Method for creating three-dimensional cell aggregate having linear shape

Abstract

Agglomeration and deformation that occur during culture of a three-dimensional cell aggregate are suppressed to create a three-dimensional cell aggregate having a linear shape. A method for producing artificial cartilage tissue, the method comprising: agglomerating cartilage progenitor cells to form cartilage progenitor spheroids; seeding the cartilage progenitor spheroids along grooves in a membrane having the grooves; culturing the cartilage progenitor spheroids in a state in which a culture medium is supplied from the obverse side and the reverse side of the membrane to fuse the spheroids together; and then maturing the spheroids into cartilage-like tissue and / or long bone progenitor cartilage organoids.

Description

A method for creating a three-dimensional cell aggregate with a linear shape.

[0001] This invention relates to a method for creating a three-dimensional cell aggregate with a linear shape.

[0002] Until now, the only curative treatments for bone defects in the craniofacial region and limbs have been autologous bone grafting and artificial bone grafting. However, these methods have drawbacks, such as limitations on the bone tissue that can be harvested, postoperative pain at the harvest site, and insufficient ossification of the artificial bone. Therefore, a method has been developed to culture stem cells and differentiate them into bone tissue in vivo (Non-Patent Literature 1). However, problems remain, such as the lack of cortical bone, insufficient ossification, and the limitation of the resulting tissue to a round shape.

[0003] Implantation of Human-Induced Pluripotent Stem Cell-Derived Cartilage in Bone Defects of Mice. Yuki Iimori, Miho Morioka, Saeko Koyamatsu, and Noriyuki Tsumaki. Tissue Engineering Part A 2021 27:21-22, 1355-1367

[0004] Traditionally, when culturing a three-dimensional cell aggregate in a linear shape, cell aggregation occurs, causing the aggregate to deform, shrink, and deviate from its linear shape. Furthermore, if a template is used to avoid this, cells in contact with the template cannot receive culture medium or gas exchange, leading to necrosis.

[0005] The present invention aims to suppress aggregation and deformation that occur during the culture of three-dimensional cell aggregates, thereby creating three-dimensional cell aggregates with a linear shape.

[0006] The inventors considered creating linear long bone organoids from chondrocytes via long bone precursor cartilage organoids by aggregating chondrocytes to form small spheroids, and then arranging these spheroids on a membrane of a cell culture insert. However, when chondrocytes are aggregated on a plane to form spheroids, the aggregation results in a curved tissue rather than a linear shape, and the compression necessary for bone differentiation is not achieved. To solve these problems, the inventors considered arranging the fabricated spheroids along grooves created in a membrane. As a result, the fabricated tissue is sandwiched between linear grooves, limiting horizontal curvature and enabling culture along a straight line. At the same time, the tissue is compressed as it is placed in the grooves. Using this method, after 15 to 30 days of culture, the tissue progressed to cartilage-like tissue, and after 56 to 90 days of culture, it became long bone precursor cartilage organoids, which are bone precursors. When this tissue was transplanted into a living organism, it became a linear long bone organoid with cortical bone on the outside and cancellous bone on the inside. This invention was completed based on these findings.

