Three-dimensional tissue body and method for producing same

Abstract

A method for producing a three-dimensional tissue body, the method comprising a step for culturing a steric structural body formed by layering cell clusters while applying stretching stimuli and / or compression stimuli thereto.

Description

Three-dimensional structure and method for producing the same

[0001] The present invention relates to a three-dimensional tissue and a method for producing the same, and more particularly to a meniscus tissue or the like having excellent biocompatibility and a method for producing the same.

[0002] Injuries to ligaments, tendons, menisci, and other tissues are often caused by sports and are among the most common orthopedic disorders. Meniscus and anterior cruciate ligament (ACL) injuries are particularly common. Current treatments for these injuries primarily involve ACL reconstruction using autologous tendon grafts, such as those harvested from the semitendinosus muscle. However, applied research into regenerative medicine has demonstrated ACL reconstruction techniques that involve the transplantation of ligament-like structures formed using artificial biomaterials (e.g., synthetic fibers and collagen) instead of autologous tendon grafts. Recently, attempts have been made to use 3D cell structures formed by fusing spheroids (cell aggregates) as models of endocrine tissues or organs or as materials for regenerative medicine (Patent Document 1).

[0003] However, tissues that develop orthopedic diseases are constantly subjected to repeated stresses of extension (loading) and relaxation (unloading), making it difficult to reproduce the tissues affected by conventional treatments and, ultimately, to enable the tissues to function properly.

[0004] For example, meniscus tissue damage is one of the most common orthopedic diseases. The number of patients is increasing, especially with the rise in the number of athletes. Like cartilage, tendons, and ligaments, the meniscus has a very poor vascular distribution, and once damaged, the torn area cannot naturally repair itself to its original tissue structure.

[0005] Meniscectomy has been performed for many years as a treatment for meniscal injuries. Although meniscal resection temporarily relieves discomfort and pain, even partial resection significantly impairs meniscal function. Furthermore, there is a high possibility of secondary osteoarthritis (OA) of the knee.

[0006] For this reason, treatment that preserves the meniscus as much as possible by suturing the meniscus is becoming mainstream. However, according to estimates by the Ministry of Health, Labor and Welfare (NDB), the probability of re-rupture after meniscus suturing is as high as 30%. In addition, meniscus scaffold transplants using biomaterials and artificial materials are only applicable to a limited number of cases, and there are concerns about adverse events such as foreign body reactions and toxicity.

[0007] There is a need for a treatment that maintains and improves the repair results of the damaged area while preserving the meniscus as much as possible and prevents progression to OA. As a technology that can solve the above problems, many researchers are working on synthesizing three-dimensional meniscus tissues formed from biomaterials, which do not require biological or artificial materials such as scaffolds and have the same histological properties as the meniscus.

[0008] For example, one known device for forming such meniscus tissue is a tissue formation device that places multiple cells into a culture vessel filled with culture medium and interconnects the multiple cells to form a tissue, and that is formed from a frame that forms a hollow body with an opening at least at the top, and that is housed within the culture vessel and includes a regulating frame section that regulates the diffusion of the multiple cells, and a pressing and shaping section that is disposed on the upper surface of the opening of the regulating frame section and presses and shapes the multiple cells housed within the opening (Patent Document 2).

[0009] Also, a method for culturing animal cells is known, which comprises fixing a culture carrier made of plant-derived fibrous branched bodies to a culture vessel, and adhering, spreading, and growing animal cells on the culture carrier (Patent Document 3).

[0010] JP 2020-202785 A JP 2016-29 A JP 7-67626 A

[0011] However, although it is possible to form a meniscus tissue from a cell mass (spheroid) obtained by culturing multiple cells, as in Patent Documents 2 and 3, an artificial meniscus tissue that can withstand the complex and powerful mechanical loads of the meniscus has not yet been obtained.

[0012] The meniscus is located between the femur and tibia of the knee joint, which is subject to frequent physical activity. Therefore, it is constantly subjected to not only repeated stresses of extension (loading) and relaxation (unloading), but also compressive stimuli. Although the details are not yet fully understood, it is known to have a complex three-dimensional structure that combines the functions of tendons and ligaments with those of cartilage. Therefore, it is difficult to obtain a meniscus tissue with a complex structure that combines the functions of tendons and ligaments with those of cartilage using the manufacturing methods described in Patent Documents 2 and 3. In other words, a practical, strong meniscus tissue that can function as a meniscus and can sufficiently withstand both repeated stresses and compressive stimuli has yet to be obtained. Furthermore, a meniscus tissue that can function as a meniscus is highly desired because it would be useful for meniscus transplantation and as a medical research model. However, such an excellent meniscus tissue is currently unknown. Therefore, there is a need for a medical material that can withstand physical strength and be used in regenerative medicine.

[0013] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in obtaining a tissue with enhanced strength by applying stretching and / or compressive stimuli to a three-dimensional structure obtained by stacking cell clusters, thereby arriving at the present invention. Specifically, the present invention is as follows: [1] A method for strengthening the strength of a three-dimensional tissue, comprising the step of culturing a three-dimensional tissue formed by stacking cell clusters while applying stretching and / or compressive stimuli to the three-dimensional tissue. [2] The method described in [1], wherein the stretching stimulus is a stretching stimulus including uniaxial motion or multiaxial motion. [3] The method described in [2], wherein the stretching stimulus includes at least one of torsional motion, tensile motion, and vibration motion. [4] The method described in [1], wherein the compressive stimulus is applied to the three-dimensional tissue by a pressing unit having a pressing surface. [5] The method described in [1], wherein the medium for culturing the three-dimensional tissue further contains an additive factor. [6] The method described in [1], wherein the three-dimensional tissue is cultured under circulatory culture. [7] The method according to [1], wherein the cell aggregates contain mesenchymal stem cells, fibroblasts, and cells that serve as a source of a target tissue. [8] The method according to [7], wherein the source cells are precursor cells of bone, cartilage, ligament, tendon, or meniscus. [9] The method according to [8], wherein the precursor cells are sclerotome cells.

[10] The method according to [9], wherein the three-dimensional tissue is a tissue for bone, cartilage, ligament, tendon, or meniscus.

[11] A method for producing a three-dimensional tissue with enhanced strength, comprising a step of culturing a three-dimensional tissue formed by layering cell aggregates while applying a stretching stimulus and / or a compressive stimulus.

[12] The method according to

[11] , wherein the stretching stimulus is a stretching stimulus including uniaxial motion or multiaxial motion.

[13] The method according to

[11] , wherein the stretching stimulus includes at least one of a torsional motion, a tensile motion, and a vibration motion.

[14] The method according to

[11] , wherein the compressive stimulus is applied to the three-dimensional tissue by a pressing unit having a pressing surface.

[15] The method according to

[11] , wherein the medium for culturing the three-dimensional tissue further contains an additive factor.

[16] The method according to

[11] , wherein the three-dimensional tissue is cultured under circulatory culture.

[17] The method according to

[12] , wherein the cell mass contains mesenchymal stem cells, fibroblasts, and cells that will serve as the source of the target tissue.

[18] The method according to

[17] , wherein the source cells are progenitor cells of bone, cartilage, ligament, tendon, or meniscus.

[19] The method according to

[18] , wherein the progenitor cells of bone, cartilage, ligament, tendon, or meniscus are sclerotome cells.

[20] The method according to

[19] , wherein the three-dimensional tissue is a tissue for bone, cartilage, ligament, tendon, or meniscus.

[21] A three-dimensional tissue having a breaking strength of at least 0.001 N / mm, formed by culturing a three-dimensional tissue formed by layering cell aggregates while applying a stretching stimulus and / or a compressive stimulus.

[22] The tissue according to

[21] , wherein the cell aggregates contain mesenchymal stem cells, fibroblasts, and cells that serve as source cells for the target tissue.

[23] The tissue according to

[22] , wherein the source cells are progenitor cells of bone, cartilage, ligament, tendon, or meniscus.

[24] The tissue according to

[23] , wherein the progenitor cells of bone, cartilage, ligament, tendon, or meniscus are sclerotome cells.

[25] The tissue structure according to

[24] , wherein the three-dimensional tissue structure is a tissue structure for bone, cartilage, ligament, tendon, or meniscus.

[26] A culture device for culturing a three-dimensional tissue structure, comprising an application unit that applies a mechanical stimulus to the three-dimensional tissue structure.

[27] The culture device according to

[26] , wherein the mechanical stimulus applied by the application unit includes at least a stretching stimulus.

[28] The culture device according to

[26] , wherein the three-dimensional tissue structure has an opening, and the application unit includes at least two shaft members, and the application unit applies a mechanical stimulus to the three-dimensional tissue structure by relatively moving the at least two shaft members while inserted into the opening.

[29] The culture device according to

[28] , wherein the application unit applies a mechanical stimulus to the three-dimensional tissue structure by relatively translating the at least two shaft members.

[30] The culture device according to

[28] , wherein the applying unit applies a mechanical stimulus to the three-dimensional tissue structure by rotating the at least two shaft members relative to one another.

[31] The culture apparatus according to

[26] , wherein the application unit includes a vibrating body that generates vibrations, and the application unit applies a mechanical stimulus to the three-dimensional tissue by stretching and contracting the three-dimensional tissue with the vibrations generated by the vibrating body.

[32] The culture apparatus according to

[26] , wherein the mechanical stimulus applied by the application unit includes at least a compressive stimulus.

[33] The culture apparatus according to

[26] , wherein the application unit includes: a base portion formed with a recess into which the three-dimensional tissue is fitted, and a pressing portion that presses the three-dimensional tissue fitted in the recess.

[34] The culture apparatus according to

[26] , wherein the application unit includes: a tensioning portion that pulls the three-dimensional tissue, and a pressing portion that presses the three-dimensional tissue that has been given a stretching stimulus by the tensioning portion in a direction different from the pulling direction of the tensioning portion.

[35] The culture apparatus according to

[26] , further comprising a circulation unit that circulates the culture solution in the culture vessel.

[36] The culture apparatus according to

[26] , further comprising a gas regulation unit that regulates the gas in the culture vessel.

[37] The culture apparatus according to

[26] , further comprising a temperature regulation unit that regulates the temperature in the culture vessel.

[38] The culture apparatus according to

[26] , further comprising a detection unit that detects a physical quantity corresponding to the magnitude of the mechanical stimulus applied to the three-dimensional tissue by the application unit, and a control unit that controls the magnitude of the mechanical stimulus applied by the application unit based on the detection result of the detection unit.

[0014] The present invention provides a method for enhancing the strength of a three-dimensional tissue structure and an apparatus for use in the method. These tissue structures are useful for treating orthopedic diseases or in regenerative medicine.

