JP7828599B2 - Method for preparing shape-type cartilage tissue - Google Patents

Description

The present invention relates to a method for preparing a shape-type cartilage tissue.
In this specification, "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.

For the removal of malignant tumors on the face, trauma such as traffic accidents, cleft lip and palate accompanied by nasal deformities, or microtia, foreign bodies such as silicone or autologous costal cartilage are used. While the implantation of foreign bodies such as silicone carries a risk of infection and inflammation, using autologous costal cartilage carries less risk, but obtaining a sufficient amount of cartilage can be difficult.
Patent Document 1 discloses a method for obtaining cartilage from chondrocytes.

WO2021/054449

The present invention aims to provide a method for obtaining cartilage tissue in any desired shape.

The present invention provides a method for preparing cartilage tissue of the following shape type.
[1]
A method for preparing a morphologically shaped cartilage tissue, comprising seeding chondrocyte progenitor cells in a cell culture space formed by a mold on a substrate, culturing them to produce cell aggregates, and further culturing them in a cartilage differentiation induction medium.
[2]
A method for preparing a shape-type cartilage tissue as described in [1], wherein the seeding density of chondrocyte precursor cells is 1.4 × ^10⁶ to 2.8 × ^10⁶ cells/ ^cm² .
[3]
A method for preparing a shape-type cartilage tissue according to [1] or [2], comprising coating a substrate portion in the cell culture space with a material that promotes cell adhesion, and then culturing chondrocytes.
[4]
A method for preparing a shape-type cartilage tissue as described in any one of items [1] to [3], comprising culturing chondrocyte progenitor cells in a chondrocyte differentiation induction medium to form aggregates, stacking multiple aggregates and culturing them further to obtain a cartilage tissue.
[5]
A method for preparing a shape-type cartilage tissue body according to any one of items [1] to [4], wherein ossification when transplanted into the body is suppressed by culturing chondrocytes with a reduced BMP4 concentration for a certain period at the end of the culture period.
[6]
A method for preparing a shape-type cartilage tissue according to any one of items [1] to [5], comprising culturing chondrogenic cells in a chondrogenic differentiation-inducing medium, removing the shape-type cartilage tissue obtained, and treating it with glutaraldehyde and/or ethanol.
[7]
Shape-type cartilage tissue derived from human iPS cells, exhibiting a volume change rate of 25% or less when transplanted into a mammalian organism.

The present invention allows for the preparation of nasal cartilage, auricular cartilage, and other cartilages in any desired shape. Unlike foreign materials 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 deformities and defects, bone and soft tissue defects, etc., and can also be used in cosmetic surgery. Specifically, this includes cartilage deformities and defects, injuries caused by trauma, combat, fighting, sports, exercise, etc., osteoarthritis, rheumatoid arthritis, osteochondritis dissecans, achondroplasia, relapsing polychondritis, articular cartilage damage, intervertebral disc herniation, auricular malformations, otitis and microtia cartilage defects, nasal malformations, eyelid defects, and tissue defects after malignant tumor resection.

Investigation of self-aggregation conditions Investigation of self-aggregation conditions Development of small ring-shaped cartilage tissue Development of small ring-shaped cartilage tissue (using FBS-containing medium) Review of the differentiation protocol (for the first three weeks of STEP 3) Review of the differentiation protocol (for the latter three weeks of STEP 3) Examination of differentiation protocols Applications of ring-shaped cartilage tissue (schematic diagram) Applications of ring-shaped cartilage tissue (actual examples) Examples of large ring-shaped cartilage Various culture protocols and post-transplant changes Subcutaneous transplantation of glutaraldehyde-treated cultured cartilage Examples of auricular frame formation using glutaraldehyde-cultured cartilage

The substrate used in this invention is equipped 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 be any shape, but examples include rod-shaped, ring-shaped (donut-shaped), cylindrical, ellipsoidal, sheet-shaped, disc-shaped, and plate-shaped. The width of the coated area of the cell culture space is preferably about 3 to 15 mm, more preferably about 5 to 10 mm. The size of the cell culture space affects the formation of aggregates of chondrocyte precursor cells. The cell culture space may be shaped to suit the cartilage transplantation site, such as nasal cartilage, auricular cartilage, or articular cartilage. Alternatively, after forming a shaped cartilage tissue according to this invention, it may be shaped to the desired form.

By culturing chondrogenic cells in a cell culture space, aggregates of chondrogenic cells are formed. When these aggregates are removed and multiple aggregates are stacked, they easily bind together to form a single large aggregate. Further culturing of this stacked aggregate to induce cartilage formation allows for the acquisition of a larger, more shaped cartilage tissue.

