A method for creating moldable cartilage tissue without the need for a scaffold.

Abstract

Developed is a method for creating cartilage tissue to solve the following problems. 1. Cartilage differentiation is not hindered by forming. 2. The cartilage can be formed into an intended shape without using a scaffold.　A method for producing artificial cartilage tissue comprises: forming a spheroid containing cartilage precursor cells into a desired shape while seeding the spheroid onto a support; culturing the spheroid while supplying a culture medium from the front side and the rear side of the surface onto which the spheroid has been seeded, and fusing the spheroids with each other; and maturing the fused spheroid into cartilage tissue in vitro.

Description

[Technical Field]

[0001] This invention relates to a method for creating moldable and scaffold-free cartilage tissue. [Background technology]

[0002] Until now, the curative treatment for chondrodysplasia in the craniofacial region, osteochondritis dissecans in the limbs, and traumatic cartilage injury has been autologous cartilage transplantation. However, this method has limitations in the amount of cartilage that can be harvested and causes postoperative pain at the harvest site. Therefore, cultured cartilage transplantation has been developed, in which a small amount of stem cells are cultured and differentiated before transplantation.

[0003] After the development of cartilage combined with artificial scaffolding materials such as hydrogels, more physiologically natural scaffold-free cartilage that does not require scaffolding materials has been developed (Non-Patent Literature 1). However, because scaffold-free cartilage is cultured in three dimensions after the aggregation of cell aggregates, it has not been possible to create the intended shape. Since it is important that the cartilage tissue conforms to the required shape when transplanted into patients with the target disease, this is one of the problems that must be overcome. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Enomura M, et al., Int J Mol Sci. 2020, 21, 8496 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] To date, no scaffold-free, sculptable cartilage has ever been developed. This invention aims to develop a method for creating cartilage tissue that solves the following problems. 1. Cartilage differentiation is not hindered by shaping. 2. It is possible to create the desired shape without using scaffolding. [Means for solving the problem]

[0006] The inventors considered that, when creating moldable and scaffold-free cartilage tissue from chondrocytes, it would be desirable to aggregate chondrocytes to form small spheroids, and then reassemble them to create the desired shape. However, when forming spheroids by aggregating chondrocytes, if the diameter exceeds 300 μm, oxygen cannot reach the cells, leading to cell death due to nutrient deficiency. Furthermore, spheroids adhered to the bottom surface of a plate may experience nutrient and oxygen depletion at the adhesion surface. To address these issues, chondrocytes were seeded and aggregated on a micropattern plate to create a group of spheroids approximately 200 μm in diameter. The created spheroids were arranged in the desired shape and fused together. Since spheroids are approximately 100 times larger than single cells, they are visible to the naked eye, making it possible to create three-dimensional structures by stacking balls. In this process, to prevent nutrient and oxygen depletion in the spheroids in contact with the bottom surface, the work was performed on a cell culture insert, and the bottom of the insert was filled with culture medium. The cartilage tissue produced by this method allows for omnidirectional exchange of culture medium and gases. When cartilage tissue cultured using this method for 15 to 30 days was transplanted into a living organism, it matured into cartilage. Furthermore, cartilage tissue cultured using this method for 56 to 70 days became hypertrophic cartilage, and when transplanted into a living organism, it became bone tissue. This invention was completed based on these findings.

[0007] The gist of this invention is as follows: (1) A method for producing artificial cartilage tissue, comprising: seeding spheroids containing chondrocyte precursor cells onto a support and shaping them into a desired form; culturing the spheroids while supplying culture medium from the front and back sides of the surface on which the spheroids are seeded to fuse the spheroids together; and maturing the fused spheroids into cartilage tissue in vitro. (2) The method according to (1), wherein the chondrocytes are cells differentiated from embryonic stem cells and / or induced pluripotent stem cells. (3) The method described in (1), wherein the chondrogenic progenitor cells are cells obtained by differentiating chondrocytes collected from the chondrium. (4) The method according to any of (1) to (3), wherein the spheroid containing chondrocytes is 20 to 1000 μm in diameter. (5) The method according to any of (1) to (4), wherein one spheroid contains 100 to 7500 chondrocytes. (6) A spheroid containing chondrocytes is prepared by culturing chondrocytes in a culture substrate having a cell non-adherent surface, according to any of the methods in (1) to (5). (7) The spheroid containing chondrocytes is prepared by culturing chondrocytes in a medium containing TGF-β, bFGF, and a Wnt / β-catenin inhibitor, according to any of the methods in (1) to (6). (8) The method according to any one of (1) to (7), wherein fusion spheroids are cultured in a medium containing BMP to mature into cartilage tissue. (9) The method according to any of (1) to (8), wherein the culture period of the fused spheroid for maturation into cartilage tissue is 14 to 42 days. (10) The method according to any of (1) to (8), wherein the culture period of the fused spheroid for maturation into cartilage tissue is 42 to 84 days. (11) The method according to any one of (1) to (10), which includes transplanting cartilage tissue matured in vitro into a non-human animal and further maturing it. (12) An artificial cartilage tissue prepared by any of the methods described in (1) to (11), wherein the artificial cartilage tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more. (13) An artificial cartilage tissue prepared by the method described in (10), wherein, after being transplanted into a living organism, a part or all of the artificial cartilage tissue differentiates into bone tissue. (14) A composition comprising artificial cartilage tissue prepared by any of the methods described in (1) to (11), wherein the composition is used to be implanted in a living organism to compensate for a deficiency of cartilage tissue and / or bone tissue in the living organism. A composition according to (14), comprising an artificial cartilage tissue produced by the method according to (15)(9), transplanted into a living body, and used to supplement a deficiency of cartilage tissue in the living body. A composition according to (14), comprising an artificial cartilage tissue produced by the method according to (16)(10), transplanted into a living body, and used to supplement a deficiency of bone tissue in the living body. A method for producing an artificial bone tissue, comprising transplanting an artificial cartilage tissue produced by the method according to (17)(10) into a non-human animal and maturing it in bone tissue. An artificial bone tissue produced by the method according to (18)(17), wherein the artificial bone tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more. A composition comprising an artificial bone tissue produced by the method according to (19)(17), transplanted into a living body, and used to supplement a deficiency of bone tissue in the living body. [Effect of the Invention]

[0008] According to the present invention, cartilage tissue of any shape having transplantable strength can be produced in vitro. By transplanting the artificial cartilage tissue produced by the method of the present invention into a living body, it can become mature cartilage or bone tissue. This specification includes the content described in the specification and / or drawings of Japanese Patent Application No. 2021-141210, which is the basis of the priority of the present application. [Brief Description of the Drawings]