[0015] 1 is a diagram showing an example of the configuration of a culture device. (a) to (d) are diagrams illustrating the operation of the application unit 1 and the mechanical stimulation applied thereby to the three-dimensional tissue structure 100. (a) to (d) are diagrams illustrating an example of a mechanism for simultaneously performing twisting and pulling operations. (a) is a diagram illustrating the timing of vibration operations, and (b) and (c) are diagrams illustrating the position and order of vibration application. (b) are diagrams illustrating an example of the configuration of a culture device. (c) are diagrams illustrating the differentiation of iPS cells into each tissue. (d) are diagrams illustrating a flowchart of a method for producing a three-dimensional tissue structure according to an embodiment of the present invention. (c) are diagrams illustrating a schematic diagram of a meniscus. (d) are diagrams illustrating immunohistochemical staining results of an artificial tissue structure for ligament and tendon. (e) are diagrams illustrating the placement and stretching of a cell structure in an apparatus to measure the strength of the artificial tissue structure. (e) are diagrams illustrating tissues with and without a stretch stimulus for an artificial tissue structure for ligament and tendon. (f) are diagrams illustrating polarized light microscope images obtained by picrosirius red staining after a stretch stimulus (torsional stretch) for an artificial tissue structure for ligament and tendon. (f) are diagrams illustrating the configuration of a circulatory culture device in the artificial tissue culture process. 1 is a diagram showing the results of breaking load with or without circulatory culture in the culture process of an artificial tissue for ligament and tendon. FIG. 2 is a diagram showing the results of stiffness with or without circulatory culture in the culture process of an artificial tissue for ligament and tendon. FIG. 3 is a diagram showing the results of breaking load according to the type of raw material cell in the culture process of an artificial tissue for ligament and tendon. FIG. 4 is a diagram showing the results of breaking load and optical microscope images according to the type of raw material cell (with or without added factors) in the culture process of an artificial tissue for ligament and tendon. FIG. 5 is a polarized microscope image of a tissue slice after transplantation of an artificial tissue for ligament and tendon stained with picrosirius red. FIG. 6 is an optical microscope image of a tissue slice after transplantation of an artificial tissue for ligament and tendon stained with picrosirius red. FIG. 7 is a diagram showing the results of stacking cell aggregates on a pin holder. FIG. 8 is a diagram showing the results of immunostaining type I collagen of a meniscus tissue under an optical microscope. FIG. 9 is a diagram showing various staining results for a meniscus tissue. FIG. 10 is a diagram showing ring-shaped and band-shaped artificial tissue for meniscus. FIG. 11 is a diagram showing a meniscus transplant produced by bundling and suturing three band-shaped artificial tissue for meniscus. The medial femoral condyle of a pig was cut and reflected to expose the medial meniscus, and the medial meniscus was then completely removed.The meniscus implant was then placed on the upper edge of the tibia with a suture thread threaded through a bone hole created in the tibia.1 shows the results of a fluoroscopic examination of a porcine knee joint four weeks after meniscus transplantation, a macroscopic examination of an incised porcine knee joint, and a histological examination of a meniscus transplant body four weeks after meniscus transplantation.

[0016] The present invention relates to a method for strengthening a three-dimensional tissue formed by stacking cell clusters, the method comprising culturing the three-dimensional tissue while applying stretching and / or compressive stimuli to the three-dimensional tissue, and the strengthened three-dimensional tissue. The present invention is characterized in that a three-dimensional tissue composed only of cells is fabricated by stacking cell clusters using a bio-3D printer with a pinholder-shaped needle, and differentiation is then induced into a tissue with enhanced mechanical strength using the stretching culture device and compressive culture device of the present invention. The three-dimensional tissue does not use artificial materials and has sufficient mechanical strength solely from the cells, thereby preventing damage to various tissues and pathological progression over the long term.

[0017] 1. Method for Strengthening Tissue and Strengthened Tissue (1) Cell Mass In the present invention, the cell mass that constitutes the three-dimensional tissue contains mesenchymal stem cells, fibroblasts, and cells that serve as the raw material for the target tissue.

[0018] (1-1) Mesenchymal Stem Cells In the present invention, one of the components of the cell mass that constitutes the three-dimensional tissue is mesenchymal stem cells (MSCs). MSCs are pluripotent cells with self-renewal and differentiation capabilities, and have a low risk of tumor formation, making them promising tools for cell therapy and regenerative medicine. The origin of the MSCs used in the present invention is not limited, and examples include adipose tissue, bone marrow, umbilical cord, pluripotent stem cells, and deciduous dental pulp. MSCs are commercially available and can be obtained from ATCC, Evercyte, Cellsource, Gene Techno Science, and other sources.

[0019] Among the sources of MSCs, pluripotent stem cells are stem cells that have the pluripotency to differentiate into all cells present in the body and have the ability to proliferate, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Preferred pluripotent stem cells are iPS cells. ES cells are stem cells established from the cell mass of early embryos of mammals such as humans and mice. ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of the target mammal and culturing the inner cell mass on a fibroblast feeder. Human ES cell lines are also available from the Institute for Virus Research and Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0020] iPS cells are artificial stem cells derived from somatic cells that have pluripotency and the ability to proliferate through self-renewal similar to that of ES cells, and can be generated by introducing specific reprogramming factors into somatic cells (Yamanaka S. et al., Cell, 126:663-676, 2006; Okita K. et al., Nature 448, 2007; WO2007 / 069666, etc.). Examples of genes contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors can be used alone or in appropriate combination, with Oct3 / 4, Sox2, Klf4, and c-Myc being preferred.

[0021] Methods for inducing iPS cells from somatic cells are well known (Yamanaka S. et al., Cell, 126:663-676, 2006; Okita K. et al., Nature 448, 2007; WO2007 / 069666, etc.). Examples of tissues from which iPS cells used in the present invention are derived include cartilage, bone, ligament, tendon, and meniscus.

[0022] In the present invention, a method for inducing pluripotent stem cells into mesenchymal stem cells (MSCs) includes, for example, culturing iPS cells in the presence of factors such as TGFB1. MSCs derived from iPS cells can also be obtained from Kyoto University.

[0023] (1-2) Fibroblasts Fibroblasts are cells that make up connective tissue and produce dermal components such as collagen, elastin, and hyaluronic acid. In the present invention, for example, skin-derived fibroblasts (e.g., DFs) are used. In addition to the above-mentioned fibroblasts, other fibroblasts, such as stem cell-derived fibroblasts, can also be used in the present invention.

[0024] (1-3) Cells as Source of Target Tissue In the present invention, the cells as source of target tissue are precursor cells of bone, cartilage, ligament, tendon, or meniscus, such as sclerotome cells or their differentiated cells. As shown in Figure 7, during mesoderm differentiation, presomitic mesoderm first differentiates into somite cells. The ventral portion then differentiates into sclerotome cells. The sclerotome cells then differentiate into cartilage, bone, or meniscus, and into sclerotome cells, which further differentiate into ligaments and tendons. In the present invention, sclerotome cells differentiated in a single step from sclerotome cells can also be used as precursor cells of these cells, and these may be derived from iPS cells.

[0025] In the present invention, sclerotome cells, which are the source of mesodermal tissues, are used, making it possible to differentiate them into various mesodermal tissues.

[0026] (2) Spheroid Production In the present invention, mesenchymal stem cells, fibroblasts, and cells (hereinafter also referred to as "raw cells") that serve as the source of the target tissue are first mixed to obtain a mixture. This mixture is obtained, for example, by mixing these three types of cells in a culture medium to prepare a cell suspension, which is then cultured to form cell masses (cell aggregates; spheroids) in which the individual cells are aggregated together. For example, when the cell mixture is cultured on a plate that has been treated to be water-repellent or non-adhesive, such as a Teflon®-coated plate, the cells seek a foothold and adhere to each other, forming spheroids, which are cell aggregates. The culture time required for spheroid formation is 6 to 48 hours, preferably 12 to 48 hours.

[0027] As another method for forming spheroids, vessels manufactured by IWAKI (EZsphere) can be used, or low-adhesion 10 cm or 6 cm dishes manufactured by other companies can also be used. The culture medium for forming spheroids is a standard culture medium commonly used for animal cell culture, such as Dulbecco's MEM medium (DMEM / High glucose), Dulbecco's MEM / Ham's F12 medium, or RPMI-1640 medium, to which serum can be added.

[0028] It should be noted that spheroids do not necessarily have to be formed as aggregates of only the above three types of cells; as long as spheroids are formed, multiple types of other cells, such as undifferentiated cells or differentiated cells thereof, may be mixed in addition to the above three types of cells.

[0029] (3) Spheroid Lamination As described above, by further aggregating the cell aggregate mixture and making it three-dimensional, a three-dimensional tissue is formed as a scaffold-free cell structure of any shape (e.g., strap-like (string-like)). This cell aggregate can be made three-dimensional using known techniques. For example, according to the techniques described in Patent Document 1 and WO 2008 / 123614, cell aggregates are pierced by needles arranged in a pinholder-like shape on a substrate, and the cell aggregates are placed in any three-dimensional space. This allows the cell aggregates to be laminated to create a three-dimensional structure. Since an automated stacking robot for implementing the above method is already known (Bio 3D Printer "Regenova" (registered trademark), Cyfuse Co., Ltd.), three-dimensional structures can also be produced using this robot. Three-dimensional structures are typically formed firmly in any shape, primarily composed of cells and collagen exuded from the cells.

[0030] The number and shape of the spheroids are not particularly limited and can be any. The ratio of the numbers of mesenchymal stem cells, fibroblasts, and starting cells is not particularly limited, but can be, for example, 1:98:1 to 98:1:1 and 49:49:2 to 1:1:98 (mesenchymal stem cells:fibroblasts:starting cells). From the perspective of production efficiency, a low ratio of starting cells is preferable, for example, 49:49:2. In one embodiment of the present invention, this three-dimensional tissue is a three-dimensional tissue formed from iPS cell-derived mesenchymal stem cells, fibroblasts, and iPS cell-derived sclerotome cells. The ratio of the numbers of iPS cell-derived mesenchymal stem cells, fibroblasts, and iPS cell-derived sclerotome cells is also not particularly limited and can be the same as above (1:98:1 to 98:1:1 and 49:49:2 to 1:1:98 (IPS cell-derived mesenchymal stem cells:fibroblasts:IPS cell-derived sclerotome cells). From the viewpoint of production efficiency, it is preferable that the ratio of iPS cell-derived sclerotome cells is low, for example, 49:49:2.