The substrate surface exposed to the cell culture space may be coated with a material that promotes cell adhesion, and then chondrocytes may be cultured thereafter.
The exposed "surface" of the substrate refers to the surface that comes into contact with chondrocytes or culture medium, and in particular, when the substrate is part of a cell culture vessel, it refers to the bottom surface that comes into contact with chondrocytes or culture medium.

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

"Pluripotent stem cells" refer to cells that possess both self-renewal ability and the ability to differentiate into multiple other cell lineages (multipotency). "Progenitor cells" refer to cells that are in the process of differentiating from stem cells to functional cells, which are the final differentiation target. Therefore, human chondrogenic progenitor cells derived from pluripotent stem cells refer to cells that have been induced to differentiate using pluripotent stem cells as raw material cells and are in the process of differentiating into chondrocytes, the final differentiation target. Examples of pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), which are pluripotent cells (cells that have the ability to differentiate into any somatic cell and germline cell).

Furthermore, in one preferred embodiment of the present invention, the "chondrogenic progenitor cells" are PRRX1 protein-positive. PRRX1 (Paired-related homeobox 1) protein is a transcription factor possessing a homeodomain, and is known to be specifically expressed in limb buds derived from the lateral plate mesoderm and head mesoderm derived from the paraxial mesoderm during development.

The nucleotide sequence of the human (Homo sapiens) PRRX1 gene cDNA and the amino acid sequence of the PRRX1 protein are registered in GenBank, provided by the National Center for Biotechnology Information (NCBI), under the following accession numbers. If multiple revisions are registered, the most recent revision is assumed to be the one 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)

Cells positive for the PRRX1 protein can be detected using known methods. For example, detection can be performed using a reporter gene whose expression is regulated by the PRRX1 gene's transcription promoter sequence, or by immunohistochemical staining using antibodies specific to the PRRX1 protein.

PRRX1 protein-positive chondrogenic cells can be produced, for example, by a method including the following steps:
- A process for inducing differentiation of mammalian pluripotent stem cells into lateral plate mesoderm cells.
- A step of culturing the differentiated cells in 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 under an environment that activates Wnt signaling to prepare chondrogenic cells.

In the above method, the pluripotent stem cells used as raw material cells can include, for example, embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). The ES cells and iPS cells can be newly created or already established.

In the aforementioned method, pluripotent stem cells are first differentiated into lateral plate mesoderm cells. Known methods can be used to differentiate pluripotent stem cells into lateral plate mesoderm cells. For example, a method similar to that described in Loh et al, 2016, Cell, 451-467 can be employed. Specifically, pluripotent stem cells are first differentiated into the mid-primitive streak, and then differentiated from the mid-primitive streak into lateral plate mesoderm cells.

The induction of differentiation of lateral plate mesoderm cells can be confirmed, for example, by detecting the expression of the HAND1 protein, a specific marker for lateral plate mesoderm cells.
Next, the differentiated lateral plate mesoderm cells are cultured in an environment that activates the Wnt signaling pathway to induce differentiation into PRRX1-positive cells derived from lateral plate mesoderm, which are positive for the PRRX1 protein. To reduce the influence of the previous culture environment, it is preferable to wash the cells with PBS buffer or the like as appropriate before culturing them in an environment that activates the Wnt signaling pathway.

The induction of differentiation from lateral plate mesoderm cells to limb bud mesenchymal cells is preferably carried out in the absence of FGF signaling activators such as FGF2, more preferably in an environment that activates Wnt signaling, and more preferably in the presence of one, two, or three agents selected from the group consisting of TGFβ signaling inhibitors, BMP signaling inhibitors, and Hedgehog signaling inhibitors, and even more preferably in the presence of TGFβ signaling inhibitors, BMP signaling inhibitors, TGFβ signaling inhibitors, and Hedgehog signaling inhibitors.

Culturing in an environment that activates Wnt signaling can be performed, for example, by culturing in the presence of an effective amount of Wnt signaling activator. Wnt signaling activators enhance Wnt-mediated signal transduction (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. For example, 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 using CHIR99021 as a Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

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

Culturing is exemplified by methods using conditions of approximately 37°C and a carbon dioxide concentration of approximately 5%, but is not limited to these. Culturing under the above conditions can be carried out, for example, by controlling the temperature and _CO2 concentration using a known _CO2 incubator.
Cell culture can be performed using two-dimensional cell culture (planar culture). Two-dimensional cell culture can be carried out by coating the culture equipment with a coating that promotes cell adhesion as needed.