[0007] The gist of the present invention is as follows: (1) A method for producing artificial cartilage tissue, comprising: aggregating chondrocytes to form chondrocyte spheroids; seeding the chondrocyte spheroids along grooves on a membrane having grooves; culturing the chondrocyte spheroids while supplying culture medium from the front and back sides of the membrane to fuse the spheroids together, and then maturing them into cartilage-like tissue and / or long bone chondrocyte organoids. (2) The method according to (1), wherein the cross-sectional shape of the grooves is V-shaped, U-shaped, or U-shaped with an upward opening. (3) The method according to (1), wherein the membrane is supported by a support structure. (4) The method according to (1), wherein the culture of the chondrocyte spheroids is performed by shaking culture. (5) The method according to (1), wherein the chondrocytes are cells differentiated from embryonic stem cells and / or induced pluripotent stem cells. (6) The method according to (1), wherein the chondrocytes are cells differentiated from chondrocytes harvested from the perichondrium. (7) The method according to (1), wherein the cartilage precursor spheroid is 20 to 1000 μm in diameter. (8) The method according to (1), wherein one cartilage precursor spheroid contains 100 to 7500 cartilage precursor cells. (9) The method according to (1), wherein 10 to 5000 cartilage precursor spheroids are seeded per 1 cm of groove. (10) The method according to (1), wherein the cartilage precursor spheroid is produced by culturing cartilage precursor cells in a culture medium having a cell non-adherent surface. (11) The method according to (1), wherein the cartilage precursor spheroid is produced by culturing cartilage precursor cells in a medium containing TGF-β and bFGF and agglutinating them. (12) The method according to (1), wherein after fusing the spheroids together, they are cultured in a medium containing BMP to mature into long bone precursor cartilage organoids. (13) The method according to (12), wherein the culture period in the medium containing BMP is 40 to 100 days. (14) A method for producing a long bone organoid, comprising transplanting a long bone precursor cartilage organoid produced by the method described in (12) into a non-human animal and allowing it to mature into a long bone organoid. (15) The method according to (14), wherein the long bone organoid has cortical bone, periosteum, cancellous bone and bone marrow. (16) Artificial cartilage tissue produced by the method described in (1). (17) Artificial cartilage tissue according to (16), wherein the artificial cartilage tissue is a long bone precursor cartilage organoid. (18) A long bone organoid produced by the method described in (14).(19) A composition comprising artificial cartilage tissue prepared by the method described in (1), wherein the composition is used to be transplanted into a living organism to compensate for a deficiency of bone tissue in the living organism. (20) The composition according to (19), wherein the artificial cartilage tissue prepared by the method described in (1) is a long bone progenitor cartilage organoid. (21) A composition comprising a long bone organoid prepared by the method described in (14), wherein the composition is used to be transplanted into a living organism to compensate for a deficiency of bone tissue in the living organism. (22) A culture vessel for producing artificial tissue, characterized by comprising a membrane having grooves and a support structure that supports the membrane.

[0008] The present invention makes it possible to create linear long bone progenitor cartilage organoids in a test tube. The present invention also makes it possible to create linear long bone organoids having cortical bone, periosteum, cancellous bone, and bone marrow in vivo. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2024-176230, which forms the basis of the priority of this application.

[0009] An example of a membrane structure with grooves is shown. Cartilage tissue cultured with a membrane with grooves and cartilage tissue cultured with a membrane without grooves are shown. Results of comparing the linearity of both tissues using ImageJ. Gene expression of long bone progenitor cartilage organoids is shown. CT images of linear long bone organoids are shown. High-resolution CT images of axial sections of long bone organoids are shown. Histological images of long bone organoids stained with safranin O are shown. Histological images of long bone organoids stained with immunohistochemistry (CD31, CD71, αSMA, PERILIPIN) are shown. Histological images of long bone organoids stained with immunohistochemistry (c-Kit) are shown.

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

[0011] The present invention provides a method for producing artificial cartilage tissue, which includes aggregating chondrocytes to form chondrocyte spheroids, seeding the chondrocyte spheroids along grooves on a membrane having grooves, culturing the chondrocyte spheroids while supplying culture medium from the front and back sides of the membrane to fuse the spheroids together, and then allowing them to mature into cartilage-like tissue and / or long bone progenitor cartilage organoids.

[0012] Generally, in tissue culture, small tissues do not have problems with the penetration of the culture medium into the center. However, as tissues become larger, the culture medium cannot reach the center, preventing the delivery of cytokines and nutrients necessary for differentiation, which is a fatal obstacle to tissue growth. To solve this, agitation culture is used, that is, by shaking or rotating the culture vessel, to apply flow pressure to the tissue and allow nutrients to penetrate to the center of the tissue. However, these culture methods lack a support mechanism, so due to intercellular interactions, the tissue becomes close to a spherical shape, and the problem arises that the linear shape cannot be maintained. On the other hand, there is a culture method with a support mechanism, which is a prior patent, in which tissue is cultured on a flat-bottom membrane. However, when culturing tissue on a flat-bottom membrane, the tissue is above the membrane, i.e., above the liquid surface, so even if shaken, the culture medium does not come into contact with the tissue and therefore no flow pressure is applied. Furthermore, because there is no lateral support when shaken, the tissue rolls around on the membrane. Therefore, the development of a culture method that fixes the tissue while applying flow pressure to it is necessary for the creation of large linear tissues. To solve this problem, we developed the V-shaped base membrane technology. When culturing tissue with a V-shaped base membrane, the tissue is sandwiched between the membrane from the sides, so even when shaken, the tissue remains below the liquid surface, and flow pressure is applied to the tissue by shaking. Also, because both sides are sandwiched between the membrane, it does not roll. In this way, the V-shaped base membrane allows for shaking culture while maintaining a linear shape, making it an important technology for creating the ultra-large linear tissues required for practical applications. In the method of the present invention, spheroids containing chondrocyte precursor cells are preferably formed into a linear shape with high accuracy by seeding them on a membrane with a groove structure and compressing them with gravity. After fusing the spheroids together in vitro, they can be matured into linear cartilage-like tissue, and further into long bone precursor cartilage organoids (hypertrophic cartilage-like tissue). By transplanting long bone precursor cartilage organoids into the body, linear long bone organoids can be produced.