[0031] (4) Differentiation into Various Tissues and Three-Dimensional Tissues The three-dimensional structures obtained by stacking spheroids can be cultured in the presence of various differentiation factors to obtain three-dimensional tissues of various tissues. As an example of a method for producing a three-dimensional tissue, as shown in Figure 8, first, mesenchymal stem cells (e.g., iPS cell-derived mesenchymal stem cells), fibroblasts, and raw cells (e.g., iPS cell-derived sclerotome cells) are mixed to obtain a mixture (S1: mixing step). This mixture is formed, for example, by introducing these three types of cells into a culture vessel filled with culture medium and inducing self-aggregation, resulting in the formation of a cell mass in which the three types of cells aggregate with each other.

[0032] This cell mass is formed by gathering, but is not particularly limited to, 30,000 to 40,000 cells. More preferably, various differentiation-inducing factors are further mixed into the culture vessel to form a mixture. Next, the cell mass is pierced with a pinholder-shaped needle and stacked to form a three-dimensional structure (S2: stacking step).

[0033] Next, the mixture is cultured in a culture vessel under solution circulation conditions and / or with the addition of a stretching stimulus (S3: stretching step). The culture in this stretching step may be cultured only under solution circulation conditions, cultured only with the addition of a stretching stimulus, or cultured under solution circulation conditions and with the addition of a stretching stimulus.

[0034] An example of an excellent and optimal culture vessel for easily achieving this stretching step is a culture vessel (also referred to as an automatic stretching culture vessel) originally developed by the present inventors, as shown in Figure 1. In one aspect of the present invention, a compressive stimulus is optionally applied to the inner peripheral side of the three-dimensional structure before, during, or after the stretching step, or throughout the entire process from before to after the stretching step, and a three-dimensional tissue such as meniscus cells can be obtained by culturing (S4: compression step). The stretching step and compression step will be described in detail below.

[0035] Next, we will explain the differentiation induction into each tissue. (i) Bone: For differentiation into bone, additional factors such as the BMP family, TGF family, FGF, IGF, HIF, and CTGF can be used, and the cells are cultured in the presence of factors such as the BMP family. BMP family members used for differentiation into bone include BMP-2, BMP-3, BMP-4, BMP-7, and BMP8a, which can be used alone or in appropriate combinations. However, the BMP family is not limited to these.

[0036] (ii) Cartilage For differentiation into cartilage, similar to bone, additional factors such as the BMP family, TGF family, FGF, IGF, HIF, and CTGF can be used, and the cells are cultured in the presence of factors such as the BMP family. BMP-2, BMP-5, BMP6, BMP-7, and BMP8a can be used alone or in appropriate combinations as BMP family members used for differentiation into cartilage, but are not limited to these BMP families.

[0037] (iii) Ligaments or Tendons: For differentiation into ligaments or tendons, ligament or tendon differentiation inducers can be included. Known ligament or tendon differentiation inducers include the BMP family, TGF family, FGF, IGF, HIF, and CTGF. Examples of these include recombinant human TGF-β3 and growth differentiation factor 7 (human GDF-7), which can promote differentiation into ligament tenocytes and allow them to mature into stronger tissue structures. Furthermore, BMP family members used for differentiation into ligaments or tendons include BMP-12, BMP-13, and BMP-14, which can be used alone or in appropriate combinations. However, these BMP families are not limited to these.

[0038] This three-dimensional tissue contains ligament tenocytes differentiated from iPS cell-derived mesenchymal stem cells. Furthermore, this three-dimensional tissue has a structure in which the orientation of type I collagen is aligned. A structure in which the orientation of type I collagen is aligned refers to a structure in which the type I collagen tissue is oriented in a specific direction. This structure can be formed, for example, under solution circulation conditions and / or by applying a stretching stimulus. In the present invention, the artificial tissue for ligament or tendon is composed of a ligament or tendon formed from a plurality of sclerotome cells. The number of sclerotome cells is not particularly limited, but can be, for example, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more.

[0039] (v) Meniscus The meniscus tissue is preferably formed further containing a meniscus differentiation inducer. Known meniscus differentiation inducers include the BMP family, TGF family, FGF, IGF, HIF, and CTGF, among which recombinant human TGF-β3 and BMP-12 are examples, and can promote differentiation into meniscus cells and mature into a stronger tissue structure. In other words, this three-dimensional tissue contains meniscus cells differentiated from iPS cell-derived mesenchymal stem cells. The BMP family used for differentiation into meniscus includes BMP-2, BMP-12, BMP-13, and BMP-14, which can be used alone or in appropriate combinations. However, the BMP family is not limited to these.

[0040] The meniscus tissue has a structure in which the orientation of type I collagen is aligned. A structure in which the orientation of type I collagen is aligned refers to a structure in which the type I collagen tissue is oriented regularly or in a certain direction to a certain extent. This structure in which the orientation is aligned can be formed, for example, under solution circulation conditions and / or by applying a stretching stimulus.

[0041] Furthermore, the meniscus tissue has a structure in which the orientation of type II collagen is aligned. A structure in which the orientation of type II collagen is aligned refers to a structure in which the type II collagen tissue is oriented regularly or in a certain direction to a certain extent. This structure in which the orientation is aligned can be formed, for example, by applying a compressive stimulus, as shown in the manufacturing method described below. This promotes an increase in proteins that hold water molecules in the structural portion in which the orientation of type II collagen is aligned, enabling the tissue to exhibit properties similar to those of cartilage. This increase in proteins that hold water molecules can be confirmed by safranin 0 staining (see the Examples described below).

[0042] Regarding the relationship between the orientation of type I collagen and type II collagen, it is preferable that the orientation directions of type I collagen and type II collagen are aligned in an orthogonal orientation to each other. Due to this characteristic orientation, as shown in Figure 9, in this three-dimensional tissue 100, the orientation of type II collagen is aligned in the inner peripheral side portion 100a, giving it properties similar to cartilage, and in the outer peripheral side portion 100b, the orientation of type I collagen is aligned in an orientation direction perpendicular to the orientation direction of the type II collagen, giving it properties similar to tendon or ligament, thereby obtaining a meniscus tissue with a structure similar to that of an actual meniscus (see Examples described later).

[0043] More preferably, when forming type I collagen and type II collagen, a stretching stimulus is applied to the entire mixture that will form the three-dimensional tissue, and then a compressive stimulus is applied to the vicinity of the inner periphery of the mixture, followed by culturing. This stretching stimulus first aligns the orientation of type I collagen throughout the entire mixture, and then a compressive stimulus aligns the orientation of type II collagen only near the inner periphery. As a result, the orientation of type II collagen is aligned in the inner periphery of the meniscus tissue, and a three-dimensional tissue is formed in which the orientation of type I collagen is aligned from the inner periphery to the outer periphery.

[0044] Alternatively, a three-dimensional tissue cultured under a compressive stimulus and a three-dimensional tissue cultured under a stretching stimulus can be separately prepared, and the tissue with the properties of cartilage can be placed on the inner circumferential side, and the tissue with the properties of tendon or ligament can be placed on the outer circumferential side, and then fused to provide a meniscus tissue.

[0045] In the present invention, the meniscus tissue comprises a meniscus formed by a plurality of the meniscus cells. The number of the meniscus cells is not particularly limited, but may be, for example, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more.

[0046] The meniscus tissue of this embodiment can be used for a wide variety of purposes, and although there are no particular limitations on its uses, it can be used, for example, for meniscus transplants in medical settings, and can also be used as a model that can reproduce the function of the meniscus for medical research.

[0047] (5) Stretching Step and / or Compression Step In the present invention, to enhance the strength of a three-dimensional tissue, the tissue is cultured in a culture vessel while being subjected to stretching and / or compression stimuli. The culture in this stretching and / or compression step may involve culturing only under solution circulating conditions, culturing only with the application of stretching and / or compression stimuli, or culturing under solution circulating conditions with the application of stretching and / or compression stimuli. The stretching stimulus includes uniaxial or multiaxial motion, and includes at least one of torsional motion, tensile motion, and vibration motion. Furthermore, the compression stimulus is applied by pressing a pressing unit having a pressing surface against the three-dimensional tissue.

[0048] In addition to the above, a method for obtaining three-dimensional tissues such as meniscus tissues can also be used by separately preparing tissues cultured under a compressive stimulus and tissues cultured under a stretching stimulus, and then arranging and fusing the tissues with the properties of cartilage on the inner circumferential side and the tissues with the properties of tendon or ligament on the outer circumferential side to provide a meniscus tissue.

[0049] (5-1) Stretching Stimulation While details of an excellent culture device that is optimal for easily achieving the stretching process will be described later, an overview of the stretching process will be described using FIG. 2. Uniaxial movement refers to the movement of one opposing shaft 11 and another opposing shaft 12 stretching and contracting the three-dimensional tissue structure 100 (a tissue structure formed in a ring shape) in one dimension (linear direction), as shown in FIG. 2(a). Multiaxial movement refers to the movement of one opposing shaft 11 and another opposing shaft 12 stretching and contracting the three-dimensional tissue structure 100 in two dimensions (planar direction) or three dimensions (stereoscopic direction), as shown in FIG. 2(b). For example, the three-dimensional tissue structure 100 can be stretched and contracted in three dimensions (stereoscopic direction) by the twisting movement of at least one opposing shaft 11 and another opposing shaft 12 in the three dimensions. In the stretching step, the strength of the stretching stimulus varies depending on the type and size of the tissue, but is generally 0.001 to 10,000 [N] (1 [mN] to 10 [kN]) or 0.0001 to 1,000 [N / mm ２ (MPa)].

[0050] 2(c), the twisting motion and the pulling motion can be simultaneously performed by each of the one opposing shaft 11 and the other opposing shaft 12. For example, by each of the one opposing shaft 11 and the other opposing shaft 12 performing a two-dimensional twisting motion and a one-dimensional pulling motion, the three-dimensional tissue structure 100 can be stretched and contracted in three dimensions (stereoscopic directions). Furthermore, for example, by combining the three-dimensional twisting motion and the one-dimensional pulling motion by the one opposing shaft 11 and the other opposing shaft 12, the three-dimensional tissue structure 100 can be stretched and contracted in more three-dimensional directions (stereoscopic directions).

[0051] Furthermore, as shown in FIG. 2(d), by including a vibrating body 13 that generates vibrations, it is possible to perform vibrational motion. This vibrating body 13 causes vibrations in three-dimensional directions (stereoscopic directions), making it possible to stretch and contract the three-dimensional tissue structure 100 in three-dimensional directions (stereoscopic directions). Furthermore, by changing the vibration intensity of this vibrating body 13, it is possible to easily control the strength of the stretching and contraction. In this way, by including uniaxial motion and / or multiaxial motion in the stretching stimulation, a three-dimensional tissue structure 100 is formed that can adapt to repeated stresses of stretching (loading) and relaxation (unloading).