The period for culturing under conditions that activate the Wnt signal is not particularly limited, but can be, for example, 6 hours to 4 days, preferably 1 to 3 days, and more preferably 2 days (about 48 hours). The culture medium can be changed as needed. The culture conditions are preferably in accordance with conventional methods.
During culture, subculturing can be performed as needed. When subculturing, harvest the cells before or immediately after reaching confluence and seed them in new culture medium. The culture medium can also be changed as appropriate.

It is preferable to use serum-free media such as IMDM medium, F12 medium, or a mixture thereof as the culture medium. Differentiation induction and maintenance culture are possible in a state free of animal-derived components. Furthermore, various culture medium additives such as antibacterial agents like streptomycin and penicillin, non-essential amino acids (NEAAs), 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 can be added to the serum-free medium.

Next, the prepared limb bud mesenchymal stem cells are cultured in an environment that activates Wnt signaling to induce differentiation into PRRX1 protein-positive chondrocytes. The environment that activates Wnt signaling is the same as described above. Furthermore, differentiation from limb bud mesenchymal cells to chondrocytes is preferably carried out in the presence of an FGF signaling activator such as FGF2.
The chondrogenic progenitor cells obtained in this way can be cultured on a larger scale within the same lot and can be stocked under consistent quality control conditions.

The seeding of chondrocyte progenitor cells into the cell culture space is carried out by known methods, such as adding a cell suspension (cells suspended in culture medium) to the cell culture space. The seeding density of chondrocyte progenitor cells is preferably 1.4 × ^10⁶ to 2.8 × ^10⁶ cells/ ^cm² , and more preferably 1.8 × ^10⁶ to 2.2 × ^10⁶ cells/ ^cm² . If the seeding density is too low, the formation of cell aggregates will be slow, pores may form in the cell aggregates, and the strength of the cartilage tissue may be insufficient to maintain its shape.
Cell aggregates are obtained by culturing chondrocytes seeded in a cell culture space.
The culture method is not particularly limited as long as it can produce cell aggregates from chondrocytes, and can be appropriately selected depending on the properties of the chondrocytes.

For example, it is preferable to culture PRRX1 protein-positive chondrogenic progenitor cells derived from pluripotent stem cells under conditions that activate Wnt signaling and/or suppress TGFβ signaling.
Culturing in an environment that activates Wnt signaling can be performed, for example, by culturing in the presence of an effective amount of Wnt signaling activator. Wnt signaling activators enhance Wnt-mediated signal transduction (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. For example, 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 using CHIR99021 as a Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably about 1 to 10 μM.

Culturing cells in an environment that suppresses TGFβ signaling can be performed, for example, by culturing them in the presence of an effective amount of a TGFβ signaling inhibitor. A TGFβ signaling inhibitor suppresses (inhibits) the 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 using A-83-01 as a TGFβ signaling inhibitor, the amount added can be, for example, about 0.1 to 10 μM, preferably 0.2 to 5 μM.

While culturing chondrogenic cells is exemplified by methods using conditions of approximately 37°C and a carbon dioxide concentration of approximately 5%, it is not limited to these methods. Culturing under the above conditions can be carried out, for example, by controlling the temperature and _CO2 concentration using a known _CO2 incubator.
The incubation period is not particularly limited, but can be, for example, 6 hours to 4 days, preferably 1 to 3 days, and more preferably 2 days (approximately 48 hours). The culture medium can be changed as needed. The incubation conditions are preferably in accordance with conventional methods.

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

In this way, self-aggregated cell aggregates are obtained from human chondrocyte precursor cells. The obtained cell aggregates are then induced to differentiate into cartilage tissue. The preparation of cell aggregates and induction into cartilage tissue may be carried out continuously by changing the culture medium, or the cell aggregates may be removed from the cell culture space, multiple cell aggregates may be used to form the desired shape, and then differentiated into cartilage tissue.
In the differentiation induction process, cell aggregates are cultured to produce cartilage tissue.
The culture method is not particularly limited as long as it can produce cartilage tissue from the cell aggregates, and can be appropriately selected depending on the properties of the cell aggregates obtained from the human chondrocytes.

For example, it is preferable to culture cell aggregates obtained from pluripotent stem cell-derived PRRX1 protein-positive human chondrogenic progenitor cells using a method that includes a step of culturing them in an environment that activates the Wnt signaling pathway.

Culturing in an environment that activates Wnt signaling can be performed, for example, by culturing in the presence of an effective amount of Wnt signaling activator. Wnt signaling activators enhance Wnt-mediated signal transduction (particularly the canonical Wnt pathway). Examples of Wnt signaling activators include GSK3β inhibitors and Wnt family proteins. For example, 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 using CHIR99021 as a Wnt signal activator, the amount added can be, for example, about 0.1 to 20 μM, preferably 1 to 10 μM.

