Method for strengthening strength of three-dimensional tissue body

Abstract

This method for strengthening the strength of a three-dimensional tissue body formed by laminating cell clusters comprises a step for culturing the three-dimensional tissue body while providing extension stimulation and / or compression stimulation to the tissue body.

Description

Method for enhancing the strength of a three-dimensional structure

[0001] The present invention relates to a method for enhancing the strength of a three-dimensional structure and an apparatus for use in said enhancement method.

[0002] Injuries to ligaments, tendons, menisci, and other tissues, often caused by sports, are among the most common orthopedic disorders. Meniscus and anterior cruciate ligament (ACL) injuries are particularly common. While current treatments for these injuries primarily involve autologous tendon transplants, typically harvested from the semitendinosus muscle, ACL reconstruction techniques using artificial biomaterials (e.g., synthetic fibers and collagen) instead of autologous tendon transplants have been employed in regenerative medicine research. 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 regenerative medicine materials (Patent Document 1). However, because orthopedic tissues are constantly subjected to repeated stresses of stretching (loading) and relaxation (unloading), it is difficult to reproduce the tissues previously treated and, ultimately, to ensure their function.

[0003] JP 2020-202785 A

[0004] Therefore, there is a need to develop medical materials that can withstand physical strength and can be used in regenerative medicine.

[0005] 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 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 a three-dimensional tissue formed by stacking cell clusters, comprising the step of culturing the three-dimensional tissue while applying a stretching stimulus and / or a compressive stimulus to the 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 a torsional motion, a tensile motion, and a 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 to the tissue.

[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

[11] , 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 raw material cells are precursor cells of bone, cartilage, ligament, tendon, or meniscus.

[19] The method according to

[18] , wherein the precursor 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 formed by layering cell aggregates is cultured while being subjected to a stretch stimulus and / or a compressive stimulus, and has a breaking strength of at least 0.001 N / mm. ２

[22] The tissue according to

[21] , wherein the cell mass contains mesenchymal stem cells, fibroblasts, and cells that will serve as raw materials for the target tissue.

[23] The tissue according to

[22] , wherein the raw materials are progenitor cells for bone, cartilage, ligament, tendon, or meniscus.

[24] The tissue according to

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

[25] The tissue according to

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

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

[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 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 by moving the at least two shaft members relatively while inserted into the openings.

[29] The culture device according to

[28] , wherein the application unit applies a mechanical stimulus to the three-dimensional tissue by moving the at least two shaft members translationally relative to each other.

[30] The culture device according to

[28] , wherein the application unit applies a mechanical stimulus to the three-dimensional tissue by moving the at least two shaft members rotationally relative to each other.

[31] The culture device 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 expanding and contracting the three-dimensional tissue with the vibrations generated by the vibrating body.

[32] The culture device according to

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

[33] The culture device according to

[26] , wherein the applying unit includes: a base unit having a recess formed therein into which the three-dimensional tissue structure fits; and a pressing unit that presses the three-dimensional tissue structure fitted in the recess.

[34] The culture apparatus according to

[26] , wherein the applying unit includes a pulling unit that pulls the three-dimensional tissue, and a pressing unit that presses the three-dimensional tissue to which a stretching stimulus has been applied by the pulling unit in a direction different from the pulling direction of the pulling unit.

[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 adjustment unit that adjusts the gas in the culture vessel.

[37] The culture apparatus according to

[26] , further comprising a temperature adjustment unit that adjusts 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.

[0006] 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.