[0052] The breaking load [mN] in the stretching step is 0.001 to 100,000 [N] (1 [mN] to 100 [kN]), or 0.0001 to 10,000 [N / mm ２ (MPa)].

[0053] In the present invention, various additive factors can be added before the start of stretch stimulation to increase the strength of the tissue, and appropriate cytokines can be added. For example, the addition of bone morphogenetic proteins (BMPs) can promote the formation of the meniscus itself or the cartilage tissue that constitutes the meniscus. Examples of such bone morphogenetic proteins (BMPs) include BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14.

[0054] (5-2) Compressive Stimulation Furthermore, in the present invention, a compressive stimulus can be applied to the inner peripheral portion of the tissue during the stretching step, or before and / or after the stretching step, during culture. Before the start of this compressive stimulus, various additive factors can be added to increase the strength of the tissue, and appropriate cytokines can be added.

[0055] For example, the addition of bone morphogenetic proteins (BMPs) can promote the formation of the meniscus itself or the cartilage tissue that constitutes the meniscus, such as BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14.

[0056] The combination of additive factors and BMP families for each tissue is the same as described above. However, the additive factors are not limited to the above combinations. The pressing load [mN] in the compression step is 0.001 to 100,000 [N] (1 [mN] to 100 [kN]), or 0.0001 to 10,000 [N / mm ２ (MPa)].

[0057] (5-3) Tissue Properties Due to Stretching and Compression Stimulation Stretching stimuli align the orientation of type II collagen in the central part of the three-dimensional tissue (for example, the part that constitutes the inner peripheral part of a meniscus tissue), and simultaneously promote the increase of proteins that hold water molecules, allowing the tissue to exhibit properties similar to those of cartilage. This results in the formation of a three-dimensional tissue that can function as a shock absorber against various pressures. For example, if the three-dimensional tissue is a meniscus tissue, the inner peripheral part can perform the cartilage function that is sufficient for practical use of the meniscus.

[0058] (6) Circulatory Culture In the present invention, the strength of a three-dimensional tissue can be enhanced by circulatory culture using a circulatory culture device. Circulatory culture is culture performed during the extension process of a three-dimensional tissue. It refers to culturing by flowing the culture medium in a culture vessel, including unidirectional flow of the medium and convection of the medium within the vessel. Circulatory culture can be performed, for example, by connecting a peristaltic pump to a tube, allowing the culture medium to flow from the tube into the culture vessel. The medium that flows into the culture vessel flows within the vessel, then is discharged outside the vessel and subjected to circulatory culture again. Circulatory culture can be performed by flowing 1 mL to 100 mL of medium into the culture vessel per unit time (minute), and discharging the same amount of medium per minute. Alternatively, the culture vessel can be shaken to convect or swirl the medium.

[0059] (7) Three-dimensional tissues with enhanced strength The three-dimensional tissues produced in this manner are structures composed solely of cells, eliminating the need for a scaffold, and have higher mechanical strength than structures not treated with the present invention. The strength of the three-dimensional tissues of the present invention is a breaking strength (rigidity) of at least 0.001 N / mm, for example, 0.001 to 100,000 N (1 mN to 100 kN). The breaking load of each tissue when subjected to stretch stimulation is 0.001 to 100,000 N (1 mN to 100 kN), or 0.0001 to 10,000 N / mm (MPa).

[0060] 2. Culture Device The following describes a culture device for culturing the three-dimensional tissue structure 100. In summary, the culture device includes an application unit that applies a mechanical stimulus to the three-dimensional tissue structure 100. By applying a mechanical stimulus to the three-dimensional tissue structure 100 during culture, the mechanical properties of the three-dimensional tissue structure 100 are improved.

[0061] 1 is a diagram showing an example of the configuration of a culture apparatus 1A as an example of a culture apparatus. The culture apparatus 1A includes a culture vessel 10, an application unit 1 composed of opposing shafts 11 and 12 (shaft members), a circulation unit 2 including a circulation culture tube 2a that circulates a culture solution, a reciprocating unit 3 that moves reciprocally by a motor, and a motor 4. The culture apparatus 1A also includes a control unit 50, a detection unit 51, a gas adjustment unit 52, and a temperature adjustment unit 53.

[0062] The application unit 1 applies a stretching stimulus to the three-dimensional tissue structure 100 attached to the opposing shafts 11 and 12 by relative movement of the opposing shafts 11 and 12 in the stretching direction A. Specifically, the three-dimensional tissue structure 100 is first attached to the culture device 1A so that the opposing shafts 11 and 12 are inserted into its openings. The opposing shaft 12 is fixed to the reciprocating unit 3 and moves in the motor moving direction C as the reciprocating unit 3 moves in the motor moving direction C. In this embodiment, the stretching direction A and the motor moving direction C are parallel, and therefore this movement causes the opposing shaft 12 to move relative to the opposing shaft 11 in the stretching direction A. The movement of the opposing shaft 12 away from the opposing shaft 11 constitutes a stretching motion for the three-dimensional tissue structure 100, and a stretching stimulus is applied to the three-dimensional tissue structure 100. The application unit 1 can be configured to apply mechanical stimulation of a strength within the numerical range (or a part thereof) indicated in "1. Method for strengthening tissue and strengthened tissue (6) Stretching stimulation and / or compressing stimulation" above.

[0063] In this embodiment, the reciprocating unit 3 is a rack and pinion mechanism, and includes a movable unit 3 a having a rack and to which the opposing shaft 12 is fixed, and a connecting unit 3 b having a pinion and connected to the motor 4. However, it is sufficient for the reciprocating unit 3 to be able to move the opposing shafts 11 and 12 relative to each other, and other known techniques such as a ball screw mechanism can be used as the specific structure.

[0064] In this embodiment, the magnitude of the mechanical stimulus applied to the three-dimensional tissue structure 100 by the applying unit 1 is detected by the detecting unit 51. Then, the control unit 50 controls the magnitude of the mechanical stimulus applied by the applying unit 1 based on the detection result of the detecting unit 51. That is, the control unit 50 controls the magnitude of the mechanical stimulus applied by the applying unit 1 by feedback control so as to approach a target value.

[0065] The control unit 50 may include, for example, a microcomputer having a CPU (Central Processing Unit), ROM (Read Only Memory), RAM, input / output ports, etc., and various other circuits. The detection unit 51 detects a physical quantity corresponding to the magnitude of the mechanical stimulus (the magnitude of the stretching stimulus (traction force) in the culture device 1A). Various types of sensors can be used as the detection unit 51, as appropriate. For example, the detection unit 51 may be a strain gauge-type load cell. Alternatively, the detection unit 51 may be a torque sensor that detects the torque of the motor 4 or detects the drive current of the motor 4. That is, the detection unit 51 may be capable of detecting a physical quantity that directly or indirectly indicates the magnitude of the mechanical stimulus applied by the application unit 1. In the device 1A, the detection unit 51 detects the magnitude of the stretching stimulus (traction force) as the magnitude of the mechanical stimulus. However, depending on the configuration of the device, the detection unit may be capable of detecting a compressive force as the magnitude of the mechanical stimulus instead of or in addition to the traction force.

[0066] In this embodiment, the stretching process can be effectively performed by controlling the magnitude of the mechanical stimulus applied to the three-dimensional tissue structure 100 by the applying unit 1 based on the detection results of the detecting unit 51. For example, controlling the relative movement amount of the opposing shafts 11 and 12 can be considered as a method for controlling the operation of the applying unit 1. However, the stiffness of the three-dimensional tissue structure 100 cultured in the culture device 1A varies depending on the type, and the magnitude of the stretching stimulus applied may vary even if the relative movement amount of the opposing shafts 11 and 12 is the same. Furthermore, even when focusing on a specific three-dimensional tissue structure 100, the stiffness may change over time if the stretching stimulus is continuously applied. In this embodiment, the detecting unit 51 provides feedback on the magnitude of the mechanical stimulus, allowing an appropriate magnitude of mechanical stimulus to be applied to the three-dimensional tissue structure 100.

[0067] In this embodiment, the culture device 1A also includes a circulation unit 2 that includes a circulation culture tube 2a. Both ends of a flow path formed by the circulation culture tube 2a are connected to the culture vessel 10. Therefore, the culture medium in the culture vessel 10 is guided to the circulation culture tube 2a by a pump (not shown) and then supplied back to the culture vessel 10.

[0068] Furthermore, as an application example of the culture apparatus 1A, a gas regulator 52 that regulates the gas inside the culture vessel 10 can be further provided, allowing oxygen gas to be injected into the culture vessel 10. This allows culture under various oxygen conditions, such as 1% oxygen or 20% oxygen. The culture apparatus 1A may also be further provided with a temperature regulator 53, such as a heater, that regulates the temperature inside the culture vessel 10. This allows the culture vessel 10 to be maintained at a moderate temperature suitable for culture, enabling long-term culture, and the culture vessel 10 itself can function as an incubator. In this way, it is possible to provide stretch stimulation while circulating culture in an optimal environment.

[0069] The motion of the application unit 1 that applies the stretching stimulus may be solely uniaxial motion (translational motion in the stretching direction A) as described above, or may include multiaxial motion. Here, multiaxial motion refers to motion involving two or more movable axes. Examples include biaxial motion and triaxial motion. As the number of movable axes increases, stretching stimulus can be applied to the three-dimensional tissue structure 100 from various angles. For example, as described above, when the counter shaft 12 is connected to the motor 4, the counter shaft 12 can be configured to perform uniaxial or multiaxial motion in conjunction with the motor 4. Furthermore, without being limited to this configuration, the counter shaft 11 can also perform uniaxial or multiaxial motion, and both the counter shafts 11 and 12 can also be configured to perform uniaxial or multiaxial motion.

[0070] An example of multi-axis operation will now be described. Figures 2(a) to 2(d) are diagrams illustrating the operation of the application unit 1 and the mechanical stimulation applied thereby to the three-dimensional tissue structure 100. Figure 2(a) shows the above-mentioned uniaxial operation.

[0071] 2(b) shows a case where the operation of the applying unit 1 includes a twisting operation. Here, the operation of the applying unit 1 includes a twisting operation of the opposing shaft 12. For example, the three-dimensional structure 100 can be stretched and contracted in three dimensions (stereoscopic directions) by a three-dimensional twisting operation of at least one of the opposing shafts 11 and 12. The twisting operation can be imparted, for example, by a relative rotational movement of the opposing shafts 11 and 12.