[0013] 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.

[0014] 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 fifth 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 per week. Chondrogenic cells that have been passaged 0-5 times should be used.

[0015] 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, marmosets, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0016] 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 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 being changed 2-4 times per week. Chondroprogenitor cells that have undergone 0-5 passages should be used.

[0017] 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, marmosets, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0018] 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.

[0019] 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.

[0020] Furthermore, the bottom of the culture device should preferably have numerous hemispherical or frustoconical depressions. For example, hemispherical or frustoconical depressions (with a depression 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 leaving 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.

[0021] 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. 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 TGFβ (TGFβ1 or TGFβ3) and bFGF may be added. Furthermore, Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3) may also be added. Other substances that may be added include antibiotics / antifungal agents, ITS-X, PDGFBB, serum, L-ascorbic acid, dexamethasone, and insulin growth factor.

[0022] Culture can be carried out using any of the following methods: batch culture, semi-batch culture (fed-batch culture), or continuous culture (perfusion culture). Static culture, aerated culture, stirred culture, shaking culture, or rotation culture are also acceptable, but static culture is preferred.

[0023] The cell culture temperature for spheroid formation is preferably 30-40°C, and more preferably 37°C.

[0024] 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.

[0025] 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.

[0026] The diameter of the spheroid is often 20 to 1000 micrometers, and preferably 200 to 350 μm.

[0027] In the present invention's method for producing artificial cartilage tissue, spheroids containing chondrocyte precursors are formed linearly by seeding them along grooves on a membrane having grooves. The membrane serves as a support for the spheroids and is preferably permeable to allow for the exchange of culture medium and gases. The spheroids seeded on the membrane form linear aggregates along the grooves. Because the aggregates are sandwiched between the grooved membranes, they cannot bend to the left or right, thus maintaining their linear shape. Furthermore, because they are dropped into the grooves, the aggregates can be subjected to compression. In addition, the cells are in contact with the membrane, and since the membrane allows for the exchange of culture medium and gases, the cells do not die.

[0028] The membrane should have grooves at an angle of 30° to 150° from the apex (bottom end) of the bottom to the opening at the top. The cross-sectional shape of the grooves should be upward-opening, preferably V-shaped, U-shaped, or an upward-opening U-shape (membranes with V-shaped, U-shaped, or upward-opening U-shaped cross-sections are sometimes called V-shaped membranes or V-bottom membranes). It is also preferable that the cross-section is narrower at the bottom and wider at the top. As can be seen from the growth from a small fetus to an adult, tissues dynamically change size as they proliferate and differentiate, and there are individual differences in size. For culturing tissues that change size, V-shaped membranes are preferable because they can respond to these changes in real time, making them suitable not only for large tissues but also for various growing tissues. U-shaped or upward-opening U-shaped membranes can be adapted to changes in size by adjusting the curvature of the U-shape or the bottom area of ​​the U-shape, but the adjustment is more complicated than that of V-shaped membranes. The membrane should be folded to create V-shaped, U-shaped, or upward-opening concave grooves. Furthermore, the membrane is preferably supported by a support structure. The support structure is preferably a structure that can support the membrane while maintaining the shape of the grooves, and has a shape and strength such that the membrane can contact the culture medium from both its front and back sides at least at the bottom of the grooves where the spheroids are seeded. For example, it is a frame that surrounds and supports the membrane having grooves. The material of the support structure may be any material that has sufficient strength to support the membrane, such as polyester, polypropylene, polyvinyl chloride, aluminum, or stainless steel. The membrane having grooves is placed in the culture medium while supported by the support structure, and the spheroids are preferably seeded in a straight line along the grooves.

[0029] The present invention also provides a culture vessel for producing artificial tissue, characterized by comprising a membrane having grooves and a support structure for supporting the membrane. In addition to the membrane and the support structure for supporting the membrane, the culture vessel of the present invention may also include a container for holding a culture medium.