[0009] [Figure 1] Shows the gene expression over time of human chondrocyte progenitor cells via human mesoderm cells from human iPS cells. [Figure 2A] Shows the results of spheroid morphology evaluation using Cell3iMager Duos. Bright-field image of chondrocyte progenitor spheroids. [Figure 2B] Shows the results of spheroid morphology evaluation using Cell3iMager Duos. Results of measuring the diameter of each spheroid with Cell3iMager Duos [Figure 2C]The results of spheroid morphology evaluation using Cell3iMager Duos are shown. The circularity of each spheroid was measured using Cell3iMager Duos. [Figure 2D] Histological staining of human cartilage precursor spheroids is shown. [Figure 2E] This shows the marker positivity rate based on histological staining of human cartilage precursor spheroids. [Figure 3] It demonstrates the ability to fuse human cartilage precursor spheroids. [Figure 4] Macroscopic images of shaped cartilage are shown in vitro. [Figure 5A] Histological staining of shaped cartilage in vitro is shown. [Figure 5B] This shows the marker positivity rate in histological staining of reshaped cartilage in vitro. [Figure 6] This shows the in vitro gene expression of shaped cartilage. [Figure 7] This shows an ELISA in vitro of reshaping cartilage. [Figure 8A] Macroscopic view of reshaping cartilage after transplantation. Reshaping cartilage at the time of excision (arrowhead: outline of the cartilage). [Figure 8B] Macroscopic view of reshaping cartilage after transplantation. Reshaping cartilage after excision. [Figure 9] The image shows a CT scan after transplantation of the shaped skeleton. [Figure 10] The histological images obtained by immunohistochemical staining after transplantation of a morphogenetic skeleton are shown. [Figure 11] This image shows a macroscopic in vitro image of shaped cartilage using chondroprogenitor cells derived from auricular perichondrium. [Modes for carrying out the invention]

[0010] The present invention will be described in detail below.

[0011] The present invention provides a method for producing artificial cartilage tissue, which includes shaping spheroids containing chondrocyte precursor cells into a desired shape while seeding them on a support, culturing the spheroids while supplying culture medium from the front and back sides of the seeded surface to fuse the spheroids together, and maturing the fused spheroids into cartilage tissue in vitro.

[0012] Chondroprogenitor cells can be obtained by differentiating embryonic stem cells (ES cells) and / or induced pluripotent stem cells (iPS cells), or by differentiating chondrometrial cells collected from the chondrium.

[0013] Chondroprogenitor cells can be differentiated from embryonic stem cells and / or induced pluripotent stem cells. One example of this method is described below. First, embryonic stem cells and / or induced pluripotent stem cells are differentiated into mesoderm cells using the method described in Cell, July 14, 2016, vol. 166, 451-467. Briefly, a basic culture medium of Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12) supplemented with 1% B27 and 1% Glutamax is used, and different additives such as Activin, bFGF, and Wnt promoters (CHIR and WNT3A) are added daily, with the type of additive changing each day. Mesoderm cells are obtained on the 5th day after the start of differentiation induction. Next, the obtained mesoderm cells are passaged and cultured in a flat surface at 36-37°C in Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12) supplemented with TGFβ inhibitors (A8301 or SB431542), PDGFBB, IGF, etc. The medium is changed every other day, and chondrogenic cells can be obtained in 3-5 days. Chondrogenic cells should preferably express SOX9, CD44, CD73, and CD105. Chondrogenic cells obtained by the above method should be cultured in a flat surface at 36-37°C in Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12) supplemented with TGFβ inhibitors (A8301 or SB431542), PDGFBB, IGF, etc. The medium should be changed 2-4 times a week. Chondroprogenitor cells with 0 to 5 passages are recommended.

[0014] Embryonic stem cells and / or induced pluripotent stem cells should primarily be of human origin, but they may also be derived from animals other than humans (for example, animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0015] Chondroprogenitor cells can be differentiated from perichondrium cells. One example of this method is described below. First, perichondrium present in tissues such as auricular cartilage and costal cartilage is collected using the method described in PNAS, August 20, 2011, vol. 108, no. 35, 12279-14484. Briefly, the perichondrium collected from cartilage tissue such as auricle and costal cartilage is cut, treated with collagenase to separate the perichondrium cells, and recovered by filtration. The perichondrium cells obtained by the above method are cultured in a flat surface at 36-37°C in Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12) supplemented with TGFβ inhibitors (A8301 or SB431542), PDGFBB, IGF, etc., and the medium is changed every other day, and chondroprogenitor cells can be obtained in 3-5 days. Chondroprogenitor cells should preferably express SOX9, CD44, CD73, and CD105. Chondroprogenitor cells obtained by the above method should be cultured in a flat state at 36-37°C in Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12) supplemented with TGFβ inhibitors (A8301 or SB431542), PDGFBB, IGF, etc., with the medium changed 2-4 times per week. Chondroprogenitor cells that have undergone 0-5 passages should be used.

[0016] Chondrocytes should primarily be derived from humans, but they may also be derived from other animals (for example, animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0017] Spheroids containing chondrocytes are best prepared without using a matrix or other cell-adhering surface (matrix-free). For example, they can be prepared by culturing chondrocytes in a culture medium that has a non-cell-adhering surface.

[0018] Culture equipment having a cell-non-adherent surface is preferably treated with a low-adsorption surface treatment, for example, by coating the culture surface with a cell-non-adherent polymer. Examples of cell-non-adherent polymers include phospholipids, phospholipid-polymer complexes, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, and photocrosslinked superhydrophilic polymers. Examples of culture equipment having a cell-non-adherent surface include Elplasia plate (Corning), Elplasia RB 500 400 NA (Kuraray), and 96-well U-bottom plate or V-bottom plate (Sumitomo Bakelite), which can be suitably used in the present invention.

[0019] Furthermore, the bottom of the culture device should preferably have numerous hemispherical or frustoconical depressions. For example, hemispherical or frustoconical depressions (with a volume of 0.068 mm²). 3 Using a 6-well plate culture system with 2885 cells per well, chondrocyte progenitor cells 5-7 x 10¹⁶ were cultured. 6 By adding 4-5 ml of a suspension containing the cells to one well and incubating it in an incubator for 1-5 days, spheroids measuring 200 μm can be formed. The spheroids in the microplate can be floated by pipetting, collected in a Falcon tube, and then centrifuged to collect the spheroids at the bottom of the tube. The supernatant can then be aspirated, leaving only the spheroids.