Furthermore, it is preferable to carry out the culture in multiple stages. Specifically,
It is preferable to carry out the process in three steps: (i) culturing in the presence of a Wnt signaling activator and an FGF signaling activator; (ii) culturing in the presence of an FGF signaling activator (and preferably in the absence of a Wnt signaling activator); and (iii) preferably in the absence of a Wnt signaling activator and an FGF signaling activator.

Culturing cells in an environment that activates FGF signaling can be performed, for example, by culturing them in the presence of an effective amount of FGF signaling activator. FGF signaling activators enhance fibroblast growth factor (FGF) signaling. Examples of FGF signaling activators include FGF1/aFGF and FGF2/bFGF. When using FGF2 as the FGF signaling activator, the amount added can be, for example, about 0.1 to 100 ng/mL, preferably 1 to 50 ng/mL.

When performing the three-stage culture described above, steps (i) and (ii) can be carried out using two-dimensional cell culture (planar culture) or three-dimensional culture. Two-dimensional cell culture can be performed by coating the culture equipment with a coating that promotes cell adhesion as needed. Step (iii) is preferably carried out using three-dimensional culture. Furthermore, between each stage of culture, it is preferable to wash the cells with PBS buffer or the like as appropriate to reduce the influence of the previous culture environment.

Culturing can be carried out in a suitable container for storing cells and culture medium. Suitable culturing methods include, but are not limited to, culturing under conditions of approximately 37°C and a carbon dioxide concentration of approximately 5%. Culturing under these conditions can be carried out, for example, by controlling the temperature and _CO2 concentration using a known _CO2 incubator.

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

The culture medium used in the differentiation induction process 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, to which L-ascorbic acid, ITS (insulin-transferrin-sodium selenite medium supplement), GDF5, and/or BMP4 are added, can also be used. Additionally, various known culture medium additives such as antibacterial agents (e.g., streptomycin, penicillin), non-essential amino acids (NEAA), ROCK inhibitors (e.g., Y-27632), cell growth factors (e.g., EGF, TGFβ), hormones (e.g., insulin), proteins (e.g., transferrin, albumin), lipids, polyvinyl alcohol, and monothioglycerol can be added as needed.

In a particularly preferred embodiment of the present invention, differentiation induction from cell aggregates to cartilage tissue can be carried out by, for example, the following steps 1 to 3.
(i) Step 1
The incubation period is 3 to 6 days, and the incubation temperature is around 37°C. The culture medium components include CHIR99021, FGF2, Ascorbic acid, ITS (Insulin, transferrin, selenium), and FBS (fetal bovine serum). The culture medium is changed as needed. In step 1 and step (i), it is preferable to use FBS at a concentration of approximately 1-10% (w/v) to maintain the shape of the aggregates.

(ii) Step 2
The culture period is 3 to 6 days, and the culture temperature is around 37°C. The culture medium components include BMP4, TGFb1, GDF5, FGF2, Ascorbic acid, ITS, and FBS. The medium is changed as needed. In step 2 and process (ii), using FBS at a concentration of approximately 1-10% (w/v) allows for the formation of smooth, uniform cartilage tissue of the desired thickness without deformation of the cartilage tissue. Histologically, the production of cartilage matrix stained red with safranin O and blue with Alcian blue can be confirmed.

(iii) Step 3
The culture period is 6 to 8 weeks, and the culture temperature is around 37°C. The culture medium components include BMP4, TGFb1, GDF5, Ascorbic acid, ITS, and FBS. The medium is changed as needed. In step 3 and step (iii), it is preferable to use FBS at a concentration of approximately 1-10% (w/v) to obtain cartilage tissue of the desired shape, uniformity, and smoothness. A differentiation induction medium containing 10% (w/v) FBS may be referred to as "STEP3 + 10% FBS" below. The obtained cartilage tissue stains red with safranin O and blue with Alcian blue, allowing for confirmation of cartilage matrix production.

The inventors prepared culture media that did not contain TGFb1 or BMP4 and performed differentiation induction. The culture period for STEP 3 was divided into the first 3 weeks and the second 3 weeks, and culture was performed in each period using STEP 3 + 10% FBS, STEP 3 + 10% FBS - TGFb1, and STEP 3 + 10% FBS - BMP4.
In the first three weeks, using STEP3 + 10% FBS-TGFb1 resulted in expansion of the cartilage tissue. This expansion became more pronounced with larger cartilage tissue.
On the other hand, such deformation did not occur when STEP3+10%FBS and STEP3+10%FBS-BMP4 were used during the first three weeks. Therefore, TGFb1 is considered 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 during the first three weeks, hypertrophic cartilage markers such as RUNX2, Col10, and IHH were elevated, but not when STEP3+10%FBS or STEP3+10%FBS-BMP4 were used. This suggests that BMP4 acts on cartilage hypertrophy, while TGFb1 suppresses it.
Based on the above, the preferred culture medium for the first three weeks is a medium consisting of BMP4, TGFb1, GDF5, Ascorbic acid, ITS, and FBS, or a medium consisting of TGFb1, GDF5, Ascorbic acid, ITS, and FBS.