[0030] In order to solve the problems of the present invention, a membrane (pore membrane) having pores through which a medium and gas can be exchanged is bent so as to form V-shaped (V-bottom), U-shaped (round bottom), or concave-shaped (concave bottom) grooves, and cells are seeded along the grooves. The seeded cells can form a linear aggregate along the grooves.

[0031] The membrane may be one through which medium components can pass, is non-toxic to spheroids, and through which spheroids cannot pass. For example, if the membrane has a porous membrane structure, it is considered advantageous for culturing after fusion in terms of the ability to supply nutrients to the fusion-type spheroids from above and below and oxygen supply. Examples of the membrane include those whose surface has been negatively charged and made hydrophilic by atmospheric corona discharge or vacuum gas plasma polymerization treatment (cell adhesion surface treatment), those whose surface has been gelatin-treated, those coated with an extracellular matrix (such as collagen, laminin, fibronectin, etc.) or mucopolysaccharide (such as heparin sulfate, hyaluronic acid, chondroitin sulfate, etc.), those coated with a basic synthetic polymer (such as poly-D-lysine, etc.), those having a synthetic nanofiber surface, those having a surface of a hydrophilic and neutral hydrogel layer, collagen membranes (Koken), etc. When the membrane has a porous membrane structure, the pore size is preferably 0.4 to 8 μm. As the membrane, those obtained by bending a membrane (it4ip) etc. can be preferably used. The spheroids can be seeded along the grooves on the membrane using a pipette or a spatula. The material of the membrane is preferably polycarbonate, polyester, or polyimide, and the thickness of the membrane is preferably 6 to 50 μm.

[0032] The spheroids may be seeded on the membrane at a high density. High density means, for example, in the case of spheroids with a diameter of 150 μm, the number of spheroids present per 1 cm 3 area is 9.5 x 10 4 to 5 3.8 x 10 5 and is preferably 5 1.9 x 10 5 to 5 3.8 x 10 3 and more preferably ５ 2.9 x 10 TM to ６ 3.8 x 10 TM cells.

[0033] The number of spheroids is preferably 2 or more. By increasing the number of spheroids, larger fused spheroids can be produced. It is advisable to seed 10 to 5000 spheroids per 1 cm of the groove.

[0034] In the method of the present invention, after shaping into a linear shape while seeding spheroids containing chondrogenic progenitor cells along the grooves on the membrane, the spheroids are cultured while supplying a culture medium from the front and back sides of the surface where the spheroids containing chondrogenic progenitor cells are seeded, thereby fusing the spheroids together.

[0035] For example, after seeding spheroids containing chondrogenic progenitor cells along the grooves of the membrane, a culture medium is added to the lower part of the membrane, and the culture plate is left stationary in an incubator with the spheroids on the membrane immersed in the culture medium to fuse the spheroids together. [[ID=1st]]

[0036] "Fusion of spheroids" means that a plurality of spheroids form a continuous structure, and disappearance of the contours of individual spheroids is confirmed. By fusing the spheroids together, the spheroids become larger. Therefore, by maturing the fused spheroids (that is, inducing differentiation of chondrogenic progenitor cells in the fused spheroids into chondrocytes), a large cartilage-like tissue can be produced. [[ID=1st]]

[0037] The medium used for the fusion of spheroids only needs to be suitable for the fusion of spheroids, and it is preferable to use the medium for the three-dimensional culture of the above chondrogenic progenitor cells. 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), F-10 Ham with TGF-β (TGFβ1 or TGFβ3) and bFGF added. In addition, antibiotics-antifungal agents, ITS-X, PDGFBB, serum, L-ascorbic acid, dexamethasone, Insulin Growth Factor, Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3) may be added.

[0038] The culture may be any of static culture and shaking culture, but shaking culture is preferable.