[0020] The culture medium used for spheroid formation can be any medium that promotes spheroid formation, and it is preferable to use a medium for three-dimensional culture of chondrocyte progenitor cells. Examples include Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12), Dulbecco's Modified Eagles's Medium (DMEM), F12-Ham, Roswell Park Memorial Institute (RPMI-1640), Eagle's minimum; essential medium (EMEM), alpha Modified Eagle Minimum Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium (IMDM), and F-10 Ham, to which TGFβ (TGFβ1 or TGFβ3), bFGF, and Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3, etc.) have been added. Other substances that may be added include antibiotics / antifungal agents, ITS-X, PDGFBB, serum, L-ascorbic acid, dexamethasone, and insulin growth factor.

[0021] The culture can be carried out using any of the following methods: batch culture, semi-batch culture (fed-batch culture), or continuous culture (perfusion culture). In addition, any of the following methods may be used: static culture, aerated culture, stirred culture, shaking culture, or rotation culture, but static culture is preferred.

[0022] The cell culture temperature for spheroid formation is preferably 30-40°C, and more preferably 37°C.

[0023] The cell culture period for spheroid formation should preferably not exceed 5 days, and more preferably 1 to 5 days. The culture medium should be changed once a day.

[0024] A single spheroid is often composed of 100 to 7500 cells (preferably 1000 to 3000 cells), and the cells that make up the spheroid include chondrocytes.

[0025] The diameter of the spheroid is preferably 20 to 1000 micrometers, and more preferably 200 to 350 μm. The roundness of the spheroid is appropriately 0.5 to 1.0, and more preferably 0.8 to 1.0. The diameter and roundness of the spheroid are determined by the Cell 3 Measurement can be performed using iMager Duo. The SOX9 positivity rate in spheroids is preferably 60% or higher, more preferably 70-100%, and more preferably 80-100%. The SOX9 positivity rate of spheroids can be calculated by cropping the tissue portion from the captured image using ImageJ, separating the three primary colors (Red, Green, Blue), setting a threshold for the measurement range, and using the area of ​​DAPI (blue) as the denominator and the area of ​​SOX9 (Green) as the numerator.

[0026] In the present invention's method for producing cartilage tissue, spheroids containing chondrocyte precursor cells are seeded onto a support and shaped into a desired form.

[0027] The support should be permeable to culture medium components, non-toxic to spheroids, and impassable to spheroids. For example, if the support has a porous membrane structure, it is considered advantageous for post-fusion culture in terms of nutrient supply from above and below and oxygen supply to fused spheroids. Examples of supports include those whose surface is negatively charged and hydrophilic due to corona discharge under air or vacuum gas plasma polymerization treatment (cell adhesion surface treatment), those whose surface is gelatinized, those coated with extracellular matrix (collagen, laminin, fibronectin, etc.) or mucopolysaccharides (heparin sulfate, hyaluronic acid, chondroitin sulfate, etc.), those coated with basic synthetic polymers (poly-D-lysine, etc.), those with a synthetic nanofiber surface, those with a hydrophilic and neutral hydrogel layer surface, and collagen membranes (KOKEN). If the support has a porous membrane structure, the pore size should be 0.4 to 8 μm. Suitable support materials include Falcon cell culture plates (Corning), Falcon multi-cell culture plates (Corning), and Falcon cell culture inserts (Corning).

[0028] Spheroids can be seeded onto a support using a pipette or spoon. Desired shapes include those that can repair malformed or damaged cartilage tissue (e.g., auricular cartilage, epiglottic cartilage, costal cartilage, articular cartilage, epiphyseal cartilage, nasal cartilage, tracheal cartilage, pharyngeal cartilage, intervertebral discs, labrum, meniscus, pubic symphysis) (e.g., rod-shaped, plate-shaped, spherical). If more detailed or complex shapes are required, a pre-made mold may be placed on the support and spheroids seeded into it using a pipette or spoon.

[0029] Spheroids should be seeded at high density on the support. High density means, for example, 1 cm of space for spheroids with a diameter of 150 μm. 3 The number of spheroids present in each area is 9.5 x 10 4 ~3.8 x 105 It is preferably several, preferably 1.9 x 10 5 ~3.8 x 10 5 pieces, more preferably 2.9 x 10 5 ~3.8 x 10 5 pieces.

[0030] The number of spheroids is preferably 2 or more. By increasing the number of spheroids, larger fused spheroids can be produced.

[0031] In the method of the present invention, after shaping the spheroids containing chondrogenic progenitor cells into a desired shape while seeding them on a support, the spheroids are cultured while supplying a culture medium from the front and back sides of the surface on which the spheroids containing chondrogenic progenitor cells are seeded, thereby fusing the spheroids together.

[0032] For example, after seeding spheroids containing chondrogenic progenitor cells on the membrane of a cell culture insert in a desired shape, a culture medium is added to the lower part of the membrane, and the culture plate is left in an incubator with the spheroids on the membrane immersed in the culture medium to fuse the spheroids together.

[0033] "Fusion of spheroids" means that a plurality of spheroids form a continuous structure, and disappearance of the contour of each individual spheroid is confirmed. By fusing the spheroids together, the spheroids become larger, so by maturing the fused spheroids (that is, inducing differentiation of chondrogenic progenitor cells in the fused spheroids into chondrocytes), a large cartilage tissue can be produced.

[0034] The culture medium used for spheroid fusion can be any medium suitable for spheroid fusion, and it is preferable to use the above-mentioned culture medium for three-dimensional culture of chondrocytes. For example, Dulbecco's Modified Eagles's Medium / Nutrient Mixture F12 (DMEM / F12), Dulbecco's Modified Eagles's Medium (DMEM), F12-Ham, Roswell Park Memorial Institute (RPMI-1640), Eagle's minimum essential medium (EMEM), alpha Modified Eagle Minimum Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium (IMDM), or F-10 Ham, to which TGF-β (TGFβ1 or TGFβ3), bFGF, and Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3) are added. Other substances that may be added include antibiotics / antifungal agents, ITS-X, PDGFBB, serum, L-ascorbic acid, dexamethasone, and insulin growth factor.

[0035] The culture method may be either static culture or shaking culture, but static culture is preferred.

[0036] The culture temperature for spheroid fusion is preferably 30-40°C, and more preferably 37°C.

[0037] The culture period for spheroid fusion is preferably 12 hours to 4 days, and more preferably 12 hours to 1 day. The culture medium should be changed every two days.