[0007] 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) is a diagram illustrating an example of a mechanism for simultaneously performing twisting and pulling operations. (a) is a diagram illustrating the timing of the vibration operation, and (b) and (c) are diagrams illustrating the position and order of vibration application. (b) is a diagram showing an example of the configuration of a culture device. (c) is a diagram showing the differentiation of iPS cells into each tissue. (c) is a schematic diagram of a meniscus. (d) is a diagram showing the differentiation of iPS cells into each tissue. (b) is a diagram of a meniscus. (c) is a diagram showing the differentiation of iPS cells into each tissue. (d) is a diagram showing the differentiation of iPS cells into each tissue. (c) is a diagram showing the immunohistochemical staining results of an artificial tissue structure for ligament and tendon. (e) is a diagram showing the placement of a cell structure in an apparatus and stretching it. (f) is a diagram showing tissues with and without a stretch stimulus for an artificial tissue structure for ligament and tendon. (f) is a diagram showing polarized microscope images obtained by picrosirius red staining after a stretch stimulus (torsional stretch) for an artificial tissue structure for ligament and tendon. (f) is a diagram showing the image results of polarized microscope images obtained by picrosirius red staining after a stretch stimulus (torsional stretch) for an artificial tissue structure for ligament and tendon. (f) is a diagram showing the configuration of an apparatus for circulatory culture in the artificial tissue culture process. (f) is a diagram showing the results of the breaking load with and without circulatory culture in the artificial tissue culture process for ligament and tendon. 1 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. 1 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. 2 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. 3 is a polarized microscope image of a tissue slice stained with picrosirius red after transplantation of an artificial tissue for ligament and tendon. FIG. 4 is an optical microscope image of a tissue slice stained with picrosirius red after transplantation of an artificial tissue for ligament and tendon. FIG. 5 is a diagram showing the results of stacking cell aggregates on a pin holder. FIG. 6 is a diagram showing the results of immunostaining of type I collagen in a meniscus tissue under an optical microscope. FIG. 7 is a diagram showing various staining results for a meniscus tissue. FIG. 8 is a diagram showing details of a method for transplanting a meniscus tissue. FIG. 9 is a diagram showing a meniscus tissue prepared for transplantation. FIG. 10 is a diagram showing the preparation of a meniscus tissue injury (resection) model. FIG. 11 is a diagram showing the actual transplantation of a meniscus tissue. FIG. 12 is an X-ray image of a knee joint 29 days (approximately 4 weeks) after meniscus tissue transplantation. 1 shows the results of an autopsy performed 29 days (approximately 4 weeks) after meniscus tissue transplantation. 2 shows the results of tissue staining performed 29 days (approximately 4 weeks) after meniscus tissue transplantation. 3 shows the mechanical properties of a cell construct in a compression test.FIG. 10 is a diagram showing the mechanical properties of a cell structure obtained by a compression test.

[0008] The present invention relates to a method for strengthening a three-dimensional tissue, which comprises culturing a three-dimensional tissue formed by stacking cell clusters while applying a stretching stimulus and / or a compressive stimulus to the three-dimensional tissue. The method involves stacking cell clusters using a bio-3D printer with a pinholder-shaped needle to create a three-dimensional tissue composed only of cells, and then using the stretching culture device and compressive culture device of the present invention to induce differentiation into a tissue with enhanced mechanical strength. The three-dimensional tissue does not use artificial materials and has sufficient mechanical strength solely from the cells, which allows for long-term avoidance of damage to various tissues and pathological progression.

[0009] 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.

[0010] (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.

[0011] 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).

[0012] 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.

[0013] 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.

[0014] 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.

[0015] (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.

[0016] (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 7-1, 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.

[0017] 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.

[0018] (2) Preparation of Spheroids In the present invention, mesenchymal stem cells, fibroblasts, and cells (hereinafter also referred to as "raw cells") that will serve as the source of the target tissue are first mixed to obtain a mixture. This mixture can be prepared, 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 interconnected. 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.

[0019] 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.

[0020] 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.

[0021] (3) Spheroid Lamination A known spheroid lamination method involves arranging cells in an arbitrary three-dimensional space to create a cellular three-dimensional structure (three-dimensional structure) (WO 2008 / 123614). This method involves arranging needle-shaped objects on a substrate in a pinholder shape, and then piercing the needle-shaped objects to place the spheroids. In the present invention, the above method is used to laminate spheroids to create a three-dimensional structure (three-dimensional structure). Since an automated lamination robot for implementing the above method is already known (Bio 3D Printer "Regenova" (registered trademark), Cyfuse Co., Ltd.), it is also possible to create a three-dimensional structure using this robot.

[0022] 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 raw material 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:raw material cells). From the viewpoint of production efficiency, a low ratio of raw material cells is preferable, for example, 49:49:2.

[0023] In this way, a three-dimensional structure composed only of cells can be obtained. "Composed only of cells" means that the components forming the three-dimensional structure are cells alone, without adding any artificial materials such as synthetic materials or biomaterials. However, this does not exclude the inclusion of extracellular matrix (e.g., collagen) secreted from the cells or cell masses that form the three-dimensional structure. Furthermore, the three-dimensional structure obtained in this way can have sufficient mechanical strength with only the cells.