On the other hand, in the latter three weeks, there was almost no enlargement of the cartilage tissue even when TGFb1 was removed. In terms of cartilage tissue thickness, it increased in the order of STEP3 + 10% FBS - TGFb1, STEP3 + 10% FBS, and STEP3 + 10% FBS - BMP4, suggesting that BMP4 promotes cartilage hypertrophy, while TGFb1 inhibits it.
Based on the above, the preferred culture medium for the latter three weeks is a medium consisting of BMP4, GDF5, Ascorbic acid, ITS, and FBS; a medium consisting of BMP4, TGFb1, GDF5, Ascorbic acid, ITS, and FBS; or a medium consisting of TGFb1, GDF5, Ascorbic acid, ITS, and FBS.
The volume change rate of the shape-type 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.

The substrate surface may be flat or uneven, but a flat surface 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 commonly used for cell culture, such as Petri dishes, tissue culture dishes, and multi-dishes; flasks such as cell culture flasks and spinner flasks; bags such as plastic bags, Teflon® bags, and culture bags; plates such as microplates, microwell plates, multi-plates, and multi-well plates; chamber slides, tubes, trays, and roller bottles.

Examples of substrate materials include glass, metal, metal-containing compounds or metalloid-containing compounds, activated carbon, or resin. 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; noble metals: Cu, Ag, Au, etc.). Examples of metal-containing compounds or metalloid-containing compounds include ceramics (sintered bodies formed by heat treatment at high temperatures with a metal oxide as the basic component), semiconductors such as silicon, inorganic solid materials such as molded bodies of metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), metal borides or metalloid borides, etc.), aluminum, nickel-titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

The resin constituting the substrate may be a natural resin or its derivative, or a synthetic resin. Preferred natural resins or their derivatives include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), cellulose immobilized with dextran sulfate, etc. Preferred synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), 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®.
The substrate used in the present invention for manufacturing shaped cartilage tissue does not require high-temperature processing, and therefore, resins with low heat resistance can also be used. The substrate material may be one type or a combination of two or more types.

The substrate may have a surface that suppresses cell adhesion. In this case, it is preferable that the substrate surface exposed to the cell culture space is coated with a material that promotes cell adhesion. Such a coating material is given by the following formula (I):
[In the formula,
A repeating unit derived from a monomer represented by [U ^a1 and U ^a2 each independently represent 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, and R ^a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms], and the following formula (II):

[In the formula,
Examples include copolymers containing repeating units derived from monomers represented by [ ^Rb represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].
Preferably, the molar ratio of repeating units derived from the monomer represented by formula (I) to the total of repeating units derived from the monomer represented by formula (I) and repeating units derived from the monomer represented by formula (II) is 99 mol% to 51 mol%.
The above copolymer is further divided into the following formula (III):

[In the formula,
The structure may include a structural unit derived from a monomer represented by [ ^Rc and ^Rd each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, ^Re represent a linear or branched alkylene group having 1 to 5 carbon atoms, and n represent a number from 1 to 50].

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

[In the formula,
U ^a11 , U ^a12 , U ^b11 , U ^b12 , and U ^b13 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and ^An- represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions.
It is preferable that the coating film containing the above is included on at least a portion of its surface.

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 an aqueous solution using a method known to that effect. Such a primer is useful for promoting cell aggregate formation.

Aqueous solutions include water, saline solution, or phosphate buffer solution containing salts, or mixed solvents combining water or a salt-containing aqueous solution with an alcohol. Examples of alcohols include alcohols with 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, and 1-hexanol. Examples include 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 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, which may be used individually or as mixed solvents of these.

Furthermore, in addition to the copolymer and solvent, the primer may contain other substances as needed, provided that they do not impair the performance of the resulting primer film. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers to improve adhesion to containers or substrates, antifungal agents, and sugars.
The above solution can be applied to a substrate for 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 tissue are formed.

Furthermore, the coating on the surface of the substrate obtained by this method can be used in the manufacturing method of the present invention either as is without a drying process, or after washing with water or a medium for samples subjected to cell culture (e.g., water, buffer solution, culture medium, etc.).
The substrate may be subjected to a drying process after coating. The drying process is carried out under air or vacuum, preferably at a temperature in the range of -200°C to 200°C. By removing the solvent during the drying process, the coating is completely fixed to the substrate.