[0038] After confirmation of fusion between spheroids, the fused spheroids are matured into cartilage tissue in vitro. To mature the fused spheroids into cartilage tissue, it is recommended to continue culturing by changing the culture medium supplied from both the front and back sides of the surface on which the spheroids were seeded to a cartilage differentiation medium. Any cartilage differentiation medium that can mature fused spheroids into cartilage tissue is acceptable, such as Dulbecco's Modified Eagle's Medium / Nutrient Mixture F12 (DMEM / F12), Dulbecco's Modified Eagle's Medium (DMEM), F12-Ham, Roswell Park Memorial Institute (RPMI-1640), Eagle's minimum; essential medium (EMEM), alpha Modified Eagle Minimum Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium (IMDM), or F-10 Ham with added BMP (BMP4 or BMP2). Other additives may include antibiotics-antifungal agents, ITS-X, TGF-β (TGFβ1 and TGFβ3), bFGF, PDGFBB, Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3, etc.), serum, L-ascorbic acid, dexamethasone, and insulin growth factor. Furthermore, L-proline may be added.

[0039] Cartilage can be confirmed by HE staining, Alcian blue staining, and immunohistochemical staining (type II collagen, type I collagen).

[0040] Cartilage tissue matured with fused spheroids exhibits improved hardness, widespread positivity for type II collagen (a cartilage tissue marker) immunohistochemically, positivity for type I collagen (a perichondrium marker) mainly around the tissue periphery, and increased gene expression of SOX9 (a cartilage precursor marker) and COL11A2 (a cartilage marker). In immunohistochemical staining, the positivity rate for type II collagen in cartilage tissue is often 60% or higher, preferably 70-90%, while the positivity rate for type I collagen is often 20% or lower, preferably 5-15%. The positivity rates for type II and type I collagen in cartilage tissue can be calculated by cropping the tissue portion from an image using ImageJ, separating the three primary colors (Red, Green, Blue), setting a threshold for the measurement range, and using the area of ​​Blue as the denominator and the areas of Red (type II collagen) and Green (type I collagen) as the numerators.

[0041] The present invention allows for the production of artificial cartilage tissue with a diameter (φ) of 2 mm or more, 6 mm or more, 40 mm or more, 80 mm or more, and a thickness of 0.5 mm or more, 1 mm or more, 5 mm or more, and 15 mm or more. The present invention provides artificial cartilage tissue produced by the above method, wherein the artificial cartilage tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more. Artificial cartilage tissue with a diameter of 6 mm or more and a thickness of 0.5 mm or more can be produced from 10 to 10,000 spheroids with a diameter of 20 to 500 μm.

[0042] Artificial cartilage tissue with a diameter of 0.5 to 15 mm and a length of 2 to 80 mm can be produced from 100 to 30,000 spheroids with a diameter of 20 to 1,000 μm. Artificial cartilage tissue with a diameter of 2 to 5 mm and a length of 4 to 40 mm can be produced from 500 to 1,500 spheroids with a diameter of 200 to 350 μm.

[0043] The hardness of the artificial cartilage tissue is appropriate at 0.2 to 1.0 MPa, and preferably at 0.4 to 0.6 MPa. The hardness of the artificial cartilage tissue can be measured using a benchtop testing machine (Shimadzu Corporation EZ-Test EZ-SX jig S346-57829-02).

[0044] Cartilage tissue matured in vitro can be transplanted into non-human animals and further matured. Similarly, cartilage tissue matured in vitro can be transplanted into non-human animals and matured into bone tissue. Examples of non-human animals include mice, rats, monkeys, and pigs. Cartilage tissue obtained by short-term culture of fused spheroids (shape-formed cartilage) can become further matured cartilage when transplanted into a living organism. Cartilage tissue obtained by long-term culture of fused spheroids (shape-formed hypertrophic cartilage) can become bone tissue when transplanted into a living organism. In shape-formed hypertrophic cartilage, hypertrophic chondrocytes with enlarged cytoplasm are observed. Hypertrophic chondrocytes can generally be observed using type X collagen as a marker by immunohistochemical staining.

[0045] The culture temperature for fused spheroids to mature into shaped cartilage is preferably 30-40°C, and more preferably 37°C.

[0046] The culture period for fused spheroids to mature into shaped cartilage is preferably 14 to 42 days, and more preferably 21 to 28 days. The culture medium should be changed every 2 to 3 days. Subcutaneous transplantation of the shaped cartilage can lead to further maturation. The transplantation period is preferably 14 to 182 days, and more preferably 28 to 56 days. Cartilage maturation can be confirmed by the appearance of cartilage lucencies in HE staining and the disappearance of type I collagen in immunohistochemical staining.

[0047] The culture temperature for fused spheroids to mature into shaped hypertrophic cartilage is preferably 30-40°C, and more preferably 37°C.

[0048] The culture period for fused spheroids to mature into shaped hypertrophic cartilage is preferably 42 to 84 days, and more preferably 56 to 70 days. The culture medium should be changed every 2 to 3 days. For maturation into bone tissue and the osteochondral transition zone, the shaped hypertrophic cartilage should be subcutaneously transplanted. The transplantation period for shaped hypertrophic cartilage to mature into bone tissue is 28 days or more, preferably 56 days or more. The obtained bone tissue can be confirmed by CT imaging and histological staining, and the osteochondral transition zone can be confirmed by histological staining. The diameter of the shaped hypertrophic cartilage is preferably 1 mm to 20 mm, preferably 5 mm to 10 mm, and the thickness of the shaped hypertrophic cartilage is preferably 2 mm to 100 mm, preferably 20 mm to 40 mm. The present invention also provides artificial cartilage tissue produced by the above method, in which part or all of the artificial cartilage tissue differentiates into bone tissue after transplantation into a living organism, i.e., shaped hypertrophic cartilage.

[0049] The present invention also provides a method for producing artificial bone tissue, which includes transplanting the artificial cartilage tissue produced by the above method into a non-human animal and allowing it to mature into bone tissue. By the method of the present invention, artificial bone tissue can be produced with a diameter (φ) of 2 mm or more, 6 mm or more, 40 mm or more, 6 mm or more, 80 mm or more, and a thickness of 0.5 mm or more, 1 mm or more, 5 mm or more, and 15 mm or more. The present invention also provides artificial bone tissue produced by the above method, wherein the artificial bone tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more.

[0050] Artificial cartilage tissue (which may be transplanted into a living body or not) and / or artificial bone tissue produced by the method of the present invention can be used in the treatment of chondrodysplasia in the craniofacial region, treatment of osteoarthritis, and other regenerative medicine in which shape is important. The present invention provides a composition comprising artificial cartilage tissue produced by the above method, which is used to supplement a deficiency of cartilage tissue and / or bone tissue in a living body by transplantation. If the artificial cartilage tissue is shaped cartilage, it can be transplanted into a living body to supplement a deficiency of cartilage tissue in a living body. If the artificial cartilage tissue is shaped hypertrophic cartilage, it can be transplanted into a living body to supplement a deficiency of bone tissue in a living body. The present invention also provides a composition comprising artificial bone tissue produced by the above method, which is used to supplement a deficiency of bone tissue in a living body by transplantation.