[0072] This twisting and stretching action caused by multi-axis movement causes the fibers of the three-dimensional tissue structure 100 to align (align) in multiple directions, making it easier to form more complex three-dimensional shapes, such as longitudinal ridges and three-dimensional patterns of bumps. Histological findings have confirmed that the structure accurately reproduces characteristics similar to those of actual ligaments, making it possible to obtain shapes that are easily adapted to the tendons and muscles of the actual human body.

[0073] 2(c) shows a case in which the twisting motion and the pulling motion are simultaneously performed. For example, the opposing shafts 11 and 12 can perform a two-dimensional twisting motion and a one-dimensional pulling motion, respectively, thereby causing the three-dimensional tissue structure 100 to expand and contract in three dimensions (stereoscopic directions). Furthermore, for example, by combining the three-dimensional twisting motion and one-dimensional pulling motion by the opposing shafts 11 and 12, the three-dimensional tissue structure 100 can be subjected to a more three-dimensional expansion and contraction motion in three dimensions (stereoscopic directions).

[0074] FIG. 3 is a diagram showing an example of a mechanism for simultaneously performing twisting and pulling operations. Here, the opposing shaft 11 is fixed, and the opposing shaft 12 is provided in a mechanism unit 30. The mechanism unit 30 includes a movable unit 30a having a rack and on which the opposing shaft 12 is provided, and a connecting unit 30b having a pinion and connected to a motor 41. The opposing shaft 12 is rotatable relative to the movable unit 30a by a motor 42. Note that the mechanism unit 30 is merely an example, and any known technology capable of relatively translating and rotating the opposing shafts 11 and 12 may be appropriately adopted. Furthermore, the rotation direction of the opposing shaft 12 is not limited to around one axis, and the opposing shaft 12 may be rotatable relative to the opposing shaft 11 around multiple axes. Although the opposing shaft 11 is fixed here, the opposing shaft 11 may also be movable.

[0075] 2(d) shows a case where a vibrating operation is performed by including a vibrating body 13 that generates vibrations. This vibrating body 13 causes vibrations in three-dimensional directions (stereoscopic directions), making it possible to expand and contract the three-dimensional tissue structure 100 in three-dimensional directions (stereoscopic directions). Furthermore, by changing the vibration intensity of this vibrating body 13, it is possible to easily control the strength of the expansion and contraction.

[0076] The vibrator 13 is not particularly limited as long as it vibrates, but for example, sonic vibrations such as ultrasonic vibrations and audible sonic vibrations can be used. As ultrasonic vibrations, low-intensity pulsed ultrasonic waves (LIPUS) can be used, and in this case, low-intensity pulsed ultrasonic waves are preferably applied to the three-dimensional tissue structure 100, since gentle vibrations at low output are applied to the three-dimensional tissue structure 100.

[0077] The timing of vibration generation by the vibrator 13 is not particularly limited, but as shown in FIG. 4( a), it is preferable to perform the vibration during a rest period after the stretching operation of the three-dimensional tissue structure 100 and before the next stretching operation, during which the stretching operation is stopped. For example, after a 30-minute stretching operation, ultrasonic vibration is performed during the 23.5-hour rest period during which the stretching operation is stopped. This ultrasonic vibration may be continuous or intermittent. After this rest period, the ultrasonic vibration stops, and the 30-minute stretching operation resumes. Similar operations are repeated thereafter. By cyclically repeating the stretching operation and ultrasonic vibration over time in this manner, ultrasonic vibration is applied without the stretching operation, and various types of vibration are applied to the three-dimensional tissue structure 100 in a complex and well-timed manner, making it possible to efficiently form a strong three-dimensional tissue structure 100. In addition to ultrasonic vibration, sound can also be used as the vibrator 13.

[0078] 4(b) and 4(c), the vibration applied to the three-dimensional structure 100 by the vibrator 13 can be alternately vibrated at different locations on the upper and lower surfaces of the three-dimensional structure 100 in the order of upper surface location D, lower surface location E, upper surface location F, and lower surface location G. In other words, it is preferable that the upper surface locations D and F, and the lower surface locations E and G are each at a different position.

[0079] This vibration applies a three-dimensional motion to the three-dimensional tissue 100 that is as if it were being twisted, and during the culture process, a variety of complex multi-axial motions consisting of tensile motions (extension motions), vibration motions, and twisting motions are alternately applied to the three-dimensional tissue 100 continuously and multifacetedly, which is advantageous as it enables the formation of a stronger three-dimensional tissue 100.

[0080] The above is an example in which the stretching operation and the vibration operation are performed alternately, but the present invention is not limited to this, and it is also possible to perform the stretching operation and the vibration operation on the three-dimensional tissue structure 100 simultaneously.

[0081] For example, in the early stages of the culture process, the three-dimensional tissue structure 100 may be subjected to gentle vibrations, such as low-intensity pulsed ultrasound (LIPUS), during the rest period, and then, as the culture process progresses, the vibration intensity may be increased, or the stretching and vibration operations may be performed simultaneously without the rest period.

[0082] In this way, by increasing the intensity of the stretching and vibration movements over time, in the early stages of the culture process, a gentle stimulus is applied to the immature and weak three-dimensional tissue 100 without destroying its shape, thereby strengthening the foundation of the three-dimensional tissue 100, and from the middle to late stages of the culture process, a stronger stimulus is applied through the stretching and vibration movements to the three-dimensional tissue 100, which has become more stable and mature in maintaining its shape, thereby effectively increasing the strength of the three-dimensional tissue 100, and the strength of the three-dimensional tissue 100 can be increased effectively and stably.

[0083] In this way, by including uniaxial and / or multiaxial stretching stimuli, a three-dimensional tissue structure 100 is formed that can adapt to repeated stresses of stretching (loading) and relaxation (unloading), i.e., an artificial tissue structure for ligaments and tendons that performs optimal function for ligaments and tendons is reliably obtained.

[0084] Furthermore, for example, in the stretching process, the intensity of the uniaxial movement and / or the multiaxial movement may be controlled based on the traction force applied to the three-dimensional tissue structure 100. The traction force is a force applied to the three-dimensional tissue structure 100 mainly by the stretching stimulus.

[0085] Furthermore, for example, in the extension process, when the traction force applied to the three-dimensional tissue structure 100 becomes higher than a threshold, the intensity of the uniaxial movement and / or the multiaxial movement may be controlled to be weakened. Furthermore, for example, when the traction force applied to the three-dimensional tissue structure 100 becomes lower than a threshold, the intensity of the uniaxial movement and / or the multiaxial movement may be controlled to be strengthened.

[0086] Furthermore, after the above-mentioned stretching step, a compressive stimulus is applied to the inner peripheral portion of this mixture and cultured to obtain a three-dimensional tissue structure 100 containing meniscus cells.

[0087] Before the start of this compressive stimulation, various additives can be added to increase the strength of the tissue, such as appropriate cytokines. For example, bone morphogenetic proteins (BMPs) can be added to promote the formation of cartilage tissue that constitutes the meniscus. Examples of such BMPs include BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14, as described above. Among these, BMP-12 can be added.

[0088] 5 is a diagram showing an example of the configuration of a culture apparatus 1B as an example of a culture apparatus. The culture apparatus 1B can be used to easily realize this compression step.

[0089] The culture device 1B includes a culture vessel 5 for culturing this mixture, a circulating culture tube 6 for circulating the culture medium, a tube tip 61 at the tip of the circulating culture tube 6 for supplying the culture medium to the culture vessel 5, a motor control device 7 for controlling the compression of the mixture, a motor 71 controlled by the motor control device 7, a base 8 for compression-molding the mixture, and a machine base 9 for fixing the culture device 1B on a flat table.

[0090] 5 , the culture vessel 5 connected to the motor 71 operates in the motor moving direction, causing a compression action in the compression direction D, which is a direction in which the three-dimensional tissue structure 100 is pressed down, and the central portion of the three-dimensional tissue structure 100 (the portion constituting the inner peripheral side of the meniscus tissue structure) is fitted into the structure installation groove 81, which is configured as a recess in the base portion 8, and is compressed, thereby enabling a compressive stimulus to be accurately applied to the central portion of the three-dimensional tissue structure 100. That is, in the culture device 1B, the culture vessel 5 and the base portion 8 function as an application unit that applies a compressive stimulus, which is an example of a mechanical stimulus, to the three-dimensional tissue structure 100.

[0091] FIG. 6 is a diagram showing an example of the configuration of a culture apparatus 1C as an example of a culture apparatus. The culture apparatus 1C includes a pressing unit 21 that presses the three-dimensional tissue structure 100 with a pressing surface 21a, and a fixing unit 22 having L-shaped hooks 22a and 22b that hook and fix the sheet-like or ring-shaped three-dimensional tissue structure 100 in an extended state. Similar to the culture apparatus 1A shown in FIG. 1, the culture apparatus 1C includes a circulation unit 2 including a circulation culture tube 2a that circulates the culture medium, and a reciprocating unit 3 connected to a motor 4 that moves the pressing unit 21 linearly toward the fixing unit 22. As shown in the enlarged view of FIG. 6, the three-dimensional tissue structure 100 is hooked vertically between two parallel-arranged L-shaped hooks 22a and 22b. In the culture apparatus 1C, the sheet- or ring-shaped three-dimensional tissue 100, which is wound between the two hooks and hooked in a fully stretched state, is pressed against the wall 22c of the fixing part 22 in the compression direction E by the pressing surface 21a. This makes it possible to apply a necessary and sufficient compressive stimulus to the three-dimensional tissue 100. That is, in the culture apparatus 1C, the pressing part 21 and the fixing part 22 function as an applying part that applies a mechanical stimulus to the three-dimensional tissue 100.

[0092] Here, the L-shaped hooks 22a and 22b in the fixing unit 22 may be relatively movable in the extension direction F. This makes it possible to apply a compressive stimulus to the three-dimensional tissue structure 100 while applying any extension stimulus to the three-dimensional tissue structure 100 in the extension direction F. In other words, the culture apparatus 1C may include a tensioning unit (fixing unit 22) that pulls the three-dimensional tissue structure 100, and a pressing unit 21 that presses the three-dimensional tissue structure 100, to which an extension stimulus has been applied by the tensioning unit, in a direction different from the pulling direction of the tensioning unit. Furthermore, the relative movement of the L-shaped hooks 22a and 22b in the extension direction F makes it possible to use the culture apparatus 1C for three-dimensional tissue structures 100 of various sizes and shapes.

