.

[0055] The aqueous solution may be water, a salt-containing aqueous solution such as physiological saline or phosphate buffer solution, or a mixed solvent of water or a salt-containing aqueous solution with an alcohol. Examples of the alcohol include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, and the like. Examples of suitable solvents include 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These solvents may be used alone or in combination.

[0056] In addition to the copolymer and solvent, other substances may be added to the primer as needed, provided that they do not impair the performance of the resulting primer film, such as pH adjusters, crosslinkers, preservatives, surfactants, primers that improve adhesion to the container or substrate, antifungal agents, and sugars. The solution can be applied to a substrate to form a coating. The concentration of the copolymer in the solution is preferably 0.1 to 200 μg / ml, more preferably 1 to 100 μg / ml, and even more preferably 6.25 to 50 μg / ml. Within this concentration range, good cell aggregates and cartilage tissues can be formed.

[0057] Furthermore, the coating on the surface of the substrate obtained by such a method can be used in the manufacturing method of the present invention either as is without a drying step or after washing with water or the medium of the sample to be subjected to cell culture (e.g., water, buffer solution, culture medium, etc.). After coating, the substrate may be subjected to a drying process. The drying process is carried out in air or under vacuum, preferably at a temperature in the range of −200° C. to 200° C. The drying process removes the solvent, thereby completely adhering the coating to the substrate.

[0058] The coating can be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, e.g., 25°C), but to form spots more quickly, drying at, for example, 40°C to 50°C may be used. If the drying temperature is below -200°C, an uncommon refrigerant must be used, making it less versatile, and drying takes a long time due to solvent sublimation, making it inefficient. If the drying temperature is above 200°C, thermal decomposition of the copolymer occurs. A more preferred drying temperature is 10°C to 180°C, and even more preferred is 20°C to 150°C. The maximum and minimum thicknesses of the coating are in the range of 1 to 1000 nm, preferably 5 to 500 nm.

[0059] In one preferred embodiment, the substrate used in the present invention is manufactured using a substrate having cell adhesion-inhibiting properties. Such substrates may include commercially available cell culture dishes that have been treated to reduce cell adhesion, cell culture vessels that have cell adhesion-inhibiting properties, and the like, including, but not limited to, the cell culture vessels described in JP 2008-61609 A. Whether or not a cartilage tissue prepared according to the present invention has been obtained can be confirmed by, for example, positive staining with safranin O staining, alcian blue staining, safranin O staining, or toluidine blue staining.

[0060] <Use of cartilage tissue> The cartilage tissue obtained by the production method of the present invention can be used for cartilage transplantation in plastic surgery when deformation or defect of cartilage tissue in the nose or ear occurs congenitally or due to trauma, surgery, etc. Furthermore, since cartilage tissue of a desired shape can be obtained, it can be transplanted into damaged or defective cartilage sites in joints, etc. [Example]

[0061] The present invention will be described in more detail below with reference to examples. Example 1 A method for obtaining a self-aggregated aggregate of chondroprogenitor cells in the preparation method of the present invention is schematically shown in Figure 1. In Figure 1, ExpLBM refers to human chondroprogenitor cells induced from human iPS cells as described in the Examples of WO2021 / 054449. First, we investigated the optimum conditions for cell seeding density and coating concentration for self-aggregation. The substrate used was a culture dish (diameter: 35 mm) with cell adhesion inhibitory properties (Sumitomo Bakelite Co., Ltd., MS9035X), and the CAT coating in Figure 1 was an aqueous solution of a copolymer of 2-(N,N-dimethylamino)ethyl methacrylate and methacrylic acid (hereinafter referred to as "CAT polymer") (the molar ratio of 2-(N,N-dimethylaminoethyl) methacrylate to all monomers was 90%).

[0062] First, as a basic study, we aimed to induce ring-shaped aggregation of ExpLBM seeded on a culture surface equipped with a silicone mold and supports by coating it with a CAT polymer. The details of the method are as follows. A circular silicone sheet was attached to a low-adhesion culture dish, and a silicone support was placed in the center. CAT polymer solution of various concentrations (6.25–100 μg / ml) was added to the donut-shaped area (cell culture space) surrounded by the silicone. After leaving it for 5 minutes, the polymer solution was completely aspirated to create a coated area (Figure 1). ExpLBM was applied to this coated area at various densities (17.5–280 × 10 4 cells / cm2 The cells were seeded in STEP1 medium, which is used to induce chondrogenic differentiation of ExpLBM. The results are shown in Figure 2.