[0051] Specifically, the artificial cartilage tissue produced by the present invention is transplanted into areas of cartilage hypoplasia such as saddle nose and microtia to treat these conditions. Furthermore, the present invention can be used to treat deformities of the auricle and nose caused by traffic accidents and sports injuries by transplanting cartilage tissue that reproduces more complex shapes. In addition, the artificial cartilage tissue produced by the present invention can be transplanted into cartilage defects on the joint surface in osteoarthritis and other conditions to treat these conditions. The artificial bone tissue of the present invention can be used for "transplantation into facial bone defects caused by trauma," "cosmetic surgery such as transplantation into the nasal bone to raise the bridge of the nose, transplantation into the cheekbone to raise the cheekbone, and transplantation into the mandible to form the jawline," "bone transplantation into pseudoarthrosis for nonunion after fracture," and "bone transplantation for bone defects that occur during tumor excision surgery such as osteosarcoma." [Examples]

[0052] The present invention will be described in more detail below with reference to examples. [Example 1] Methods for culturing human iPS cells 1.5 ml of AK02 medium (Ajinomoto) containing 7 μl of iMatrix-511 (Nippi) and 1.5 μl of Y-27632 (Fujifilm Wako Pure Chemical Industries) was added to each well of a 6-well plate and incubated in a 37°C incubator for 1 hour. 5 x 10⁶ samples were then taken from each well of the plate. 3 Human iPS cells (Kyoto University Center for iPS Cell Research and Application, 1383D6) were seeded. The culture medium was changed daily with 1.5 ml of AK02 medium (Ajinomoto), and multiple colonies were observed on day 7. For subculturing, the iPS cells were washed with PBS, 500 μl of Accutase (ICT) solution was added, and the cells were incubated at 37°C for 6 minutes. After detaching the cells by pipetting, 5 ml of AK02 medium (Ajinomoto) was added, and centrifugation was performed at 900 rpm for 5 minutes. The cells were then suspended in AK02 medium (Ajinomoto), and the iPS cells were cultured again or differentiated into chondrocytes.

[0053] Method for inducing differentiation from human iPS cells to human mesoderm cells 7 μl of iMatrix-511 (Nippi) and 1.5 ml of AK02 medium (Ajinomoto) containing 1.5 μl were added to each well of a 6-well plate, and the plate was incubated at 37°C for 1 hour. Human iPS cells were then placed in each well of the plate at a rate of 1 to 1.5 x 10⁶. 5The cells were seeded. The next day, the medium was replaced with DMEM / F12 Ham (1:1) (Sigma-Aldrich) containing 1% Glutamax, 1% B27, 4 μM CHIR (CAYMAN), 100 nM PIK90 (EMD Millipore), 30 ng / ml Activin, and 20 ng / ml bFGF. The following day, the medium was replaced with DMEM / F12 Ham (1:1) (Sigma-Aldrich) containing 1% Glutamax, 1% B27, 3 μM CHIR (CAYMAN), 250 nM DMH1 (Selleck), and 20 ng / ml bFGF (Wako). The following day, the culture medium was changed to DMEM / F12 Ham(1:1)(Sigma-Aldrich) containing 1% Glutamax, 1% B27, 1 μM A8301 (TOCRIS), 250 nM DMH1 (Selleck), 250 nM PD0325901 (TOCRIS), and 1 μM C59 (Cellagen Tech). The next day, the medium was changed again to DMEM / F12 Ham(1:1)(Sigma-Aldrich) containing 1% Glutamax, 1% B27, 1 μM C59 (Cellagen Tech), and 5 nM SAG21K (TOCRIS), and the cells were cultured for 2 days. Gene expression analysis was then performed using human mesodermal cell markers (HOXB5, FOXF1, etc.).

[0054] Method for harvesting human auricular chondrocytes Human perichondrium specimens (using perichondrium obtained from surplus human auricular cartilage during surgery with the consent of the patient or their parents; approved by the ethics committees of Kanagawa Children's Medical Center and Yokohama City University Hospital) were cut with scissors until the solid mass disappeared, and the specimens were shaken in 0.2% collagenase solution (Worthington) at 37°C and 600 rpm for 2 hours to isolate human perichondrium cells. The resulting suspension was filtered through a 40 μm cell strainer and centrifuged at 1500 rpm for 5 minutes to collect human perichondrium cells.

[0055] Method for inducing differentiation of human chondrocytes 7 ml of 0.1% gelatin was administered to a 10 cm dish and left to stand in a 37°C incubator for 1 hour. The supernatant was removed from the dish and mixed with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) in DMEM / F12 Ham (1:1) (Sigma-Aldrich). TM 8 ml of a medium containing 1 μM A8301 (TOCRIS), 20 ng / ml bFGF (Wako), 30 ng / ml PDGFBB (Peprotech), 1 μM WntC59 (Cellagen Tech), 4% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich) was added to the dish. Human mesoderm cells or human auricular chondrocytes were placed in the dish in a 1.2 x 10⁶ 6 Individual seeds were seeded and placed in an incubator at 37°C. After 48 hours, the culture medium was changed, and after another 24 hours, confluence of the dish was confirmed. Gene expression analysis was then performed using human chondrocyte markers (SOX9, CD44, CD73, CD105).

[0056] Human chondrocyte spheroid formation by three-dimensional culture of human chondrocytes Chondroprogenitor cells (derived from either human iPS cells or human auricular cartilage) seeded in a 10cm dish were washed with PBS and then detached by treatment with trypsin solution for 3 minutes. The trypsin solution containing the chondroprogenitor cells was inactivated using DMEM / F12 containing 10% fetal bovine serum (Biowest), equivalent to three times the volume of the trypsin solution, and collected in a Falcon tube. This was then centrifuged at 400G for 3 minutes. After removing the cell supernatant, the cells were mixed with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) in DMEM / F12 Ham (1:1) (Sigma-Aldrich). TMUsing a medium containing 15 ng / ml TGFβ1 (Peprotech), 15 ng / ml bFGF (Wako), 10 ng / ml PDGFBB (Peprotech), 1 μM WntC59 (Cellagen Tech), 4% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich), 1.4 x 10⁶ doses were taken. 6 The suspension was prepared to a concentration of 7x10 / ml. 6 Chondrothoncrine progenitor cells were administered to one well of an Elplasia plate (Corning, 6-well standard) and left to stand in an incubator at 37°C. The next day, 4 ml of culture medium was changed, and the cells were cultured for a further 24 hours.