[0039] The culture temperature for spheroid fusion is preferably 30-40°C, and more preferably 37°C. The culture period for spheroid fusion is preferably 12 hours to 4 days, and more preferably 12 hours to 1 day. It is good to change the culture medium every 3 days, and after confirmation of spheroid fusion, it is good to change the culture medium with the same medium as above for 3 weeks. If the culture is continued after the spheroids have fused, they will mature into linear cartilage-like tissue. Cartilage-like tissue is tissue that has chondrocytes and extracellular matrix such as proteoglycans surrounding them. Further maturation of the cartilage-like tissue will result in long bone progenitor cartilage organoids (hypertrophic cartilage-like tissue). To mature cartilage-like tissue into long bone progenitor cartilage organoids (hypertrophic cartilage-like tissue), it is best to culture it in a medium that can mature it into hypertrophic cartilage tissue. Examples include 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), and F-10 Ham, to which BMP (BMP4 or BMP2) has been added. In addition, antibiotics-antifungal agents, ITS-X, TGF-β (TGFβ1 or TGFβ3), bFGF, PDGFBB, serum, L-ascorbic acid, dexamethasone, and insulin growth factor may also be added. Furthermore, L-proline and Wnt / β-catenin inhibitors (Wnt-C59, IWP1, IWP2, IWP3) may be added. Long bones are elongated, tubular bones found in the limbs. The outer periphery consists of dense cortical bone, while the interior contains cancellous bone, adipocytes, and bone marrow (hematopoietic cells and their surrounding hematopoietic niche). The cortical bone is covered by the periosteum. Long bones arise from the ossification of tissue composed of rod-shaped, mature cartilage (hypertrophic cartilage).Long bone progenitor cartilage organoids (hypertrophic cartilage-like tissue) are cartilage that mimics mature, rod-shaped cartilage and has the potential to differentiate into long bone organoids in the future.

[0040] The culture temperature for cartilage-like tissue to mature into long bone precursor cartilage organoids is preferably 30-40°C, and more preferably 37°C. The culture period for cartilage-like tissue to mature into long bone precursor cartilage organoids is preferably 40-100 days, and more preferably 60-80 days. The culture medium should be changed every 2-3 days.

[0041] Maturation into long bone progenitor cartilage organoids can be confirmed by qPCR through the expression of the ACAN, COL10A1, and RUNX2 genes.

[0042] The present invention allows for the production of cylindrical artificial cartilage tissue (e.g., long bone progenitor cartilage organoids), with a diameter of 0.5 mm or more, 2 mm or more, 10 mm or more, or 30 mm or more, with an upper limit of approximately 50 mm. The length can be any length, for example, 2 mm or more, 30 mm or more, 100 mm or more, or 300 mm or more, with an upper limit of approximately 500 mm. The present invention also provides artificial cartilage tissue produced by the above method, such as cartilage-like tissue or long bone progenitor cartilage organoids. The long bone progenitor cartilage organoids of the present invention preferably have a diameter of 1 mm or more with an upper limit of approximately 50 mm, and the length can be any length, but preferably 30 mm or more with an upper limit of approximately 500 mm. Long bone progenitor cartilage organoids, which have a diameter of 1 mm or more with an upper limit of approximately 50 mm and a length of 30 mm or more with an upper limit of approximately 500 mm, can be fabricated from 10 to 50,000 spheroids with a diameter of 20 to 500 μm.

[0043] The appropriate sinuosity index for long bone progenitor cartilage organoids is 1.0 to 1.1. The linearity of long bone progenitor cartilage organoids is determined by cutting out a tissue portion from an image using ImageJ and identifying the central axis of the tissue using ImageJ's "Skeletonize" function. The sinuosity index is calculated by dividing the length of this central axis by the length of the straight line connecting the two ends of the central axis.

[0044] Long bone progenitor cartilage organoids matured in vitro can be transplanted into non-human animals and matured into long bone organoids. Examples of non-human animals include mice, rats, pigs, monkeys, and marmosets. Subcutaneous transplantation of long bone progenitor cartilage organoids can further develop into long bone organoids containing cortical bone, cancellous bone, and bone marrow. The transplantation period is often 30 days or longer, preferably 120 days or longer. The maturation from long bone progenitor cartilage organoids to long bone organoids can be confirmed by the detection of rod-shaped bone tissue by micro-CT imaging, the presence of cortical bone surrounding the periphery and cancellous bone inside, and the presence of cortical bone and trabeculae within it in tissue staining. Furthermore, long bone organoids may possess hematopoietic function. This can be confirmed by the presence of bone marrow-specific CD31-positive sinusoidal structures, clusters of CD71-positive erythroblasts, αSMA-positive arterioles, c-Kit-positive hematopoietic progenitor cells, and PERILIPIN-positive adipocytes. Long bone organoids are elongated, tubular bone tissues that have the characteristics of long bones, with a dense cortical bone outer layer surrounded by a periosteum, and containing cancellous bone, adipocytes, and bone marrow (hematopoietic cells and their surrounding hematopoietic niche) inside.

[0045] The method of the present invention makes it possible to produce long bone organoids having widths of 2 mm or more, 6 mm or more, 40 mm or more, 60 mm or more, and 80 mm or more, with an upper limit of 100 mm, and thicknesses of 0.5 mm or more, 1 mm or more, 5 mm or more, and 15 mm or more, with an upper limit of 100 mm. The length can be any length, for example, 2 mm or more, 30 mm or more, 100 mm or more, and 300 mm or more, with an upper limit of approximately 500 mm.