[0063] STEP 1 medium: CDM2 basal medium + 3 μM CHIR99021 + 10 ng / mL FGF2 + 50 μg / mL ascorbic acid + 1×ITS When the seeding density of ExpLBM was low, aggregation itself was slow, and holes were formed in the aggregates and the rings were torn off. 4 cells / cm 2 It was found that the smoothest and cleanest aggregation occurred when the concentration of CAT polymer was 100 μg / ml. Furthermore, it was found that aggregation was inhibited at concentrations of 6.25 to 50 μg / ml, although this depended on the seeding density. Good aggregation was confirmed at concentrations of 6.25 to 50 μg / ml. A high seeding density was also required to achieve clean, band-like aggregation. From the above results, the CAT concentration was 12 μg / ml, and the seeding density was 200 × 10 4 cells / cm 2 The following experiments were carried out under the optimum conditions.

[0064] Next, we investigated whether the ring-shaped aggregates could be differentiated into cartilage. The differentiation protocol used the media in STEP 1 above and STEPs 2 to 3 below. STEP2 medium: CDM2 basal medium + 10ng / mL FGF2 + 50μg / mL ascorbic acid + 30ng / mL BMP4 + 10ng / mL TGFβ1 + 10ng / mL GDF5 + 1×ITS STEP 3 medium: CDM2 basal medium + 50 μg / mL ascorbic acid + 30 ng / mL BMP4 + 10 ng / mL TGFβ1 + 10 ng / mL GDF5 + 1×ITS The culture period for each step is as follows: STEP 1: 3-6 days, STEP 2: 3-6 days, STEP 3: 42 days or more (6 weeks or more). STEP 3 medium is particularly effective in promoting cartilage matrix production, and it takes 6 weeks or more to produce mature cultured cartilage.

[0065] When cultured in conventional STEP 1-3 medium, the ring-shaped aggregates expanded in diameter and grew into irregular cartilage with protrusions on the edges (Figure 3). Histologically, the production of cartilage matrix was confirmed, staining red with Safranin O and blue with Alcian blue. Next, chondrogenic differentiation of the ring-shaped aggregates was induced using STEP3 medium supplemented with various concentrations of FBS (0, 0.1, 1, 10%). When STEP 3 + 10% FBS was used, no deformation of the ring was observed, and smooth, uniform ring-shaped cartilage with a thickness of 1-2 mm was formed. Histologically, the production of cartilage matrix was confirmed, staining red with Safranin O and blue with Alcian blue (Figure 4). We also investigated the addition of FBS in STEP 1 and STEP 2, and found that adding 10% FBS in STEP 1 made aggregation smoother and more uniform, while adding a low concentration of FBS in STEP 2 made it easier to maintain the shape of the aggregates (data not shown).In subsequent experiments, 10% FBS was added to STEP 1 medium, 1% to STEP 2 medium, and 10% to STEP 3 medium.

[0066] Next, we investigated the culture conditions for inducing more mature cartilage. While the conventional STEP3 medium contains the growth factors TGFb1, BMP4, and GDF5, we created a medium that did not contain TGFb1 or BMP4 and used it for differentiation induction. The STEP3 culture period was divided into two periods: the first 3 weeks and the second 3 weeks, and cultures were performed using STEP3 + 10% FBS, STEP3 + 10% FBS-TGFb1, and STEP3 + 10% FBS-BMP4 for each period. During the first 3 weeks, when STEP3 + 10% FBS-TGFb1 was used, the ring diameter increased, which was more pronounced when creating larger rings, making it difficult to control the shape of the rings themselves (Figure 5).

[0067] On the other hand, when STEP3 + 10% FBS and STEP3 + 10% FBS-BMP4 were used, such deformation did not occur (Figure 5). From the above, it is thought that TGFb1 is essential for maintaining the ring morphology during the first three weeks. Furthermore, gene expression analysis by qPCR showed that when STEP3 + 10% FBS-TGFb1 was used for the first three weeks, hypertrophic cartilage markers such as RUNX2, Col10, and IHH were elevated, but this did not occur when STEP3 + 10% FBS or STEP3 + 10% FBS-BMP4 were used. This suggests that BMP4 promotes cartilage hypertrophy, while TGFb1 inhibits it. On the other hand, in the last three weeks, even when TGFb1 was omitted, there was almost no increase in ring diameter. The cartilage thickness increased in the following order: STEP3 + 10% FBS-TGFb1, STEP3 + 10% FBS, and STEP3 + 10% FBS-BMP4. Here again, the results suggest that BMP4 promotes cartilage hypertrophy, while TGFb1 inhibits it (Figure 6).