[0057] Induction from human cartilage precursor spheroids to human-shaped cartilage Cartilage precursor spheroids from the wells of an Elplasia plate (Corning, 6-well standard) were collected into a Falcon tube by pipetting. Centrifuge was performed at 1000 rpm for 2 minutes, and the supernatant was removed. The remaining cartilage precursor spheroids were collected by pipette and seeded in the intended shape onto a membrane in one well of a 0.4 μm pore cell culture insert (Falcon, 6-well standard). At the bottom of the membrane, DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) was added. TM), 15 ng / ml TGFβ1 (Peprotech), 15 ng / ml bFGF (Wako), 10 ng / ml PDGFBB (Peprotech), 1 μM WntC59 (Cellagen Tech), 4% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml Insulin Growth Factor (Sigma-Aldrich) were added to 3 ml of culture medium, and the mixture was left standing in an incubator at 37°C. In this report, rod-shaped cartilage was used as an example. The seeded cartilage precursor spheroids fused with each other in about 12 hours, and the disappearance of the spheroid outlines was confirmed under a microscope. The entire volume (3 ml) of culture medium at the bottom of the membrane was replaced the following day. Three days later, DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) TM The medium was changed to one containing 5 ng / ml TGFβ1 (Peprotech), 10 ng / ml bFGF (Wako), 5 ng / ml PDGFBB (Peprotech), 20 ng / ml BMP4, 1 μM WntC59 (Cellagen Tech), 2% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich). Three days later, the medium was changed to DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TMThe medium was changed to one containing 2.5 ng / ml TGFβ1 (Peprotech), 1 ng / ml bFGF (Wako), 20 ng / ml BMP4 (R&D), 1% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml Insulin Growth Factor (Sigma-Aldrich), and 40 μg / ml L-proline (Sigma-Aldrich). Three days later, the medium was changed to DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM The culture medium was changed to one containing 10 ng / ml BMP4, 0.5% fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml insulin growth factor (Sigma-Aldrich), and 40 μg / ml L-proline (Sigma-Aldrich), and the medium was changed every 3 days thereafter. By culturing the membrane for 20 days using the above method, cartilage tissue expressing type II collagen and Alcian blue was obtained.

[0058] Induction of human cartilage precursor spheroids into human-shaped hypertrophic cartilage Cartilage precursor spheroids from the wells of an Elplasia plate (Corning, 6-well standard) were collected into a Falcon tube by pipetting. Centrifuge was performed at 1000 rpm for 2 minutes, and the supernatant was removed. The remaining cartilage precursor spheroids were collected by pipette and seeded in the intended shape onto a membrane in one well of a 0.4 μm pore cell culture insert (Falcon, 6-well standard). At the bottom of the membrane, DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) was added.TM ), 15 ng / ml TGFβ1 (Peprotech), 15 ng / ml bFGF (Wako), 10 ng / ml PDGFBB (Peprotech), 1 μM WntC59 (Cellagen Tech), 4% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml Insulin Growth Factor (Sigma-Aldrich) were added to 3 ml of culture medium, and the mixture was left to stand in an incubator at 37°C. In this report, rod-shaped cartilage, mimicking the shape of the nose and ear, was used as an example. The seeded cartilage precursor spheroids fused with each other in about 12 hours, and the disappearance of the spheroid outlines was confirmed under a microscope. The entire volume (3 ml) of culture medium at the bottom of the membrane was replaced the following day. Three days later, DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco) TM The medium was changed to one containing 5 ng / ml TGFβ1 (Peprotech), 10 ng / ml bFGF (Wako), 5 ng / ml PDGFBB (Peprotech), 20 ng / ml BMP4, 1 μM WntC59 (Cellagen Tech), 2% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich). Three days later, the medium was changed to DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TMThe medium was changed to one containing 2.5 ng / ml TGFβ1 (Peprotech), 1 ng / ml bFGF (Wako), 20 ng / ml BMP4 (R&D), 1% Fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml Insulin Growth Factor (Sigma-Aldrich), and 40 μg / ml L-proline (Sigma-Aldrich). Three days later, the medium was changed to DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM The culture medium was changed to one containing 10 ng / ml BMP4, 0.5% fetal bovine serum (Biowest), 40 μg / ml L-ascorbic acid (Sigma-Aldrich), 40 μg / ml dexamethasone (Sigma-Aldrich), 10 ng / ml insulin growth factor (Sigma-Aldrich), and 40 μg / ml L-proline (Sigma-Aldrich), and the medium was changed every 3 days thereafter. By culturing the membrane seeding for 50 days using the above method, hypertrophic cartilage was obtained, characterized by hypertrophic chondrocytes with hypertrophied extracellular matrix and cytoplasm that were positive for type II collagen and Alcian blue.

[0059] Transplantation and excision of reshaped cartilage Mice (NOD / SCID) or rats (IL2rg-KO) were anesthetized by inhaled administration of isoflurane (Pfizer 1 ml / 1 ml). Hair was removed from the transplantation site, and skin incisions were made with scissors and hooked forceps according to the transplant sample. Samples that had undergone 3D culture for 15-30 days were placed subcutaneously and the wounds were closed with sutures at 2 mm intervals using size 6-0 threads. For excision, anesthesia was also performed by inhaled administration of isoflurane, and the transplant samples were excised with scissors and hooked forceps. The samples were stored in PBS or formalin solution according to the requirements for analysis.

[0060] Induction from shaping hypertrophic cartilage to shaping skeleton Mice (NOD / SCID) or rats (IL2rg-KO) were anesthetized by inhaled administration of isoflurane (Pfizer 1 ml / 1 ml). Hair was removed from the transplantation site, and skin incisions were made with scissors and hooked forceps according to the transplant sample. Samples that had undergone 3D culture for 56-70 days were placed subcutaneously and the wounds were closed with sutures at 2 mm intervals using size 6-0 sutures. Ossification was confirmed in samples at 1 month and 2 months post-transplantation using micro-CT. Sample extraction was also performed under anesthesia by inhaled administration of isoflurane, and the transplanted samples were extracted with scissors and hooked forceps. Samples were stored in PBS or formalin solution according to the analysis requirements.

[0061] Gene expression analysis Chondrogenic cells obtained from 10cm dishes were collected, and RNA was purified using the PureLink RNA mini kit (Thermo Fisher Scientific). cDNA synthesis was performed using the High capacity cDNA reverse transcription kit (Thermo Fisher Scientific). 18S rRNA (Applied Biosystems) was used as an internal standard for gene quantification. Gene amplification and detection were performed using the Light Cycler® 480 (Roche Life Science).