[0024] (4) Differentiation into various tissues and three-dimensional tissues The three-dimensional structure obtained by layering spheroids can be cultured in the presence of various differentiation factors to obtain three-dimensional tissues of various tissues. However, in this specification, there is no need to clearly define the terms "three-dimensional structure" and "three-dimensional tissue" to distinguish between them. The terms "three-dimensional structure" and "three-dimensional tissue" can be used arbitrarily during the process from the structure immediately after layering spheroids to the final three-dimensional tissue, which is then cultured in the presence of added factors and subjected to physical stimulation.

[0025] (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.

[0026] (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.

[0027] (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.

[0028] This three-dimensional tissue contains ligament tenocytes differentiated from the 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, as described below.

[0029] 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, 50 or more, 100 or more, 1,000 or more, or 10,000 or more.

[0030] (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, which promote differentiation into meniscus cells and allow them to mature into stronger tissue structures. Examples of BMP family members used for differentiation into meniscus include 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.

[0031] This 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, as described below.

[0032] Furthermore, this three-dimensional 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, making it possible to exhibit properties similar to those of cartilage. This increase in proteins that hold water molecules can be confirmed by safranin O staining or alcian blue staining (see the Examples described below).

[0033] Regarding the relationship between the orientation of the type I collagen and the type II collagen, it is preferable that the orientation directions of the type I collagen and the type II collagen are aligned in an orthogonal orientation to each other. Due to this characteristic orientation, as shown in Fig. 7-2, in the three-dimensional tissue 100, the orientation of the 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 the type I collagen is aligned in an orientation direction perpendicular to the orientation direction of the type II collagen, giving it properties similar to tendons and ligaments, thereby obtaining a meniscus tissue with a structure similar to that of an actual meniscus (see Examples described later).

[0034] More preferably, when forming type I collagen and type II collagen, a stretching stimulus is applied to the entire mixture (three-dimensional structure) 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, a three-dimensional tissue is formed in which the orientation of type II collagen is aligned at the inner periphery of the meniscus tissue, and the orientation of type I collagen is aligned from the inner periphery to the outer periphery.

[0035] 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.

[0036] 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, 50 or more, 100 or more, 1,000 or more, or 10,000 or more.

[0037] (5) Stretching Stimulus and / or Compression Stimulus In the present invention, to enhance the strength of a three-dimensional tissue, the tissue is cultured in a culture vessel under solution circulation conditions and / or with the application of a stretching stimulus. The culture in this stretching step may involve culturing only under solution circulation conditions, culturing only with the application of a stretching stimulus, or culturing under solution circulation conditions with the application of a stretching stimulus. 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.

[0038] (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.

[0039] 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)].

[0040] In one aspect of the present invention, the strength of the stretching stimulus may have a lower limit, but is not limited to, for example, 0.001 [N], 0.01 [N], or 0.1 [N], and an upper limit, but is not limited to, for example, 10,000 [N], 1,000 [N], 100 [N], 10 [N], or 1 [N]. In one aspect of the present invention, the range of the strength of the stretching stimulus is, for example, 0.0001 to 1,000 [N], 0.0001 to 100 [N], 0.0001 to 1 [N], 0.001 to 1,000 [N], 0.001 to 100 [N], 0.001 to 10 [N], 0.001 to 1 [N], 0.01 to 1,000 [N], 0.01 to 100 [N], 0.01 to 10 [N], 0.01 to 1 [N], 0.1 to 1,000 [N], 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N]. In a preferred embodiment of the present invention, the strength of the stretching stimulus is, for example, 0.1 [N], 0.2 [N], 0.3 [N], 0.4 [N], 0.5 [N], 0.6 [N], 0.7 [N], 0.8 [N], 0.9 [N], or 1.0 [N].

[0041] 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).

[0042] 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).

[0043] 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 bone, tendons, ligaments, the meniscus itself, or the cartilage tissue that constitutes the meniscus. Examples of such bone morphogenetic proteins (BMPs) include, but are not limited to, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14.

[0044] 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)]. In one embodiment of the present invention, the breaking load has a lower limit, but is not limited to, for example, 0.001 [N], 0.01 [N], or 0.1 [N], and an upper limit, but is not limited to, for example, 100,000 [N], 10,000 [N], 1,000 [N], 100 [N], 10 [N], or 1 [N]. In one embodiment of the present invention, the breaking load ranges, for example, 0.001 to 100,000 [N], 0.001 to 10,000 [N], 0.001 to 1,000 [N], 0.001 to 100 [N], 0.001 to 10 [N], 0.001 to 1 [N], 0.01 to 100,000 [N], 0.01 to 10 ,000 [N], 0.01 to 1,000 [N], 0.01 to 100 [N], 0.01 to 10 [N], 0.01 to 1 [N], 0.1 to 100,000 [N], 0.1 to 10,000 [N], 0.1 to 1,000 [N], 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N]. In a preferred embodiment of the present invention, the breaking load is, for example, 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N].