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 drying may be performed at, for example, 40°C to 50°C to form spots more quickly. If the drying temperature is below -200°C, it is necessary to use uncommon refrigerants, which is inconvenient, 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 preferable drying temperature is 10°C to 180°C, and a more preferable drying temperature is 20°C to 150°C.
The coating thickness is such that the maximum and minimum thicknesses are in the range of 1 to 1000 nm, preferably in the range of 5 to 500 nm.

In one preferred embodiment, the substrate used in the present invention is manufactured using a substrate having cell adhesion inhibitory properties. As such a substrate, commercially available cell culture dishes with low cell adhesion treatment, cell culture vessels having cell adhesion inhibitory properties, etc., may be used, but are not limited to, the cell culture vessel described in Japanese Patent Application Publication No. 2008-61609.
The fact that cartilage tissue prepared according to the present invention has been obtained can be confirmed, for example, by a positive result of safranin O staining, Alcian blue staining, safranin O staining, or toluidine blue staining.

<Use of cartilage tissue>
The cartilage tissue obtained by the manufacturing method of the present invention can be used for reconstructive surgery cartilage grafting when deformation or defects of cartilage tissue in the nose or ear occur due to congenital conditions, trauma, surgery, etc. Furthermore, since cartilage tissue of a desired shape can be obtained, it can be transplanted to damaged or defective areas of cartilage, such as in joints.

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

As a first step, we aimed to induce ring-shaped aggregation of ExpLBM seeds by coating the culture surface, which was fitted with a silicone mold and support structure, with 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. Various concentrations (6.25–100 μg/ml) of CAT polymer solution were added to the donut-shaped area (cell culture space) enclosed by the silicone, and after standing for 5 minutes, the polymer solution was completely aspirated to create a coated area (Figure 1). ExpLBM cells were seeded into this coated area at various densities (17.5–280 × ^10⁴ cells/ ^cm² ) and cultured. STEP1 medium, used for inducing chondrogenic differentiation of ExpLBM, was used as the culture medium. The results are shown in Figure 2.

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 was slow, and the aggregates developed holes or the rings broke apart. The smoothest and cleanest aggregation occurred at a seeding density of 200 × ^10⁴ cells/ ^cm² . Regarding the CAT polymer concentration, if it was too high, such as 100 μg/ml, aggregation was suppressed, depending on the seeding density. Good aggregation was observed at concentrations between 6.25 and 50 μg/ml. Furthermore, a high seeding density was necessary to achieve clean, band-like aggregation.
Based on these results, subsequent experiments were conducted under optimal conditions: a CAT concentration of 12 μg/ml and a seeding density of 200 × ^10⁴ cells/ ^cm² .

Next, we investigated methods for inducing differentiation of the ring-shaped aggregates into cartilage.
The differentiation protocol used the culture media described in STEP 1 above and STEP 2-3 below.
STEP2 medium: CDM2 basal medium + 10 ng/mL, FGF2 + 50 μg/mL, ascorbic acid + 30 ng/mL, BMP4 + 10 ng/mL, TGFβ1 + 10 ng/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). In particular, the STEP 3 medium has the effect of promoting cartilage matrix production, and it takes 6 weeks or more to produce mature cultured cartilage.

When cultured in conventional STEP 1-3 medium, the ring-shaped aggregates expanded in inner diameter and grew into irregular cartilage with protrusions at the edges (Figure 3). Histologically, the production of cartilage matrix stained red with safranin O and blue with Alcian blue was confirmed.
Next, ring-shaped aggregates were subjected to cartilage differentiation induction using STEP 3 medium supplemented with various concentrations of FBS (0, 0.1, 1, 10%).
In the case using STEP3 + 10% FBS, no ring deformation was observed, and a smooth, uniform ring-shaped cartilage with a thickness of 1-2 mm was formed. Histologically, the production of cartilage matrix stained red with safranin O and blue with Alcian blue was confirmed (Figure 4).
Furthermore, the addition of FBS was investigated in STEP 1 and STEP 2. In STEP 1, it was shown that adding 10% FBS resulted in smoother and more uniform aggregation, while in STEP 2, a lower concentration of FBS 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.