[0062] High-precision bright-field analysis of cartilage precursor spheroids Cell 3 An Elplasia plate was placed in an iMager Duos (SCREEN) and high-precision bright-field analysis was performed to automatically measure the roundness (0-1.0) and diameter (μm) of each spheroid.

[0063] Evaluation of cartilage precursor spheroid fusion Immediately after seeding cartilage precursor spheroids into cell culture inserts, 12 hours later, and 10 days later, the margins of the shaped cartilage were observed under a microscope (Olympus IX73) at 20x magnification.

[0064] Hardness measurement and analysis of reshaped cartilage tissue The hardness of shaped cartilage tissue was measured using a benchtop testing machine (Shimadzu Corporation EZ-Test EZ-SX, fixture S346-57829-02). The elastic modulus (MPa) was determined at a pressure of 3 mm / min between 0.2 mm and 0.6 mm.

[0065] Functional evaluation of morphogenetic cartilage tissue using ELISA Capture, Detection, and Standard samples from DuoSet (R&D) were prepared in PBS solution according to the Certificate of Analysis procedure. 100 μl of Capture solution was added to each well of a 96-well plate. The plate was wrapped in plastic wrap and left to stand overnight at room temperature. The Capture solution was removed from each well, and 200 μl of PBS-Tween solution was added to each well, repeating this process three times. The plate was then placed on a Kimwipe to remove excess moisture. 300 μl of Block Ace (DC Farmer) solution was added to each well, the plate was wrapped in plastic wrap, and left to stand at room temperature for 1 hour. The solution was removed from the plate, and 200 μl of PBS-Tween solution was added to each well, repeating this process three times. The plate was then placed on a Kimwipe to remove excess moisture. 100 μl of Standard, Sample, and Blank samples were added to each well. The plate was wrapped in plastic wrap and left to stand at room temperature for 2 hours. The solution was removed from the plate, and 200 μl of PBS-Tween solution was added to each well, repeating this process three times. The plate was placed on a Kimwipe to remove moisture. 100 μl of Detection solution was added to each well. The solution was removed from the plate, and 200 μl of PBS-Tween solution was added to each well, repeating this process three times. The plate was placed on a Kimwipe to remove moisture. The required amount of Streptavidin (requires light protection) was added to each well, adjusted to the concentration specified in the Certificate of Analysis's Working Concentration. The solution was added to each well. The plate was wrapped in aluminum foil and allowed to stand at room temperature for 20 minutes. The solution was removed from the plate, and 200 μl of PBS-Tween solution was added to each well, repeating this process three times. The plate was placed on a Kimwipe to remove moisture. 50 μl of TMB one Solution (requires light protection) was added to each well. The plate was wrapped in aluminum foil and allowed to develop color at room temperature for several minutes while observing the results. After color development was confirmed, 50 μl of HCl was added to each well. Absorbance was measured using a plate reader at 450 nm, and also measured at 540 nm or 570 nm as a reference. Calibration curves were drawn and concentrations were calculated.

[0066] Paraffin sectioning method The collected samples were fixed by standing them overnight in formalin (Fujifilm Wako Pure Chemical Industries) solution. The fixed samples were washed with PBS solution at room temperature for 30 minutes and then with 70% ethanol at room temperature for 30 minutes. Using an automated embedding machine, the samples were dehydrated with 100% ethanol for 1 hour x 7 times, then immersed and embedded in xylene for 1 hour x 3 times and in 100% paraffin for 1 hour x 4 times. The embedded samples were sectioned to a thickness of 2-4 μm using a microtome and dried at 42°C.

[0067] Hematoxylin and eosin staining Paraffin sections were deparaffinized and hydrophilized in the following order: xylene, 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, and Milli-Q water. They were stained with hematoxylin solution (Muto Chemical) for 20 minutes, washed with running water for 10 minutes, and then stained with eosin solution (Muto Chemical) for 2 minutes. After washing with running water, dehydration was performed stepwise using ethanol, and after clearing with xylene solution, the samples were mounted with a non-aqueous mounting medium and a coverslip.

[0068] Alcian Blue dyeing Paraffin sections were deparaffinized and hydrophilized in the following order: xylene, 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, and Milli-Q water. After treatment with 3% aqueous acetic acid for 1 minute, staining was performed with pH 2.5 Alcian blue solution (Wako) for 40 minutes. After treatment with 3% aqueous acetic acid for 5 minutes, washing was performed with running water for 5 minutes. After staining with Kern Ehrlot solution (Muto Chemical) for 5 minutes, washing was performed with running water for 1 minute. Dehydration was performed stepwise using ethanol, and after clearing with xylene solution, the samples were mounted with a non-aqueous mounting medium and a coverslip.

[0069] Immunohistological analysis Paraffin sections were deparaffinized and hydrophilized in the following order: xylene, 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, and Milli-Q water. After washing the samples twice with 0.1% TBS-tween solution for 5 minutes each, pepsin (Abcam) was added to the samples and antigen retrieval was performed at room temperature for 20 minutes. After washing with 1% TBS-tween solution for 5 minutes, the samples were treated with a protein blocker (Dako) at room temperature for 30 minutes. Diluted primary antibodies (Anti-Collagen Type II Antibody, clone 6B3 (Merck), Anti-Collagen type I, Human, rabbit-polyclonal (ACRIS)) were reacted with the samples at room temperature for 2 hours or overnight at 4°C. After washing three times with 1% TBS-tween solution for 5 minutes each, the samples were reacted with fluorescently labeled secondary antibodies at room temperature for 1 hour. After washing three times for 5 minutes with 1% TBS-tween solution, the DAPI was mounted with apatic mounting medium containing the solution and a coverslip, and then observed under a microscope. The positivity rate of each antibody was calculated in ImageJ as the percentage of the positive area of ​​each antibody relative to the positive area of ​​the DAPI.

[0070] Experimental results • Differentiation induction from human iPS cells to human mesoderm cells, and from human mesoderm cells to human chondrocytes. Human iPS cells (day 0) were differentiated into human mesodermal cells (day 5) and then into human chondrocyte progenitor cells (day 8), and gene expression was confirmed day by day (Figure 1). Undifferentiated markers OCT4 and NANOG showed high expression at day 0 and decreased expression over time. Human mesodermal markers also showed increases in BRACHURY, MESOGININ1, FOXF1, and HOXB5 in that order from early to late stages, consistent with the developmental order of increase. Furthermore, at day 8, chondrocyte progenitor cells were obtained that showed high expression of pre-chondrial markers SOX9 and CD44, as well as mesenchymal markers CD73 and CD105 (n=7) (Figure 1).