[0045] (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.

[0046] For example, the addition of a bone morphogenetic protein (BMP) can promote the formation of bone, the meniscus itself, or the cartilage tissue that constitutes the meniscus. Examples of such bone morphogenetic proteins (BMPs) include, but are not limited to, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14. The combinations of additional factors for each tissue and the combinations of BMP families are the same as those described above. However, the additional factors are not limited to the above combinations. The compression 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)].

[0047] In one aspect of the present invention, the strength of the compressive stimulus (pressure load) can have a lower limit of, but is not limited to, 0.001 [N], 0.01 [N], or 0.1 [N], and an upper limit of, but is not limited to, 10,000 [N], 1,000 [N], 100 [N], 10 [N], or 1 [N].

[0048] In one aspect of the present invention, the range of the strength of the compressive stimulus is, for example, 0.0001 to 1,000 [N], 0.0001 to 100 [N], 0.0001 to 1 [N], 0.001 to 1,000 [N], 0.001 to 100 [N], 0.001 to 10 [N], 0.001 to 1 [N], 0.01 to 1,000 [N], 0.01 to 100 [N], 0.01 to 10 [N], 0.01 to 1 [N], 0.1 to 1,000 [N], 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N]. In a preferred embodiment of the present invention, the strength of the compressive stimulus is, for example, 0.1 [N], 0.2 [N], 0.3 [N], 0.4 [N], 0.5 [N], 0.6 [N], 0.7 [N], 0.8 [N], 0.9 [N], or 1.0 [N].

[0049] The breaking load [mN] in the pressing step is 0.001 to 100,000 [N] (1 [mN] to 100 [kN]), or 0.0001 to 10,000 [N / mm ２ (MPa)]. In one embodiment of the present invention, the breaking load has a lower limit, but is not limited to, for example, 0.001 [N], 0.01 [N], or 0.1 [N], and an upper limit, but is not limited to, for example, 100,000 [N], 10,000 [N], 1,000 [N], 100 [N], 10 [N], or 1 [N]. In one embodiment of the present invention, the breaking load ranges, for example, 0.001 to 100,000 [N], 0.001 to 10,000 [N], 0.001 to 1,000 [N], 0.001 to 100 [N], 0.001 to 10 [N], 0.001 to 1 [N], 0.01 to 100,000 [N], 0.01 to 10 ,000 [N], 0.01 to 1,000 [N], 0.01 to 100 [N], 0.01 to 10 [N], 0.01 to 1 [N], 0.1 to 100,000 [N], 0.1 to 10,000 [N], 0.1 to 1,000 [N], 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N]. In a preferred embodiment of the present invention, the breaking load is, for example, 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N].

[0050] (6) Circulating Culture In the present invention, the strength of a three-dimensional tissue can be enhanced by circulating the tissue using a circulating culture device. Circulating culture is culture performed during the extension process of a three-dimensional tissue, and refers to culturing by flowing the culture medium in a culture vessel, including unidirectional flow of the culture medium and convection of the culture medium within the vessel. Circulating 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 culture medium that flows into the culture vessel flows through the vessel, then is discharged outside and subjected to circulating culture again.

[0051] Circulation culture is preferably performed by allowing 1 mL to 100 mL of medium to flow into the culture vessel per unit time (minute) and discharging the same amount of medium. Alternatively, the culture vessel can be shaken to create convection or vortex of the medium.

[0052] (7) Strength-enhanced three-dimensional tissues. 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 strength-enhanced three-dimensional tissue when subjected to a stretching stimulus or a compressive stimulus is 0.001 to 100,000 N (1 mN to 100 kN), or 0.0001 to 10,000 N / mm (MPa).