[0068] Based on the above results, it was found that cartilage that maintained its shape and grew thicker could be produced by using STEP3 + 10% FBS or STEP3 + 10% FBS-BMP4 for the first three weeks, and STEP3 + 10% FBS-TGFb1 for the second half (Figure 7). Of these, the combination of STEP3 + 10% FBS-BMP4 → STEP3 + 10% FBS-TGFb1 produced particularly thick cartilage, and the results showed that it was possible to create mature cartilage both histologically (Safranin O, Alcian blue) and in terms of gene expression (Col2) (Figure 7).

[0069] As mentioned above, we developed a method for producing mature small cartilage rings, and then applied this method to produce giant cartilage rings (Figure 8). The silicone frame and support were changed to larger ones, and CAT coating, seeding of ExpLBM, and induction of cartilage differentiation were performed using the same procedures as described above. Furthermore, ring-shaped aggregates of ExpLBM prepared using the same procedure as above were detached from the supports at the appropriate time, placed on top of a silicone tube, and pierced with a silicone tube while differentiation was induced, producing tubular cartilage with fused rings. The results are shown in Figure 9. By using struts with inner diameters of 14 mm and 20 mm, it was possible to produce ring-shaped cartilage with a uniform thickness of 1 to 2 mm, as shown in Figure 9. By increasing the size of the strut, it is possible to produce ring-shaped cartilage of any size.

[0070] In the production of tubular cartilage, six ring-shaped aggregates were collected on the 9th day after the start of aggregation, passed through a silicone tube, and continued to be cultured. As a result, all of the aggregates fused together to form a tubular cartilage approximately 5 mm long and 1 mm thick in wall. The right side of Figure 9(b) shows a cut fragment of the tubular cartilage. In both types of cartilage, the production of cartilage matrix was confirmed by staining with Safranin O and Alcian blue. By using an elliptical silicone frame and support, it is possible to create an elliptical ring cartilage using the same procedure. Figure 10 (top) shows an elliptical cartilage with a diameter of 2 mm, a major axis of 30 mm, and a minor axis of 15 mm.

[0071] It is also possible to create straight cartilage by using a track-shaped (with straight sections) silicone frame and support (Figure 10 (bottom)). By overlapping these linear cartilages, it is also possible to create plate-shaped cartilage whose length and width can be adjusted. It has been confirmed that subcutaneous transplantation of cultured cartilage leads to greater maturation of the cartilage matrix and increased strength, but there are concerns about the risk of hypertrophy and ossification. To prevent this, the culture period in STEP 3 of the differentiation protocol consisting of STEPs 1 to 3 was divided into three periods: 0 to 3 weeks, 3 to 9 weeks, and 9 to 15 weeks, and culture was performed using the differentiation medium shown in Figure 11. 1.0 x 10 cells in a 96-well U-bottom culture plate (low cell adhesion). 5 Cells were seeded at 1000 cells / well and centrifuged at 2000 rpm for 5 minutes to prepare a cell pellet, which was then induced to differentiate using the media used in Steps 1 to 3. From 0 to 3 weeks of STEP 3, STEP 3 + 10% FBS was used, from 3 to 9 weeks, STEP 3 + 10% FBS-TGFb1, STEP 3 + 10% FBS-BMP4, STEP 3 + 10% FBS, and STEP 3 + 10% FBS (containing 10 times the amount of TGFb1 and BMP4) were used, and from 9 to 15 weeks, DMEM (HG) + 10% FBS was used.

[0072] The chondrocytes prepared using these protocols were transplanted subcutaneously into the backs of immunodeficient mice (NOD scid), and changes in morphology and volume after transplantation were observed using MRI T2 fat-suppressed images. In MRI T2 fat-suppressed images, the cultured cartilage was visualized as a high-signal area, but after transplantation, the area where ossification was observed changed to a low-signal area. Based on this change in signal, the volumes of the cartilage and ossification areas were calculated.

[0073] In the STEP 3 + 10% FBS-TGFb1 treated sample (No. 9) from weeks 3 to 9 of STEP 3, the tissue was absorbed after implantation and its volume shrank to less than 10%, but the other samples maintained their volume up to 13 weeks after implantation without hypertrophy or absorption. Furthermore, ossification tended to progress depending on the BMP4 content: in No. 14 (containing 10 times the amount of BMP4), almost all tissue ossified, while in No. 13 (containing the normal amount of BMP4), only 30% ossified, and in No. 10 (containing no BMP4), no ossification was observed. These results suggest that culturing the cells in DMEM(HG) + 10% FBS at the end of the culture period can prevent hypertrophy after transplantation, and that reducing the BMP4 content can prevent or reduce ossification (Figure 11).