[0046] Artificial cartilage tissue (e.g., long bone precursor cartilage organoids) and long bone organoids produced by the method of the present invention can be used for the treatment of bone defects and bone deformities throughout the body, cosmetic surgery for the purpose of facial augmentation, and treatment of hematopoietic dysfunction using the hematopoietic capacity of the bone marrow region. The present invention also provides compositions containing the above-mentioned artificial cartilage tissue (e.g., long bone precursor cartilage organoids) or long bone organoids.

[0047] Specifically, the artificial cartilage tissue (e.g., long bone progenitor cartilage organoid) or long bone organoid produced by the method of the present invention is transplanted into areas of bone defects such as congenital facial deformities or facial trauma caused by traffic accidents to treat the condition. It can also be used to replace bone in mechanically severed fingers and to treat bone defects that occur during bone resection in osteosarcoma.

[0048] The present invention will be described in more detail below with reference to examples. [Examples] In vertebrate development, limb buds, which are the precursors of cartilage, differentiate and grow into linear cartilage, which then matures into cartilage (hypertrophic cartilage) that forms the basis of long bones, and when it ossifies, it becomes a long bone. In this example, chondrocyte precursors were aggregated to form chondrocyte precursor spheroids, and the chondrocyte precursor spheroids were seeded along the grooves on a membrane having grooves. After fusing the spheroids together by supplying culture medium from the front and back sides of the membrane, the culture was further continued to mature them into linear cartilage-like tissue, and then further matured into long bone precursor cartilage organoids (hypertrophic cartilage-like tissue). These were then transplanted into rats and implanted, successfully causing them to ossify into long bone organoids. The long bone organoids were rod-shaped, with cortical bone on the outer circumference, and contained cancellous bone, adipocytes, and bone marrow (hematopoietic cells and their surrounding hematopoietic niche) inside. The cortical bone was covered with periosteum, mimicking long bones. Long bones are formed when tissue composed of rod-shaped mature cartilage (hypertrophic cartilage) ossifies. Long bone progenitor cartilage organoids (hypertrophic cartilage-like tissue) are tissues that mimic this rod-shaped mature cartilage and possess the ability to differentiate into long bone organoids.

[0049] Human iPS cell culture method: 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 10 cells were cultured per 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 the 7th day. 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 chondrocyte precursor cells.

[0050] Method for inducing differentiation from human iPS cells to human mesodermal cells: 1.5 ml of AK02 medium (Ajinomoto) containing 7 μl of iMatrix-511 (Nippi) and 1.5 μl of Y-27632 (Wako) was added to each well of a 6-well plate and incubated in a 37°C incubator for 1 hour. 1 to 1.5 x 10⁶ human iPS cells were then added to each well of the plate. ５The seeds were sown. 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 next 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 replaced with 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 culture medium was replaced with 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 incubated for 2 days.

[0051] Differentiation induction method for 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 DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM 8 ml of a medium containing 20 ng / ml bFGF (Wako), 30 ng / ml PDGFBB (Peprotech), 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 chondromal cells were placed in the dish at a rate of 1.2 x 10⁶ ６Individual seeds were sown and placed in an incubator at 37°C. After 48 hours, the culture medium was changed, and confluence of the dish was confirmed after another 24 hours.

[0052] Human Chondrocyte Spheroid Formation by Three-Dimensional Culture of Human Chondrocytes Chondrocytes (derived from either human iPS cells or human auricular chondromal cells) seeded in a 10 cm dish were washed with PBS and then detached by treatment with trypsin solution for 3 minutes. The trypsin solution containing the chondrocytes 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 centrifuged at 400G for 3 minutes. After removing the cell supernatant, the solution was mixed with DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM ), 10 ng / ml TGFβ1 (Peprotech), 10 ng / ml bFGF (Wako), 10 ng / ml PDGFBB (Peprotech), 4% Fetal bovine serum (Biowest), 0.2 mM L-ascorbic acid (Sigma-Aldrich), 10 -7 Using a medium containing M dexamethasone (Sigma-Aldrich) and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich), 1.4 x 10 ６ The suspension was prepared to a concentration of 7 x 10 / ml. 5 ml of this chondrocyte suspension (7 x 10) ６ (containing chondrocytes) was added 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 medium was changed and the cells were cultured for a further 24 hours.