[0053] In one embodiment of the present invention, the range of the breaking load is, for example, 0.001 to 100,000 [N], 0.001 to 10,000 [N], 0.001 to 1,000 [N], 0.001 to 100 [N], 0.001 to 10 [N], 0.001 to 1 [N], 0.01 to 100,000 [N], 0.01 to 10 ,000 [N], 0.01 to 1,000 [N], 0.01 to 100 [N], 0.01 to 10 [N], 0.01 to 1 [N], 0.1 to 100,000 [N], 0.1 to 10,000 [N], 0.1 to 1,000 [N], 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N]. In a preferred embodiment of the present invention, the breaking load is, for example, 0.1 to 100 [N], 0.1 to 10 [N], or 0.1 to 1 [N].

[0054] 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.

[0055] 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.

[0056] 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 a mechanical stimulus with a strength within the numerical range (or a part thereof) indicated in the above section "1. Tissue Strengthening Method and Strengthened Tissue (6) Stretching Stimulus and / or Compression Stimulus." While the opposing shafts 11 and 12 have been described as shaft members in the present invention, other methods besides the method of stretching using a shaft member can also be employed, such as a method of grasping (clamping) the three-dimensional structure and stretching it, or a method of adhering the three-dimensional structure to a stretching member. Furthermore, while the embodiment in which the shaft member is inserted into the opening of the three-dimensional structure has been described, a needle-shaped shaft member may be inserted into the three-dimensional structure without being inserted into the opening. Furthermore, the stretching direction may be not only perpendicular to the opening axis, but also coaxial (parallel) with the opening axis.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] Furthermore, after the 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 (S3: compression step).

[0081] 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, but are not limited to, BMP-2, BMP-4, BMP-10, BMP-11, BMP-12, BMP-13, and BMP-14, and among these, BMP-12 can be added.

[0082] 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.

[0083] 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 portion 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 pressing portion 8 and a base portion 10 for compression-molding the mixture, and a machine base 9 for fixing the culture device 1B on a flat table.

[0084] As shown in the enlarged view of the circled portion in FIG. 5 , the culture vessel 5 connected to the motor 71 operates in the motor movement direction, causing a compression motion along the compression direction D, which is a direction in which the three-dimensional tissue structure 100 (not shown) is pressed down. 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 and compressed by the structure installation groove 81, which is configured as a recess in the base 10, thereby enabling accurate application of a compressive stimulus to the central portion of the three-dimensional tissue structure 100. That is, in the culture device 1B, the pressing unit 8 and the base 10 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. Here, the culture vessel 5 is cylindrical, but only the lid of the culture vessel 5 is depicted in FIG. 5 . The base 10 is located on the bottom (not shown) of the culture vessel 5, and a tube tip 61 protrudes from the side wall of the culture vessel 5, penetrating into the interior of the culture vessel 5, allowing the culture medium to be circulated through the circulating culture tube 6.

[0085] 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.

[0086] In addition, 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 the 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.

[0087] 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.

[0088] 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.

[0089] 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."

[0090] 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."

[0091] <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."

[0092] 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."

[0093] <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.

[0094] <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

[0095] (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 ５iPSC-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.

[0096] 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 ４ The 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 pin holder (Cyfuse, NA1029).

[0097] 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.

[0098] Stretching stimulation was performed during the three-dimensional tissue culture process. 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, 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.

[0099] 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.

[0100] The obtained image results are shown in Figure 8. From the obtained results, as shown in Figure 8, under the experimental conditions, Mkx-positive cells (Figure 8(a)) and Scx-positive cells (Figure 8(b)) were confirmed, and furthermore, the presence of Tenm-positive cells was certainly confirmed. This confirms the literature (Taiki Nakajima et al., Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture, NATURE COMMUNICATIONS, 2021, Based on the description in https: / / doi.org / 10.1038 / s41467-021-25328-6, it was confirmed that the three-dimensional tissue according to this Example 1-1 certainly existed as a ligament tendon.

[0101] (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. 9, 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. 10(a) shows an image of the three-dimensional tissue without stretching stimulation. The tensile test results in FIG. 10(a) confirmed that the orientation of type I collagen was not aligned, and individual cell clusters were randomly present. FIG. 10(b) shows an image of the three-dimensional tissue when stretching stimulation was applied. The results in FIG. 10(b) confirmed that the orientation of type I collagen was aligned in a fixed direction along the direction of extension in the figure.

[0102] (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.

[0103] After the stretching stimulation, the collagen fibers on the tissue sections of the three-dimensional tissue were stained with Picro-Sirius Red stain. A polarized microscope image of the three-dimensional tissue stained with Picro-Sirius Red is shown in Figure 11(a). Enlarged views of the left and right sides of this three-dimensional tissue are shown in Figure 11(b) and (c), respectively.