[0093] Incidentally, as components corresponding to the control unit 50, detection unit 51, gas adjustment unit 52, and temperature adjustment unit 53 of the culture apparatus 1A, the culture apparatus 1B may include a control unit 50b, a detection unit 51b, a gas adjustment unit 52b, and a temperature adjustment unit 53b. Furthermore, as components corresponding to the control unit 50, detection unit 51, gas adjustment unit 52, and temperature adjustment unit 53 of the culture apparatus 1A, the culture apparatus 1C may include a control unit 50c, a detection unit 51c, a gas adjustment unit 52c, and a temperature adjustment unit 53c. In this case, the detection unit 51b may detect the magnitude of a compressive stimulus as the mechanical stimulus. Furthermore, the detection unit 51c may detect the magnitude of a stretching stimulus and / or a compressive stimulus as the mechanical stimulus.

[0094] EXAMPLES The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0095] The following reagents, media, and cells were used in the examples. <Recombinant human fibroblast growth factor solution> Recombinant human fibroblast growth factor (Wako, 060-04543, 100 μg, Fibroblast Growth Factor Human Recombinant) was dissolved in 0.1% BSA-PBS to prepare a 10 μg / ml rhFGF solution. Hereinafter, this solution will be referred to as "FGF solution."

[0096] Recombinant Human GDF-7 Solution: Recombinant Human GDF-7 / BMP-12 (R&D, 8386-G7-050, 50 μg, Growth Differentiation Factor-7) was dissolved in 4 mM hydrochloric acid to prepare a 500 μg / ml BMP-12 solution. Hereinafter, this solution will be referred to as "BMP-12 solution."

[0097] <Recombinant human CTGF solution> Recombinant human CTGF (R&D, 9190-CC-050, 50 μg, Connective Tissue Growth Factor) was dissolved in PBS to prepare a 500 μg / ml CTGF solution. Hereinafter, this solution will be referred to as "CTGF solution."

[0098] Recombinant human TGF-β3 solution: Recombinant human TGF-β3 (PT-4124, 2 μg, Tissue Growth Factor-3, manufactured by Lonza) was dissolved in 4 mM hydrochloric acid to prepare a 20 μg / ml TGF-β3 solution. Hereinafter, this solution will be referred to as "TGF-β3 solution."

[0099] <Culture media> α-MEM, manufactured by Nacalai Tesque, 21444-05 (supplemented with 10% FBS, 1% antibiotics, and 5 ng / ml FGF solution) Fibroblast culture kit: FGM-2, manufactured by Lonza, CC-3132 Vascular endothelial cell culture kit: EGM-2, manufactured by Lonza, CC-3162 Construct production medium: FBS was removed from FGM-2, manufactured by Lonza, CC-3132 and EGM-2, manufactured by Lonza, CC-3162, and the medium was mixed at a 1:1 ratio.

[0100] <Cells> Fibroblasts (DFs): CC-2509, manufactured by Lonza iPS cell-derived mesenchymal stem cells (iPSC-MSCs): Obtained from Kyoto University iPS cell-derived ligament-tendon progenitor cells (iPSC-LPCs): Obtained from Kyoto University

[0101] (Example 1) [Preparation of artificial tissue] (Example 1-1) Preparation of ligament and tendon tissue constructs NHDF were placed in a 150 mm culture dish (FALCON, 353025) at 8 x 10 ５ iPSC-MSCs were seeded onto a 150 mm culture dish (FALCON, 353025) at 1 × 10 ６ The iPSC-LPCs were collected on day 19 after the start of induction of ligament-tendon progenitor cells from iPS cells.

[0102] The cells were mixed at a ratio of 10% iPSC-LPCs, 45% NHDF, and 45% iPSC-MSCs, suspended in construct preparation medium, and cultured at 3.5 x 10 ４The cells were seeded at 1000 cells / well in a 96-well plate (Sumitomo Bakelite, MS-9096U) to form spheroids. Two days later, the spheroids were layered using a three-dimensional cell layering system (Cyfuse, Regenova). The spheroids were layered in 10 layers at 500 μm intervals on a 7 mm diameter circular pinholder (Cyfuse, NA1029).

[0103] After culturing for 7 days while circulating the medium for constructing the structure using a Peristaltic Pump (registered trademark), the laminate was removed from the pinholder to obtain a circular three-dimensional structure. This Peristaltic Pump (registered trademark) is a tube pump (roller pump) that uses a roller to squeeze a tube such as silicone to deliver liquid.

[0104] Stretching stimulation was performed during the three-dimensional tissue culture process. Two metal rods were inserted into the hollow of the circular three-dimensional tissue, which was then placed in the device and stretched. Stretching began seven days after removal from the pin holder, with a stretch rate of 5%, a stretch frequency of 0.5 Hz, and a stretch time of 30 minutes per day. Stretching stimulation was applied for five consecutive days, followed by no stretching for two days. Tendon-ligament differentiation-inducing factors were added during the three-dimensional tissue culture process. BMP-12 solution, CTGF solution, and TGF-β3 solution were each added at 10 ng / ml. These solutions were added one month after removal from the pin holder, and the tissues were cultured for one week.

[0105] The three-dimensional structures obtained above were fixed in 10% neutral buffered formalin, dehydrated, defatted, and then thinly sectioned into paraffin blocks. The antibodies used were rabbit anti-human MKX (ATLAS ANTIBODIES, HPA006927) or rabbit anti-human SCXA (abcam, ab58655), and the reaction was carried out overnight at 4°C. Color development was carried out using the DAB polymer method.

[0106] The obtained image results are shown in Figure 10. From the obtained results, as shown in Figure 10, under the experimental conditions, Mkx-positive cells (Figure 10(a)) and Scx-positive cells (Figure 10(b)) were confirmed, and furthermore, the presence of Tenm-positive cells was certainly confirmed. Therefore, this is in accordance with the literature (Taiki Nakajima et al., Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture, NATURE Based on the description in COMMUNICATIONS, 2021, https: / / doi.org / 10.1038 / s41467-021-25328-6, it was confirmed that the three-dimensional tissue according to this Example 1-1 indeed exists as a ligament tendon.

[0107] (Example 1-2) Confirmation of Stretching Stimulus The presence or absence of stretching stimulation during the culture process of the three-dimensional tissue was confirmed. As shown in FIG. 11, two metal rods serving as the opposing axes 1 were inserted parallel to each other into the lumen of the three-dimensional tissue that had become a tubular structure, and this three-dimensional tissue was placed in the device (container) of FIG. 1 and stretched. FIG. 12(a) shows an image of the three-dimensional tissue without stretching stimulation. The tensile test results in FIG. 12(a) confirmed that the orientation of type I collagen was not aligned, and individual cell clusters were randomly present. FIG. 12(b) shows an image of the three-dimensional tissue when stretching stimulation was applied. The results in FIG. 12(b) confirmed that the orientation of type I collagen was aligned in a fixed direction along the direction of extension in the figure.

[0108] (Example 1-3) Confirmation of Stretching Stimulation (Twisting Stretching) Since uniaxial movement was confirmed in Example 1-2 above, stretching stimulation by multiaxial movement was next confirmed. Stretching stimulation (twisting stretching) was performed in the culture process of a three-dimensional tissue using the same procedure as in Example 1-2 above. Two metal rods serving as the opposing axes 1 were inserted parallel to each other into the lumen of the three-dimensional tissue that had become a tubular structure, and this three-dimensional tissue was placed in the device (container) shown in Figure 1 and stretched while twisting.

[0109] After the stretching stimulation, the collagen fibers on the tissue sections of the three-dimensional tissue were stained with Picro-Sirius Red. A polarized microscope image of the three-dimensional tissue stained with Picro-Sirius Red is shown in Figure 13(a). Enlarged views of the left and right sides of this three-dimensional tissue are shown in Figure 13(b) and (c), respectively.

[0110] The polarized light microscope images obtained confirmed that the number of brightly lit areas was reduced compared to the results shown in Figure 10 obtained in Example 1-2. Generally, the more brightly lit areas in a polarized light microscope image, the stronger the tendency toward unidirectionality. Therefore, it was confirmed that the torsional extension using multiaxial motion in this example aligned collagen fibers in multiple directions while maintaining a certain degree of unidirectionality overall in this three-dimensional tissue. Furthermore, as shown in Figure 13, the three-dimensional tissue subjected to torsional extension using multiaxial motion formed a more three-dimensionally complex shape, such as longitudinal ridges and three-dimensional patterns of unevenness. Histological findings confirmed that the characteristics of actual ligaments were well reproduced.

[0111] (Example 1-4) Confirmation of Circulation Culture The presence or absence of circulation culture during the culture process of the three-dimensional tissue was confirmed using the same procedure as in Example 1-1 above. As shown in Figure 14, cell aggregates were stacked in a tubular shape on a pin holder 4 and then placed in the device (container) of Figure 1. A tensile test was performed to confirm the presence or absence of a tube pump 5, such as a Peristaltic Pump (registered trademark), that enables circulation culture within the container, as shown in Figure 14(a) (static culture), and a tube pump 5, such as a Peristaltic Pump (registered trademark), that enables circulation culture within the container, connected to a tube, as shown in Figure 14(b) (circulation culture). This pin holder 4 is a pin holder approximately 3.4 mm square with nine needles arranged vertically and horizontally at 400 μm intervals.

[0112] Tensile test: Measurements were performed using a force gauge (Imada, ZTA-500N) attached to an electric test stand (Imada, MX2-500N-FA). Two metal rods were passed through the lumen of the three-dimensional tissue, one fixed to the stand and the other passed through the force gauge, and the force gauge side was pulled at a speed of 50 mm / min to determine the load at which the three-dimensional tissue broke.

[0113] The breaking load [mN] for static culture is shown in Fig. 15(a) and the breaking load [mN] for circulating culture is shown in Fig. 15(b). The results in Fig. 15 confirmed that the breaking load [mN] for circulating culture (1310 [mN]) was more than twice as strong as that for static culture (581 [mN]).

[0114] Regarding stiffness [N / mm], the results for static culture are shown in Figure 16(a) and the results for circulating culture are shown in Figure 16(b). From the results in Figure 16, it was confirmed that the breaking load [mN] in circulating culture (748 [mN / mm]) was more than twice as strong as that in static culture (302 [mN / mm]).

[0115] (Example 1-5) Confirmation of Raw Material Cells The type of raw material cells used in the mixing step was confirmed using the same procedure as in Example 1-1 above. Two types of three-dimensional tissues (artificial tissues for ligaments and tendons) were confirmed: one using iPS cell-derived mesenchymal stem cells as raw material cells in addition to fibroblasts, and the other using somatic stem cells.