[0071] • High-precision bright-field evaluation of chondrocyte spheroids from human chondrocyte progenitor cells. Numerous spheroids were formed 48 hours after seeding chondrocyte progenitor cells onto an Elplasia plate (Figure 2A). 3 The diameter and circularity of each spheroid were measured using iMager Duos. Of the 37,344 spheroids, 34,261 (91.7%) had a diameter of 200-300 μm, and 36,262 (97.1%) had a circularity of 0.8-1.0. (Figures 2B, C)

[0072] • In vitro histological analysis of human chondrocyte precursors Human cartilage precursor spheroids were confirmed to be positive for the cartilage precursor markers SOX9 and type I collagen by immunohistochemical staining (Figure 2D). The positivity rate for SOX9 within the spheroids was 69%, and the positivity rate for type I collagen was 74% (Figure 2E).

[0073] • Fusion ability of human cartilage precursor spheroids Spheroids prepared on an Elplasia plate were seeded onto a cell culture insert in an arbitrary shape (a rod shape in this example). Under a microscope, spheroids of approximately 200 μm in size were observed on the cell culture insert immediately after seeding. After 12 hours, the boundaries between each spheroid disappeared, and fusion of the spheroids was confirmed (Figure 3).

[0074] ·In vitro histological analysis of shaped cartilage Three-dimensional culture was performed for 20 days using cell culture inserts, and as a result, shaped cartilage was obtained with high reproducibility in macroscopic images (n=24) (Figure 4). The obtained shaped cartilage stained with Alcian blue, which stains the extracellular atmosphere of cartilage tissue, and type II collagen, a marker of cartilage tissue, was widely positive, with approximately 80% being positive. In addition, type I collagen, a marker of the perichondrium, also showed positivity mainly around the shaped cartilage, with approximately 5% being positive (n=4) (Figure 5A, B).

[0075] ·In vitro functional analysis of shaped cartilage Quantitative RT-PCR revealed that the cartilage precursor marker SOX9 and the cartilage marker COL11A2 increased over time in cartilage precursor spheroids (10 days after culture initiation), shaped cartilage (20 days after culture initiation), and shaped cartilage (30 days after culture initiation) (n=7-29) (Figure 6). These results showed even higher gene expression when BMP was administered compared to when it was not administered (Figure 6). Using ELISA, the secretion levels of human hyaluronic acid and human melanoma inhibitory activity were measured every 10 days. It was confirmed that secretion levels increased in shaped cartilage (20 days after the start of culture) and could be maintained thereafter until shaped hypertrophic cartilage (50 days after the start of culture) (n=6-20) (Figure 7).

[0076] • Histological analysis after transplantation of reshaped cartilage After subcutaneous transplantation for one month, shaped cartilage with maintained shape was obtained in macroscopic images (Figures 8A, B).

[0077] CT image analysis of the shaped skeleton Subcutaneous grafting of shaped hypertrophic cartilage revealed ossification on CT scans at 1 and 2 months post-grafting (Figure 9).

[0078] • Histological analysis of the morphological skeleton Subcutaneous grafting of shaped hypertrophic cartilage revealed an osteochondral transition zone and COL1-positive trabeculae at 3 months post-transplantation by immunohistochemical staining (Figure 10). • Shaped cartilage using chondrocytes derived from human auricular chondrocytes It was confirmed that complex shapes (in this example, mimicking a nose, ear, and rod) can be created even when using cartilage precursor spheroids derived from human auricular perichondrium (Figure 11). All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0079] This invention can be used in the treatment of chondrodysplasia in the craniofacial region, the treatment of osteoarthritis, and other regenerative medicines where shape is important. It can also be used in orthopedic treatments.

Claims

1. A method for producing artificial cartilage tissue, comprising: culturing chondrocytes in a medium containing TGF-β, bFGF, and a Wnt / β-catenin inhibitor to produce spheroids containing chondrocytes; shaping the spheroids containing chondrocytes into a desired shape while seeding them on a support; culturing the spheroids while supplying medium from the front and back sides of the seeded surface to fuse the spheroids together; and maturing the fused spheroids into cartilage tissue in vitro.

2. The method according to claim 1, wherein the chondrogenic progenitor cells are cells differentiated from embryonic stem cells and / or induced pluripotent stem cells.

3. The method according to claim 1, wherein the chondrogenic progenitor cells are cells obtained by differentiating chondrocytes collected from the chondrium.

4. The method according to any one of claims 1 to 3, wherein the spheroid containing chondrocyte precursor cells has a diameter of 20 to 1000 μm.

5. The method according to any one of claims 1 to 4, wherein one spheroid contains 100 to 7500 chondrocytes.

6. The method according to any one of claims 1 to 5, wherein the spheroid containing chondrocytes is prepared by culturing chondrocytes in a culture substrate having a cell non-adherent surface.

7. The method according to any one of claims 1 to 6, wherein fusion spheroids are cultured in a culture medium containing BMP to mature into cartilage tissue.

8. The method according to any one of claims 1 to 7, wherein the culture period of the fusion spheroid for maturation into cartilage tissue is 14 to 42 days.

9. The method according to any one of claims 1 to 7, wherein the culture period of the fused spheroid for maturation into cartilage tissue is 42 to 84 days.

10. The method according to any one of claims 1 to 9, comprising transplanting cartilage tissue matured in vitro into a non-human animal and further maturing it.

11. Artificial cartilage tissue prepared by the method described in any one of claims 1 to 10, wherein the artificial cartilage tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more.

12. Artificial cartilage tissue produced by the method described in claim 9, wherein, after being transplanted into a living organism, a part or all of the artificial cartilage tissue differentiates into bone tissue.

13. A composition comprising artificial cartilage tissue prepared by any one of claims 1 to 10, the composition being used to be implanted in a living organism to compensate for a deficiency of cartilage tissue and / or bone tissue in the living organism.

14. The composition according to claim 13, comprising artificial cartilage tissue produced by the method described in claim 8, and used for transplantation into a living organism to compensate for a deficiency of cartilage tissue in the living organism.

15. The composition according to claim 13, comprising artificial cartilage tissue produced by the method described in claim 9, and used for transplantation into a living organism to compensate for a deficiency of bone tissue in the living organism.

16. A method for producing artificial bone tissue, comprising transplanting artificial cartilage tissue produced by the method described in claim 9 into a non-human animal and allowing it to mature into bone tissue.

17. Artificial bone tissue prepared by the method described in claim 16, wherein the artificial bone tissue has a diameter of 6 mm or more and a thickness of 0.5 mm or more.

18. A composition comprising artificial bone tissue prepared by the method described in claim 16, the composition being used to be implanted in a living organism to compensate for a deficiency of bone tissue in the living organism.

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