[0104] The polarized light microscope images obtained confirmed that the number of brightly lit areas was reduced. 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 the multiaxial motion of this example resulted in the alignment of collagen fibers in multiple directions while maintaining a certain degree of unidirectionality throughout the three-dimensional tissue. Furthermore, as shown in Figure 11 , the three-dimensional tissue subjected to torsional extension using the multiaxial motion developed a more three-dimensionally complex shape, including longitudinal ridges and three-dimensional patterns of unevenness. Histological findings confirmed that the characteristics of actual ligaments were well reproduced.

[0105] (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 12, 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 12(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 12(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.

[0106] Tensile tests were performed using a tissue puller (DMT, 560TP) or a testing machine consisting of a motorized test stand (Imada, MX2-500N-FA) equipped with a force gauge (Imada, ZTA-500N). Two metal rods were inserted into the lumen of the three-dimensional tissue, one of which was fixed to the stand and the other was inserted through the force gauge. The force gauge side was pulled at a speed of 50 mm / min, and the load at which the three-dimensional tissue broke was determined.

[0107] The breaking load [mN] for static culture is shown in Fig. 13(a) and the breaking load [mN] for circulating culture is shown in Fig. 13(b). The results in Fig. 13 confirmed that the breaking load [mN] for circulating culture (1310 [mN]) was more than twice as strong as that for static culture (581 [mN]).

[0108] Regarding stiffness [N / mm], the results for static culture are shown in Figure 14(a) and the results for circulating culture are shown in Figure 14(b). From the results in Figure 14, 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]).

[0109] (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.

[0110] FIG. 15( a ) shows the results of breaking load [mN] when fibroblasts and somatic stem cells were used (without stretching stimulation). The composition ratio of fibroblasts to somatic stem cells was 1:1. FIG. 15( b ) shows the results when fibroblasts and somatic stem cells were used (with stretching stimulation). The composition ratio of fibroblasts to somatic stem cells was 1:1. FIG. 15( c ) shows the results when fibroblasts and iPS cell-derived mesenchymal stem cells were used (with stretching stimulation). The composition ratio of fibroblasts to iPS cell-derived mesenchymal stem cells was 1:1.

[0111] The results in Figure 15 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.

[0112] (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.

[0113] The results of the traction of the three-dimensional structure obtained without the addition of an additive factor are shown in Figure 16(a) and the results of the traction of the three-dimensional structure obtained with the addition of an additive factor are shown in Figure 16(b).

[0114] As shown in Figure 16, 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.

[0115] (Examples 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 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 microscopy and optical microscopy of the picrosirius red-stained tissue sections are shown in Figures 17 and 18, respectively.

[0116] 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.

[0117] 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.

[0118] [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.

[0119] Stretching stimulation was performed during the three-dimensional tissue extension process. 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 three-dimensional tissue extension process. 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. The state of culture using this pin holder is shown in Figure 19. Figure 19 shows, from left to right, photographs of the cell aggregates immediately after layering 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.

[0120] Immunohistochemical Staining The three-dimensional structure obtained above was fixed in 10% neutral buffered formalin, dehydrated, defatted, and then thinly sectioned into paraffin blocks. The antibody used was 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. As shown in Figure 8, 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 structure of Example 2-1 indeed possesses the functions of a meniscus.

[0121] (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 9, 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.

[0122] Immunostaining of type I collagen was confirmed using an optical microscope. Figure 20(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 20(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.

[0123] Next, the orientation of type I collagen was confirmed using a polarizing microscope. Polarizing microscope images of the three-dimensional tissue before stretch stimulation confirmed that, in the absence of stretch stimulation, the orientation of type I collagen was not aligned, and individual cell clusters were randomly present. Polarizing microscope images of the three-dimensional tissue after stretch stimulation also confirmed that the stretch stimulation resulted in a structure in which the orientation of type I collagen was aligned in a fixed direction.

[0124] (Example 2-3) Confirmation of circulating culture Confirmation was made based on the presence or absence of circulating culture during the stretching process of the three-dimensional tissue. As in Example 1-4, as shown in Fig. 12, cell aggregates were layered in tubes, and then placed in the device of Fig. 1 (automatic stretching culture vessel). Confirmation was made by a tensile test based on the presence or absence of a peristaltic pump that enables circulating culture within the vessel (static culture) as shown in Fig. 12(a), and the presence or absence of a peristaltic pump that enables circulating culture within the vessel connected to the tube (circulating culture) as shown in Fig. 12(b).