[0116] The results of the breaking load [mN] when fibroblasts and somatic stem cells were used (with stretch stimulation) are shown in Figure 17(b). The composition ratio of fibroblasts to somatic stem cells was 1:1. The results of the breaking load [mN] when fibroblasts and somatic stem cells were used (without stretch stimulation) are shown in Figure 17(a). The composition ratio of fibroblasts to somatic stem cells was 1:1. The results of the breaking load [mN] when fibroblasts and iPS cell-derived mesenchymal stem cells were used (with stretch stimulation) are shown in Figure 17(c). The composition ratio of fibroblasts to iPS cell-derived mesenchymal stem cells was 1:1.

[0117] The results in Figure 17 confirm that when fibroblasts and iPS cell-derived mesenchymal stem cells were used (with stretching stimulation), the breaking load [mN] exceeded the detection limit, and a stronger strength was obtained than when fibroblasts and somatic stem cells were used.

[0118] (Examples 1-6) Confirmation of Raw Material Cells (with or without Added Factors) Confirmation was carried out using the same procedure as in Example 1 above, depending on the type of raw material cells (with or without added factors). That is, the raw material cells were composed of fibroblasts and iPS cell-derived mesenchymal stem cells at a 1:1 ratio, and the three-dimensional tissues (artificial tissues for ligaments and tendons) obtained with and without the addition of the above-mentioned BMP-12, CTGF, and recombinant human TGF-β3 as added factors were subjected to traction.

[0119] The results of the traction of the three-dimensional structure obtained without the addition of an additive factor are shown in Figure 18(a) and the results of the traction of the three-dimensional structure obtained with the addition of an additive factor are shown in Figure 18(b).

[0120] As shown in Figure 18, the three-dimensional tissue obtained with the addition of the additive factor exhibited high values ​​of breaking load (9.9 N) and stiffness (6.5 N / mm). These values ​​corresponded to 23.8 N and 15.5 N / mm for the size of the tissue to be transplanted into rabbits. The breaking load and stiffness were approximately 1.5 times and 3 times higher than those of the three-dimensional tissue obtained without the addition of the additive factor. Furthermore, optical microscopy confirmed that the collagen fibers in the three-dimensional tissue obtained with the addition of the additive factor were thicker, more abundantly distributed, and more unidirectionally aligned than those obtained without the addition of the additive factor.

[0121] (Example 1-7) Immunodeficient Miniature Pig Model A model was also prepared using an immunodeficient miniature pig [Itho et al. Nat Commun. 2019], and the three-dimensional tissue construct (ligament / tendon synthetic tissue construct) obtained in Example 1-6 above was transplanted. To confirm the histological characteristics of the three-dimensional tissue construct (ligament / tendon synthetic tissue construct) used as a reconstructed ligament transplanted into this immunodeficient miniature pig, tissue sections after transplantation were stained with picrosirius red. The results of polarizing microscope and optical microscope observations of the picrosirius red-stained tissue sections are shown in Figures 19 and 20, respectively.

[0122] The results showed that one month after transplantation, the transplanted ligament / tendon artificial tissues had already taken root, and numerous Sharpey's fibers were formed at the attachment site between the ligament / tendon artificial tissues and the bone, demonstrating early fusion, confirming that the fusion between the ligament / tendon artificial tissues and the bone had progressed significantly. Furthermore, one month after transplantation, the transplanted ligament / tendon artificial tissues had matured into ligaments in vivo, and the collagen fibers of the transplants were very thick and bundled, closely resembling the appearance of native ligament tissue. Furthermore, one month after transplantation, it was confirmed that the adhesion between the tissues and the residual ligament had progressed significantly. These findings confirm that the transplanted ligament / tendon artificial tissues had matured into ligaments in vivo.

[0123] Example 2 The recombinant human fibroblast growth factor solution, recombinant human GDF-7 solution, recombinant human CTGF solution, recombinant human TGF-β3 solution, medium, and cells used in Example 1 were the same as those used in Example 1.

[0124] [Preparation of artificial tissue] <Cells> (Example 2-1) Preparation of meniscus NHDF were placed in a 150 mm culture dish (FALCON, 353025) at 8 × 10 ５ The IPSC-MSCs were seeded at 1 × 10 cells / dish, cultured for 1 week using a fibroblast culture kit, and then harvested. ６ The cells were seeded in α-MEM medium at 10% IPSC-SCLs / dish, cultured for 1 week, and then harvested. IPSC-SCLs were harvested on the 19th day after the initiation of induction of meniscal progenitor cells from IPS cells. The cells were mixed at a ratio of 10% IPSC-SCLs, 45% NHDF, and 45% IPSC-MSCs, suspended in construct preparation medium, and collected at 3.5 x 10 ４ The cells were seeded at 1000 cells / well into a 96-well plate (Sumitomo Bakelite, MS-9096U) to form spheroids. Two days later, spheroids were layered using a three-dimensional cell layering system (Cyfuse, Regenova). Ten layers of spheroids were layered at 500 μm intervals on a 7 mm diameter circular pinholder (Cyfuse, NA1029). After 7 days of culture while circulating the construct preparation medium using a peristaltic pump, the layered structure was removed from the pinholder to obtain a circular three-dimensional tissue structure.

[0125] Stretching stimulation was performed during the stretching process of the three-dimensional tissue. Two metal rods were inserted into the cavity of the circular three-dimensional tissue, which was then placed in the device and stretched. Stretching began seven days after removal from the pin holder. Stretching was performed at a stretch rate of 5%, a stretch frequency of 0.5 Hz, and a stretch time of 30 minutes per day. Stretching stimulation was applied for five consecutive days, followed by two days without stretching. Meniscus differentiation inducer was added during the stretching process of the three-dimensional tissue. BMP-12 solution, CTGF solution, and TGF-β3 solution were added at 10 ng / ml each. These solutions were added one month after removal from the pin holder, and the cells were cultured for one week. Figure 21 shows the state of culture using this pin holder. From left to right, Figure 21 shows photographs of the cell aggregates immediately after layering them on the pin holder, one week later, and two weeks later. The results confirmed that tightly cohesive cell aggregates were formed as the culture progressed.

[0126] Immunohistochemical Staining: The three-dimensional tissue obtained above was fixed in 10% neutral buffered formalin, dehydrated, defatted, and then thinly sectioned into paraffin blocks. The antibodies used were rabbit anti-human MKX (ATLAS ANTIBODIES, HPA006927) or rabbit anti-human SCXA (abcam, ab58655), and the tissue was incubated overnight at 4°C. Color development was performed using the DAB polymer method. As shown in Figure 10, the presence of Mkx-positive cells, Scx-positive cells, and even Tenm-positive cells was clearly observed under these experimental conditions, confirming that the three-dimensional tissue of Example 2-1 indeed possesses the functions of a meniscus.

[0127] (Example 2-2) Confirmation of Stretching Stimulus The presence or absence of stretching stimulation during the stretching process of the three-dimensional tissue was confirmed. In the same manner as in Figure 11, two metal rods were inserted into the three-dimensional tissue that had become a sheet-like structure, and this three-dimensional tissue was placed in the device of Figure 1 (automatic stretching culture vessel) and stretched.

[0128] Immunostaining of type I collagen was confirmed using an optical microscope. Figure 22(a) shows an optical microscope image of the three-dimensional tissue before stretch stimulation. This result confirmed that no color was observed in the immunostaining of type I collagen in the absence of stretch stimulation. Figure 22(b) shows an optical microscope image of the three-dimensional tissue after stretch stimulation. This result confirmed that the immunostaining of type I collagen was observed due to stretch stimulation.

[0129] Next, the orientation of type I collagen was confirmed using a polarizing microscope. The polarizing microscope image of the three-dimensional tissue before stretch stimulation was similar to that shown in Figure 13. It was confirmed that, without stretch stimulation, the orientation of type I collagen was not aligned, and individual cell clusters were randomly present. Furthermore, the polarizing microscope image of the three-dimensional tissue after stretch stimulation was similar to that shown in Figure 13. It was confirmed that stretch stimulation resulted in a structure in which the orientation of type I collagen was aligned in a fixed direction.

[0130] (Example 2-3) Confirmation of circulatory culture Confirmation was made based on the presence or absence of circulatory culture during the extension process of the three-dimensional tissue. As in Example 1-4, as shown in Fig. 14, cell aggregates were layered in tubes, and then placed in the device of Fig. 1 (automatic expanding culture vessel). Confirmation was made by a tensile test based on the presence or absence of a peristaltic pump that enables circulatory culture within the vessel (static culture) as shown in Fig. 14(a), and the presence or absence of a peristaltic pump that enables circulatory culture within the vessel connected to the tube (circulatory culture) as shown in Fig. 14(b).

[0131] Tensile test: Measurements were performed using a force gauge (Imada, ZTA-500N) attached to an electric test stand (Imada, MX2-500N-FA). Two metal rods were passed through the lumen of the three-dimensional tissue, one fixed to the stand and the other passed through the force gauge, and the force gauge side was pulled at a speed of 50 mm / min to determine the load at which the three-dimensional tissue broke.

[0132] As for the breaking load [mN], the results of the static culture confirmed that, as in Example 1, the circulating culture exhibited a strength more than twice that of the static culture.

[0133] As with Example 1, it was also confirmed that the rigidity [N / mm] was more than twice as strong in circulating culture as in static culture.

[0134] (Example 2-4) Confirmation of Compression Cultivation The effect of compressive stimulation during the compression process of the three-dimensional tissue was confirmed. After the above-mentioned stretching process, the three-dimensional tissue was cultured by applying compressive stimulation to the three-dimensional tissue using the device (automatic compression culture vessel 1B) shown in Figure 5. The three-dimensional tissue was then stained with Safranin 0, immunostained with type I collagen, and immunostained with type II collagen. The staining results are shown in Figure 23. As shown in Figure 23, in the central portion of the three-dimensional tissue to which compressive stimulation was applied (the portion constituting the inner peripheral portion of the meniscus tissue), it was confirmed that the orientation of type II collagen was indeed aligned. Furthermore, coloring by Safranin 0 staining confirmed an increase in proteins containing water molecules, confirming that the central portion of the three-dimensional tissue exhibits properties similar to those of cartilage, such as those found in meniscus.

[0135] (Example 2-5) Confirmation of raw material cells The type of raw material cells in the mixing step was confirmed. Two cases were confirmed: one in which IPS cell-derived mesenchymal stem cells were used as raw material cells in addition to fibroblasts, and the other in which somatic stem cells were used.

[0136] The results for the breaking load [mN] when fibroblasts and somatic stem cells were used (with stretch stimulation) were similar to those shown in Figure 17. The composition ratio of fibroblasts to somatic stem cells was 1:1. The results for the use of fibroblasts and somatic stem cells (without stretch stimulation) were also similar to those shown in Figure 17. The composition ratio of fibroblasts to somatic stem cells was 1:1. The results for the use of fibroblasts and IPS cell-derived mesenchymal stem cells (with stretch stimulation) were also similar. The composition ratio of fibroblasts to IPS cell-derived mesenchymal stem cells was 1:1.