Next, we investigated culture conditions to induce more mature cartilage.
Conventional STEP3 medium contains growth factors TGFb1, BMP4, and GDF5. However, a medium without either TGFb1 or BMP4 was prepared and used for differentiation induction. The STEP3 culture period was divided into two 3-week periods, and culture was performed using STEP3 + 10% FBS, STEP3 + 10% FBS - TGFb1, and STEP3 + 10% FBS - BMP4 in each period.
During the first three weeks, using STEP3+10%FBS-TGFb1 resulted in an increase in ring diameter. This was more pronounced when creating larger rings, making it difficult to control the morphology of the ring itself (Figure 5).

On the other hand, no such deformation occurred when using STEP3 + 10% FBS and STEP3 + 10% FBS - BMP4 (Figure 5).
Based on the above, it is considered that TGFb1 is essential for maintaining the ring morphology during the first three weeks.
Furthermore, gene expression analysis using qPCR revealed that when STEP3+10%FBS-TGFb1 was used during the first three weeks, hypertrophic cartilage markers such as RUNX2, Col10, and IHH were elevated, but these were not elevated when STEP3+10%FBS or STEP3+10%FBS-BMP4 were used. This suggests that BMP4 acts on cartilage hypertrophy, while TGFb1 inhibits it.
On the other hand, in the latter three weeks, there was almost no expansion of the ring diameter even when TGFb1 was excluded. In terms of cartilage thickness, it increased in the order of STEP3 + 10% FBS - TGFb1, STEP3 + 10% FBS, and STEP3 + 10% FBS - BMP4, suggesting that BMP4 promotes cartilage hypertrophy and TGFb1 inhibits it (Figure 6).

Based on the results described above, it was found that by using STEP3 + 10% FBS or STEP3 + 10% FBS-BMP4 in the first three weeks and STEP3 + 10% FBS-TGFb1 in the latter half, it is possible to maintain the shape and produce thicker, more mature cartilage (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 could create mature cartilage both histologically (safranin O, Alcian blue) and in terms of gene expression (Col2) (Figure 7).

As described above, we developed a method for producing mature small cartilage rings, and next we applied this method to produce giant cartilage rings (Figure 8).
The method involved changing the silicone frame and support used to larger ones, and performing CAT coating, ExpLBM seeding, and cartilage differentiation induction using the same procedure as described above.
Furthermore, ring-shaped aggregates of ExpLBM, prepared using the same procedure as described above, were removed from the support at the appropriate time and differentiated by inserting them into a silicone tube, thereby creating tubular cartilage with fused rings. The results are shown in Figure 9.
By using support struts with inner diameters of 14 mm and 20 mm, it was possible to produce uniform ring-shaped cartilage with a thickness of 1 to 2 mm, as shown in Figure 9. By increasing the size of the support struts, it is possible to produce ring-shaped cartilage of any size.

In the preparation of tubular cartilage, six ring-shaped aggregates were collected on the 9th day after the start of aggregation and cultured in a silicone tube. As a result, all the aggregates fused together, forming tubular cartilage approximately 5 mm in length and 1 mm thick. Figure 9(b) on the right shows a fragment of the tubular cartilage after cutting.
In all types of cartilage, the production of cartilage matrix was confirmed by safranin O and Alcian blue staining.
By making the silicone frame and support elliptical, an elliptical ring-shaped cartilage can also be fabricated using a similar procedure. Figure 10 (top) shows an elliptical cartilage with a thickness of 2 mm, a major axis of 30 mm, and a minor axis of 15 mm.

Furthermore, by using a track-shaped (with straight sections) silicone frame and support, it is also possible to create linear cartilage (Figure 10 (bottom)).
By overlapping these linear cartilages, it is also possible to create plate-like cartilage whose length and width can be adjusted.
While it has been confirmed that transplanting cultured cartilage subcutaneously leads to greater maturation of the cartilage matrix and increased strength, there are concerns about the risk of hypertrophy and ossification.
To prevent this, the culture period in STEP 3 of the differentiation protocol (STEP 1-3) was divided into three periods: 0-3 weeks, 3-9 weeks, and 9-15 weeks, and culture was performed using the differentiation medium shown in Figure 11.
Cells were seeded at a rate of 1.0 × ^10⁵ cells/well in a 96-well U-bottom culture plate (low cell adhesion), and a cell pellet was prepared by centrifugation at 2000 rpm for 5 minutes. This pellet was then differentiated using the culture media described in STEP 1-3.
For weeks 0-3 of STEP 3, STEP 3 + 10% FBS was used; for weeks 3-9, 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 for weeks 9-15, DMEM(HG) + 10% FBS was used.

The cartilage globules produced using these protocols were transplanted subcutaneously into the dorsal region 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, cultured cartilage appeared as a high-signal area, but this changed to a low-signal area in the areas where ossification occurred after transplantation. Based on this change in signal, the volumes of the cartilage and ossified portions were also calculated.