[0125] 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.

[0126] 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.

[0127] 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.

[0128] (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 above-mentioned device (automatic compression culture vessel 1B) shown in Figure 5. The three-dimensional tissue was then stained with Safranin O, immunostained with type I collagen, and immunostained with type II collagen. The staining results are shown in Figure 21. As shown in Figure 21, 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 O 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.

[0129] (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.

[0130] The results for the breaking load [mN] when fibroblasts and somatic stem cells were used (with compressive stimulation) were similar to those shown in Figure 15. The composition ratio of fibroblasts to somatic stem cells was 1:1. The results for the use of fibroblasts and somatic stem cells (without compressive stimulation) were also similar to those shown in Figure 15. 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.

[0131] 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.

[0132] (Example 3-1) Transplantation of a meniscus tissue into a right limb medial meniscus total resection model created using an immunodeficient miniature pig In this example, a tissue regeneration experiment was performed by transplanting the meniscus tissue created in Example 2-1 into the right limb medial meniscus total resection site of an immunodeficient miniature pig. The medial side of the right knee joint of the immunodeficient miniature pig was exposed, and the meniscus was completely resected.

[0133] The details of the transplantation method are shown in Figure 22. In Figure 22, the meniscus tissue 220 (referred to as the "transplant" in this example) was sutured and reinforced with simple knots at the joint capsule adjacent to the portions corresponding to the anterior, middle, and posterior segments. 2-0 Fiber wire 221a was used for the sutures and reinforcement. At the portions corresponding to the anterior, middle, and posterior segments of the transplant, the adjacent joint capsule was sutured using a horizontal mattress. 2-0 Fiber wire 221b was used for the sutures. The 2-0 Fiber wire 221b was threaded so that it was hooked onto the reinforcing thread (2-0 Fiber wire 221a) of the joint capsule.

[0134] Both ends of the graft were sutured with a baseball suture using 2-0 fiber wire 221c, and the sutures were passed through the inside of a bone hole 222 made in the tibia, and then each suture was fixed on the medial side of the tibia. An actually prepared graft is shown in Figure 23. In Figure 23, (a) shows a ring-shaped tissue body, (b) shows a tissue body that has been partially cut and spread out, and (c) shows both ends of the tissue body sutured with 2-0 fiber wire 221c.

[0135] Figure 24 shows the preparation of a meniscus tissue injury model. Figure 25 shows the actual transplantation of a meniscus tissue. In Figure 24, (a) shows the medial femoral condyle after excision and inversion, and (b) shows the resection of at least two-thirds of the inner circumference of the medial meniscus. In Figure 25, (a) shows the confirmation of the position of the tibial bone tunnel, and (b) shows the state immediately after transplantation. The area enclosed by the dotted line in (b) is the meniscus tissue.

[0136] Figure 26 shows X-ray images of the knee joint 29 days (approximately 4 weeks) after meniscus tissue transplantation. (a) shows the right knee joint (transplanted limb), and (b) shows the left knee joint (healthy limb). As with the healthy limb, no narrowing of the joint space on the medial side of the knee joint was observed in the transplanted limb (arrow).

[0137] Figure 27 shows the results of an autopsy performed 29 days (approximately 4 weeks) after meniscus tissue transplantation. The figure shows the meniscus tissue at the time of autopsy, with the area enclosed by the dotted line representing the reconstructed meniscus. This demonstrates that the meniscus was reconstructed by the transplantation.

[0138] Figure 28 shows the results of histological staining of the meniscus tissue, showing a histologically stained cross-section of the meniscus (dotted line) shown in Figure 28(a) along the solid line. (b) and (c) show the results of hematoxylin and eosin (HE) staining, with (c) showing an enlarged image of the boxed area in (b). (d) and (e) show the results of Alcian blue (ALB) staining, with (e) showing an enlarged image of the boxed area in (d). These results demonstrate that the tissue shape of the meniscus was reconstructed. ALB staining revealed a positive reaction in part of the reconstructed meniscus (arrows), indicating that the fibrocartilage structure had been regenerated.