[0137] As a result, when fibroblasts and IPS cell-derived mesenchymal stem cells were used (with stretching stimulation), the breaking load [mN] exceeded the detection limit, confirming that a stronger strength was obtained than when fibroblasts and somatic stem cells were used.

[0138] Example 3: The recombinant human fibroblast growth factor solution, recombinant human GDF-7 solution, recombinant human CTGF solution, recombinant human TGF-β3 solution, medium, and cells were the same as those used in Example 1. In Example 3, a meniscus graft (strip-shaped) was prepared and implanted into a pig. <Method> The medial femoral condyle of a pig was dissected and inverted from the distal end of the femur without damaging the medial collateral ligament to expose the medial meniscus. Next, bone holes were created at the attachment points of the anterior and posterior roots of the medial meniscus on the superior border of the tibia and at the medial surface of the upper end of the tibia. After complete resection of the medial meniscus, threads sutured at both ends of the meniscus graft were passed through the respective bone holes, and the anterior, middle, and posterior portions of the meniscus graft were sutured to the joint capsule. The threads passed through the bone holes were then fixed to the medial surface of the upper end of the tibia. The medial femoral condyle was then returned to its original position and fixed to the distal femur with a screw. <Results> The results are shown in Figures 24 to 30. Figure 24 shows an annular and band-shaped meniscus prosthesis. By cutting and unfolding a portion of the annular meniscus prosthesis, a band-shaped meniscus prosthesis could be obtained. Figure 25 shows a meniscus implant fabricated by bundling and suturing three band-shaped meniscus prosthesis. It was possible to apply tension in opposite directions to the sutures attached to both ends. Figure 26 shows the medial femoral condyle of a pig that was cut and reflected to expose the medial meniscus, after which the medial meniscus was completely resected. Total meniscus replacement was possible in the pig's knee joint without damaging the ligaments. Figure 27 shows the placement of a meniscus transplant on the upper edge of the tibia by passing a suture through a bone tunnel created in the tibia. The transplant, which took on a crescent shape similar to that of a healthy meniscus, enabled total meniscus replacement. Figure 28 shows the results of a fluoroscopic examination of a pig's knee joint four weeks after the meniscus transplant. It was confirmed that the meniscus maintained its normal shape and functioned as a shock absorber in the knee joint, without narrowing the joint space. Figure 29 shows the results of a macroscopic examination of a pig's knee joint after incision, confirming the presence of a meniscus transplant with a shape similar to that of a meniscus.FIG. 30 shows the results of a histological examination of the meniscus implant four weeks after the meniscus was transplanted, and the same tissue morphology as that of the meniscus was confirmed.

Claims

1. A method for strengthening a three-dimensional tissue, comprising a step of culturing a three-dimensional tissue formed by stacking cell aggregates while applying a stretching stimulus and / or a compression stimulus, wherein the three-dimensional tissue is composed only of cells without artificial materials, the stretching stimulus is a stretching stimulus including uniaxial motion or multiaxial motion, the compression stimulus is applied by pressing the three-dimensional tissue with a pressing unit having a pressing surface, and the three-dimensional tissue is cultured under circulatory culture.

2. (delete)

3. The method of claim 1 , wherein the stretching stimulus comprises at least one of a twisting motion, a pulling motion, and a vibrating motion.

4. (delete)

5. The method according to claim 1 , further comprising adding a supplementary factor to the medium in which the three-dimensional tissue is cultured.

6. (delete)

7. The method according to claim 1 , wherein the cell mass comprises mesenchymal stem cells, fibroblasts, and cells that are the source of a target tissue.

8. The method according to claim 7, wherein the source cells are progenitor cells of bone, cartilage, ligament, tendon or meniscus.

9. The method of claim 8, wherein the progenitor cells are sclerotome cells.

10. The method of claim 9, wherein the three-dimensional tissue structure is a tissue structure for bone, cartilage, ligament, tendon or meniscus.

11. A method for producing a three-dimensional tissue with enhanced strength, comprising a step of culturing a three-dimensional tissue formed by stacking cell aggregates while applying a stretching stimulus and / or a compression stimulus, wherein the three-dimensional tissue is composed only of cells without artificial materials, the stretching stimulus is a stretching stimulus including uniaxial movement or multiaxial movement, the compression stimulus is applied by pressing the three-dimensional tissue with a pressing unit having a pressing surface, and the three-dimensional tissue is cultured under circulatory culture.

12. (delete)

13. The method of claim 11 , wherein the stretching stimulus comprises at least one of a twisting motion, a pulling motion, and a vibrating motion.

14. (delete)

15. The method according to claim 11 , further comprising adding a supplementary factor to the medium in which the three-dimensional tissue is cultured.

16. (delete)

17. The method according to claim 11 , wherein the cell mass comprises mesenchymal stem cells, fibroblasts, and cells that are the source of a target tissue.

18. The method according to claim 17, wherein the source cells are progenitor cells of bone, cartilage, ligament, tendon or meniscus.

19. 19. The method of claim 18, wherein the bone, cartilage, ligament, tendon or meniscus progenitor cells are sclerotome cells.

20. 20. The method of claim 19, wherein the three-dimensional tissue structure is a tissue structure for bone, cartilage, ligament, tendon or meniscus.

21. A three-dimensional tissue having a breaking strength of at least 0.001 N / mm, which is formed by culturing a three-dimensional tissue formed by stacking cell clusters while applying stretching and / or compressive stimuli, wherein the three-dimensional tissue is composed only of cells that do not contain artificial materials, the cell clusters contain mesenchymal stem cells, fibroblasts, and cells that are the raw material for the target tissue, and the culture of the three-dimensional tissue is carried out under circulatory culture.

22. (delete)

23. The tissue according to claim 21, wherein the source cells are progenitor cells of bone, cartilage, ligament, tendon or meniscus.

24. 24. The tissue of claim 23, wherein the progenitor cells of bone, cartilage, ligament, tendon or meniscus are sclerotome cells.

25. The tissue structure according to claim 24, wherein the three-dimensional tissue structure is a tissue structure for bone, cartilage, ligament, tendon or meniscus.

26. A culture device for culturing a three-dimensional tissue, An application unit that applies a mechanical stimulus to the three-dimensional tissue structure. A culture apparatus, wherein the mechanical stimulus applied by the applying unit includes at least a stretching stimulus, the three-dimensional structure has an opening; The application portion includes at least two shaft members, The application unit applies a mechanical stimulus to the three-dimensional tissue structure by relatively moving the at least two shaft members while being inserted into the opening. The apparatus.

27. (delete)

28. (delete)

29. 27. The culture device of claim 26, The application unit applies a mechanical stimulus to the three-dimensional tissue structure by the relative translational movement of the at least two shaft members. Culture device.

30. 27. The culture device of claim 26, The application unit applies a mechanical stimulus to the three-dimensional tissue structure by relatively rotating and moving the at least two shaft members. Culture device.

31. 27. The culture device of claim 26, The applying unit includes a vibration body that generates vibration, The applying unit applies a mechanical stimulus to the three-dimensional tissue structure by expanding and contracting the three-dimensional tissue structure using vibration generated by the vibrator. Culture device.

32. 27. The culture device of claim 26, The mechanical stimulus applied by the application unit includes at least a compression stimulus. Culture device.

33. 27. The culture device of claim 26, The applying unit is A base portion having a recess into which the three-dimensional structure fits; A pressing unit that presses the three-dimensional structure fitted into the recess; Including, Culture device.

34. 27. The culture device of claim 26, The applying unit is A tensioning unit that tensions the three-dimensional tissue structure; a pressing unit that presses the three-dimensional tissue structure to which a stretching stimulus has been applied by the tension unit in a direction different from the tension direction of the tension unit; Including, Culture device.

35. 27. The culture device of claim 26, Further comprising a circulation unit for circulating the culture solution in the culture vessel; Culture device.

36. 27. The culture device of claim 26, Further comprising a gas adjusting unit for adjusting the gas in the culture vessel; Culture device.

37. 27. The culture device of claim 26, Further comprising a temperature control unit for controlling the temperature inside the culture vessel; Culture device.

38. 27. The culture device of claim 26, a detection unit that detects a physical quantity corresponding to the magnitude of the mechanical stimulus applied to the three-dimensional tissue by the application unit; A control unit that controls the magnitude of the mechanical stimulus applied by the application unit based on the detection result of the detection unit; Further comprising: Culture device.

39. A method for strengthening ligament and tendon tissue, comprising a step of culturing ligament and tendon tissue formed by layering cell masses consisting of mesenchymal stem cells, fibroblasts, and ligament and tendon precursor cells, while applying a stretching stimulus, wherein the ligament and tendon tissue is composed only of cells containing no artificial material, the stretching stimulus is a uniaxial or multiaxial stretching stimulus having a strength of 0.001 to 10,000 [N], and the stretching stimulus is repeated between periods of rest in which no stretching stimulus is applied, and the ligament and tendon tissue is cultured under circulatory culture.

40. The method according to claim 39, wherein the stretching stimulation is performed for 5 days under the condition of a stretching time of 30 minutes / day.

41. 40. The method of claim 39, wherein vibration stimulation is administered during the resting period.

42. The method according to claim 39, further comprising adding a tendon ligament differentiation factor during the culture step.

43. The method according to claim 41, wherein the tendon ligament differentiation induction factors are bone morphogenetic factor-12, connective tissue growth factor and tissue growth factor-β3.

44. A method for strengthening a meniscus tissue, comprising a step of culturing a meniscus tissue formed by layering cell masses consisting of mesenchymal stem cells, fibroblasts, and meniscus progenitor cells, while applying a stretching stimulus and / or a compressive stimulus, wherein the meniscus tissue is composed only of cells containing no artificial material, the stretching stimulus is a stretching stimulus including uniaxial or multiaxial motion, and is applied with a strength of 0.001 to 10,000 [N], the stretching stimulus is repeated between a rest period in which the expansion stimulus is not applied, the compressive stimulus is applied by pressing the meniscus tissue with a pressing part having a pressing surface, and the meniscus tissue is cultured under circulatory culture.

45. The method according to claim 44, wherein the stretch stimulation is performed for 5 days under the condition of a stretch time of 30 minutes / day.

46. 45. The method of claim 44, wherein vibration stimulation is administered during the resting period.

47. The method of claim 43, further comprising adding a meniscus differentiation induction factor during the culture step.

48. The method according to claim 46, wherein the meniscus differentiation induction factors are osteoinductive factor-12, connective tissue growth factor and tissue growth factor-β3.