[0053] Induction of human long bone progenitor cartilage organoids from human cartilage progenitor spheroids: Cartilage progenitor spheroids in 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 progenitor spheroids were collected by pipette and seeded linearly onto a 0.4 μm pore membrane with grooves (it4ip). The bottom of the membrane was treated with DMEM / F12 Ham (1:1) (Sigma-Aldrich) with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM ), 10 ng / ml TGFβ1 (Peprotech), 10 ng / ml bFGF (Wako), 10 ng / ml PDGFBB (Peprotech), 4% Fetal bovine serum (Biowest), 0.2 mM L-ascorbic acid (Sigma-Aldrich), 10 -7 3 ml of culture medium containing M dexamethasone (Sigma-Aldrich) and 10 ng / ml Insulin Growth Factor (Sigma-Aldrich) was added and incubated at 37°C. In this example, rod-shaped cartilage was used as an example. The seeded cartilage precursor spheroids fused with each other in approximately 12 hours, and the disappearance of the spheroid outlines was confirmed under a microscope. The following day, the entire volume (4 ml) of culture medium at the bottom of the membrane was replaced. Culture medium was changed every three days. After three weeks, DMEM / F12 Ham (1:1) (Sigma-Aldrich) was mixed with 1% Antibiotic Antimycotic Solution (Sigma-Aldrich) and 1% ITS-X (Gibco). TM ), 20 ng / ml BMP4 (R&D), 1% Fetal bovine serum (Biowest), 0.2 mM L-ascorbic acid (Sigma-Aldrich), 10 -7The culture medium was changed to one containing M dexamethasone (Sigma-Aldrich) at 3 ng / ml Insulin Growth Factor (Sigma-Aldrich). By culturing the membrane for 60 days using the above method, linear long bone progenitor cartilage organoids positive for ACAN, COL10A1, and RUNX were obtained.

[0054] Human long bone progenitor cartilage organoid induction rats (IL2rg-KO) were anesthetized by inhaling isoflurane (Pfizer 1 ml / 1 ml). Hair removal was performed at the transplantation site, and skin incisions were made with scissors and hooked forceps according to the size of the transplant sample (long bone progenitor cartilage organoid). The sample was placed between the quadriceps femoris muscles, and the wound was closed by suturing with size 6-0 sutures at 2 mm intervals. Post-transplantation micro-CT was used to confirm ossification and to confirm that the long bone progenitor cartilage organoid matured into a long bone organoid.

[0055] The linearity of long bone progenitor cartilage organoids is evaluated by photographing the tissue from above with a camera. The linearity of the tissue is determined by cropping the tissue portion from the image using ImageJ and identifying the central axis of the tissue using ImageJ's "Skeletonize" function. The sinuosity index is calculated by dividing the length of this central axis by the length of the straight line connecting the two ends of the central axis. The sinuosity index is 1.0 ≤ straight < 1.1, 1.1 ≤ curve ≤ 1.5, and 1.5 < meandering. (Paper: Kusratmoko, E., Wibowo, A. & Ahmad Kurnia, A. Changes in the value of sinuosity index in Komering River Channel, Province South Sumatera Years 1990-2016. IOP Conf. Ser.: Earth Environ. Sci. 338, 012024 (2019))

[0056] Gene expression analysis of long bone progenitor cartilage organoids was performed by physically disrupting the tissue and purifying RNA 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).

[0057] Evaluation of ossification of long bone organoids using micro-CT: Rat transplanted with organoids were anesthetized by inhaled administration of isoflurane (Pfizer 1 ml / 1 ml), and micro-CT scans were performed. Furthermore, hydroxyapatite (phantoms) with various bone densities were imaged, and the bone density of the organoids was calculated using these as an indicator of bone density.

[0058] Histological Evaluation of Long Bone Organoids: To prepare thin sections for histological evaluation of long bone organoids, the lower limbs were amputated and fixed in 10% neutral formalin for 48 hours. Subsequently, the bone tissue was decalcified and softened over 14 days using a neutral decalcification solution. Paraffin embedding was performed using an automated embedding machine, and thin sections with a thickness of 3-7 μm were prepared.

[0059] Experimental Results - Evaluation of the linearity of long bone progenitor cartilage organoids The grooved membrane (it4ip) used in this example is shown (Figure 1). Tissue was cultured linearly on the membrane, and the linearity of the tissue was confirmed with and without grooves in the membrane (Figures 2A, B). All (100%) of the tissue cultured on the grooved membrane was linear, whereas only 60% of the tissue cultured on the grooveless membrane was linear.