In the case using STEP3+10%FBS-TGFb1 during weeks 3-9 of STEP3 (No. 9), the volume was reduced to less than 10% after transplantation due to resorption, but the other samples maintained their volume until 13 weeks post-transplantation without hypertrophy or resorption. Furthermore, there was a tendency for ossification to progress according to the BMP4 content; in No. 14 (containing 10 times the amount of BMP4), almost the entire portion was ossified, but in No. 13 (containing the normal amount of BMP4), only 30% was ossified, and in No. 10 (containing no BMP4), no ossification was observed.
Based on the above, it was suggested that culturing in DMEM(HG) + 10% FBS towards the end of the culture period could prevent hypertrophy after transplantation, and that reducing the BMP4 content could prevent or mitigate ossification (Figure 11).

By inactivating the cells contained in cultured cartilage and using them as an extracellular matrix product, it is possible to resolve the risks of changes in shape and ossification after transplantation, as well as tumorigenicity, which is a concern specific to iPS-derived products. This could result in a safer product that is more likely to gain social consensus.
For this reason, the inventors inactivated the cells by fixing the cartilage ring prepared by the aforementioned method with glutaraldehyde. Specifically, the prepared cultured cartilage was immersed in 2% Sterihyde® for one day, washed with PBS, and then transplanted subcutaneously into the dorsal region of immunodeficient mice (NOD scid). Changes in volume and shape after transplantation were evaluated using MRI T2 fat-suppressed images, but no changes such as resorption or hypertrophy were observed up to 12 weeks after transplantation. Furthermore, the cartilage removed 12 weeks after transplantation maintained the same shape as before transplantation. Histologically, there was a slight decrease in staining for safranin O, but no major structural changes were observed. Kossa staining showed diffuse punctate calcification (Figure 12).

The above findings suggest that while treating cultured cartilage with glutaraldehyde carries a risk of calcification, it may prevent changes such as resorption, hypertrophy, and ossification, allowing it to remain subcutaneously while maintaining its original shape.
Furthermore, adding high-concentration ethanol treatment after glutaraldehyde treatment is expected to remove aldehyde residues and phospholipids, thereby preventing calcification.
Specifically, the rat costal cartilage was immersed in 0.6% glutaraldehyde for one day, then in 0.2% glutaraldehyde for more than one week, followed by shaking in high-concentration ethanol for one day, and finally soaking in physiological saline for one day to remove the ethanol. Rat costal cartilage treated in this way showed no calcification at two months after transplantation into the subcutaneous tissue of mice (not shown).

It is expected that treating the cartilage with glutaraldehyde will cross-link the collagen fibers and increase its strength, but we investigated whether it could withstand processing such as suturing and fixation.
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 immersed in 0.2% glutaraldehyde for more than one week, followed by shaking in high-concentration ethanol for one day, and finally immersed in physiological saline for one day to remove the ethanol.
After fixation, the cultured cartilage was fixed to the mold in its desired shape. It was also possible to easily fix this to a silicone plate using 5-0 nylon (Figure 13).
Based on the above, it was concluded that glutaraldehyde treatment contributes to increasing the strength of cultured cartilage by cross-linking collagen, and facilitates processing such as suturing and fixation.

Claims (5)

A method for preparing a morphologically shaped cartilage tissue, comprising seeding chondrocytes in a cell culture space formed by a mold on a substrate, culturing them to create cell aggregates, and further culturing them in a cartilage differentiation induction medium , wherein ossification when transplanted into vivo is suppressed by culturing the chondrocytes with a reduced BMP4 concentration for a certain period at the end of the culture period . A method for preparing a shaped cartilage tissue according to claim 1, comprising seeding chondrocytes in a cell culture space formed by a mold provided on a substrate, culturing them to produce cell aggregates, and further culturing them in a cartilage differentiation induction medium, and removing the shaped cartilage tissue obtained by culturing the chondrocytes in the cartilage differentiation induction medium, and treating it with glutaraldehyde and/or ethanol. A method for preparing a shape-type cartilage tissue according to claim 1 or 2, wherein the seeding density of chondrocyte precursor cells is 1.4 × ^10⁶ to 2.8 × ^10⁶ cells/ ^cm² . A method for preparing a shaped cartilage tissue according to any one of claims 1 to 3, comprising coating a substrate portion in the cell culture space with a material that promotes cell adhesion, and then culturing chondrocytes. A method for preparing a shape-type cartilage tissue according to any one of claims 1 to 4 , comprising culturing chondrogenic cells in a chondrogenic differentiation-inducing medium to produce aggregates, stacking multiple aggregates and culturing them further to obtain a cartilage tissue.