[0074] By inactivating the cells contained in the cultured cartilage and using it as an extracellular matrix product, it is possible to eliminate the risks of shape change and ossification after transplantation, and also to eliminate the tumorigenicity that is a concern specific to iPS-derived products, making it possible to produce a product that is safer and more likely to gain social consensus. To achieve this, the inventors inactivated the cells in the cartilage rings prepared using the method described above by fixation with glutaraldehyde. Specifically, the cultured cartilage was immersed in 2% Sterihyde® for one day, washed with PBS, and then implanted subcutaneously into the dorsal skin of immunodeficient mice (NOD scid). Changes in volume and shape after implantation were evaluated using MRI T2 fat-suppressed images. No changes such as resorption or hypertrophy were observed up to 12 weeks after implantation. Furthermore, the cartilage removed 12 weeks after implantation maintained the same shape as before implantation. Histologically, staining with Safranin O was slightly impaired, but no gross structural changes were observed. Kossa staining revealed diffuse punctate calcification (Figure 12).

[0075] From the above, it was suggested that although there is a risk of calcification when cultured cartilage is treated with glutaraldehyde, it may be possible to prevent changes such as absorption, hypertrophy, and ossification, and allow the cartilage to remain subcutaneously while maintaining its original shape. Furthermore, by adding high-concentration ethanol treatment after glutaraldehyde treatment, aldehyde residues and phospholipids can be removed, which is expected to prevent calcification. Specifically, rat costal cartilage was immersed in 0.6% glutaraldehyde for one day, then in 0.2% glutaraldehyde for over a week, followed by shaking in high-concentration ethanol for one day and then soaking in saline for one day to remove the ethanol. Two months after subcutaneous transplantation into mice, rat costal cartilage treated in this manner showed no calcification (data not shown).

[0076] It is expected that glutaraldehyde treatment will cross-link collagen fibers and increase the strength of the cultured cartilage, but we investigated whether it could withstand processing such as suturing and fixation. A large ring-shaped cultured cartilage was prepared, fitted into a silicone mold, and treated with glutaraldehyde and high-concentration ethanol. Specifically, it was immersed in 0.6% glutaraldehyde for one day, then in 0.2% glutaraldehyde for over a week, then shaken in high-concentration ethanol for one day, and then soaked in saline for one day to remove the ethanol. After fixation, the cultured cartilage was fixed in the mold and could be easily fixed to a silicone plate using 5-0 nylon (Figure 13). From the above, it was thought that glutaraldehyde treatment contributes to increasing the strength of cultured cartilage by cross-linking collagen, and makes processing such as suturing easier.

Claims

1. A method for preparing a shaped cartilage tissue body, comprising seeding chondrocyte precursor cells into a cell culture space formed by a formwork provided on a substrate, culturing them to produce cell aggregates, and further culturing them in a cartilage differentiation induction medium, wherein the chondrocyte precursor cells are cultured with a reduced BMP4 concentration for a certain period at the end of the culture period, thereby suppressing ossification when transplanted into a living body.

2. A method for preparing a shaped cartilage tissue as described in claim 1, comprising seeding chondrocyte precursor cells into a cell culture space formed by a formwork provided on a substrate, culturing them to produce cell aggregates, and further culturing them in a cartilage differentiation induction medium, and extracting the shaped cartilage tissue obtained by culturing the chondrocyte precursor cells in the cartilage differentiation induction medium, and treating it with glutaraldehyde and / or ethanol.

3. The seeding density of chondroprogenitor cells was 1.4 × 10 6 ~2.8×10 6 cells / cm ２ 3. The method for preparing a shaped cartilage tissue structure according to claim 1 or 2, wherein the shaped cartilage tissue structure is

4. A method for preparing a shaped cartilage tissue construct according to any one of claims 1 to 3, wherein the substrate portion in the cell culture space is coated with a material that promotes cell adhesion, and then chondroprogenitor cells are cultured.

5. The method for preparing a shaped cartilage tissue according to any one of claims 1 to 4, wherein chondroprogenitor cells are cultured in a cartilage differentiation-inducing medium to produce aggregates, and a plurality of aggregates are superimposed and further cultured to obtain a cartilage tissue.

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