[0060] - Gene expression analysis of long bone precursor cartilage organoids: Quantitative RT-PCR showed that the cartilage marker ACAN and the osteogenic differentiation markers COL10A1 and RUNX2 increased over time from day 10 to day 90 of culture (n=10) (Figure 3).

[0061] - Analysis of ossification of long bone organoids: Long bone precursor cartilage organoids were transplanted into rats, and ossification was confirmed by micro-CT imaging (Figure 4).

[0062] Morphological analysis of long bone organoids: Long bone organoids were imaged using high-resolution micro-CT, and the axial images of the organoids confirmed that they were covered by cortical bone and contained cancellous bone internally (Figure 5).

[0063] Histological analysis of long bone organoids: As a result of transplantation of long bone progenitor cartilage organoids, safranin O staining at 5 months post-transplantation confirmed that the long bone organoids had cortical bone and periosteum surrounding it, and that they contained cancellous bone internally (Figure 6). Furthermore, the presence of bone marrow (clusters of CD71-positive erythroblasts, which are hematopoietic cells, surrounded by CD31-positive sinusoidal structures, which are hematopoietic niches, αSMA-positive arterioles, and PERILIPIN-positive adipocytes) was confirmed within the organoids (Figure 7). The presence of c-Kit-positive hematopoietic progenitor cells was also confirmed (Figure 8). All publications, patents, and patent applications cited herein are incorporated herein by reference as is.

[0064] This invention can be used for the treatment of bone defects and deformities throughout the body, cosmetic surgery aimed at facial augmentation, and the treatment of hematopoietic dysfunction using the hematopoietic function of the bone marrow.

Claims

1. A method for producing artificial cartilage tissue, comprising: aggregating chondrocytes to form chondrocyte spheroids; seeding the chondrocyte spheroids along grooves on a membrane having grooves; culturing the chondrocyte spheroids while supplying culture medium from the front and back sides of the membrane to fuse the spheroids together, and then maturing them into cartilage-like tissue and / or long bone progenitor cartilage organoids.

2. The method according to claim 1, wherein the cross-sectional shape of the groove is V-shaped, U-shaped, or a U-shape that opens upwards.

3. The method according to claim 1, wherein the membrane is supported by a support structure.

4. The method according to claim 1, wherein the culture of the cartilage precursor spheroid is performed by shaking culture.

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

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

7. The method according to claim 1, wherein the cartilage precursor spheroid has a diameter of 20 to 1000 μm.

8. The method according to claim 1, wherein one chondrogenic spheroid contains 100 to 7500 chondrogenic cells.

9. The method according to claim 1, wherein 10 to 5,000 of the cartilage precursor spheroids are seeded per 1 cm of groove.

10. The method according to claim 1, wherein the cartilage precursor spheroid is prepared by culturing cartilage precursor cells in a culture medium having a cell non-adherent surface.

11. The method according to claim 1, wherein the chondrogenic spheroid is obtained by culturing chondrogenic cells in a medium containing TGF-β and bFGF and then agglutinating them.

12. The method according to claim 1, wherein spheroids are fused together and then cultured in a medium containing BMP to mature into long bone progenitor cartilage organoids.

13. The method according to claim 12, wherein the incubation period in the culture medium containing BMP is 40 to 100 days.

14. A method for producing a long bone organoid, comprising transplanting a long bone progenitor cartilage organoid produced by the method described in claim 12 into a non-human animal and allowing it to mature into a long bone organoid.

15. The method according to claim 14, wherein the long bone organoid comprises cortical bone, periosteum, cancellous bone, and bone marrow.

16. Artificial cartilage tissue prepared by the method described in claim 1.

17. The artificial cartilage tissue according to claim 16, wherein the artificial cartilage tissue is a long bone progenitor cartilage organoid.

18. A long bone organoid prepared by the method described in claim 14.

19. A composition comprising artificial cartilage tissue prepared by the method described in claim 1, the composition being used to be transplanted into a living organism to compensate for a deficiency of bone tissue in the living organism.

20. The composition according to claim 19, wherein the artificial cartilage tissue produced by the method described in claim 1 is a long bone progenitor cartilage organoid.

21. A composition comprising a long bone organoid prepared by the method described in claim 14, the composition being used for implantation into a living organism to compensate for a deficiency of bone tissue in the living organism.

22. A culture vessel for producing artificial tissue, characterized by comprising a membrane having grooves and a support structure for supporting the membrane.

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