[0139] (Example 3-2) Compression Test In this example, the mechanical properties (compressive breaking strength) of cell constructs cultured while individually applying each compressive stimulus (0.0 [N], 0.1 [N], 0.2 [N]) were evaluated. The meniscus described in Example 2-1 was used as the cell construct. The compressive breaking strength was measured using a force gauge (Imada, ZTA-500N) attached to an electric measurement stand (Imada, MX2-500N-FA). The meniscus was placed on the base of the stand, and a metal rod attached directly to the force gauge was pressed against the meniscus from above at a speed of 50 mm / min to determine the load at which the meniscus broke.

[0140] The results are shown in Figures 29 and 30. In Figure 29, when compressive stimulation was applied at each force, it was observed that the peak force temporarily decreased after a predetermined distance of compression. The force at the apex of the peak (indicated by the arrow) at this time represents the maximum compressive breaking strength. Figure 30 summarizes the results of Figure 29. The maximum compressive breaking strength of the cell construct was 6.3 N (2.00 MPa) when cultured with a compressive stimulation of 0.1 N, 5.8 N (1.85 MPa) when cultured with a compressive stimulation of 0.2 N, and 4.0 N (1.27 MPa) when cultured without a compressive stimulation (0.0 N). This demonstrates that the mechanical strength of the cell construct is enhanced when cultured with a compressive stimulation. [Explanation of symbols] 1A: Culture device 1B: Culture device 1: Application unit 2: Circulation unit 3: Reciprocating unit 4: Motor 5: Culture vessel 6: Circulation culture tube 7: Motor control device 8: Pressing unit 9: Machine base 10: Base 11, 12: Counter shaft 13: Vibrator 21: Pressing unit 22: Fixing unit 30: Mechanical unit 50: Control unit 51: Detection unit 52: Gas adjustment unit 53: Temperature adjustment unit 71: Motor 100: Three-dimensional structure 220: Meniscus tissue 221: 2-0 Fiber wire 222: Bone hole

Claims

1. A method for strengthening a three-dimensional tissue formed by stacking cell clusters, comprising a step of culturing the three-dimensional tissue while applying stretching and / or compressive stimuli to the tissue.

2. The method according to claim 1, wherein the stretching stimulus is a stretching stimulus including uniaxial motion or multiaxial motion.

3. The method of claim 2, wherein the stretching stimulus includes at least one of a twisting motion, a pulling motion, and a vibrating motion.

4. The method according to claim 1, wherein the compressive stimulus is applied by a pressure member having a pressure surface against the three-dimensional tissue structure.

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. The method according to claim 1, wherein the three-dimensional tissue is cultured under circulating culture.

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

10. The method of claim 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 stacking cell clusters while applying stretching and / or compressive stimuli to the tissue.

12. The method of claim 11, wherein the stretch stimulus is a stretch stimulus that includes uniaxial motion or multiaxial motion.

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

14. The method according to claim 11, wherein the compressive stimulus is applied by a pressing portion having a pressing surface against the three-dimensional tissue structure.

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. The method according to claim 11, wherein the three-dimensional tissue is cultured under circulating culture.

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

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

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

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

21. A three-dimensional tissue formed by stacking cell aggregates and culturing the same while applying a stretching stimulus and / or a compressive stimulus, the breaking strength of which is at least 0.001 N / mm ２ A three-dimensional tissue having the structure.

22. The tissue structure according to claim 21, wherein the cell mass comprises mesenchymal stem cells, fibroblasts, and cells that are the source of the target tissue structure.

23. The tissue according to claim 22, wherein the source cells are precursor cells of bone, cartilage, ligament, tendon or meniscus.

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, which is a tissue structure for bone, cartilage, ligament, tendon or meniscus.

26. A culture apparatus for culturing a three-dimensional tissue, comprising: an application unit that applies a mechanical stimulus to the three-dimensional tissue.

27. The culture device according to claim 26, wherein the mechanical stimulus applied by the application unit includes at least a stretching stimulus.

28. A culture device as described in claim 26, wherein the three-dimensional tissue has an opening, the application unit includes at least two axial members, and the application unit applies a mechanical stimulus to the three-dimensional tissue by moving the at least two axial members relative to each other while inserted into the opening.

29. A culture device according to claim 28, wherein the application unit applies a mechanical stimulus to the three-dimensional tissue structure by translationally moving the at least two axial members relative to one another.

30. A culture device according to claim 28, wherein the application unit applies a mechanical stimulus to the three-dimensional tissue by rotating the at least two shaft members relative to one another.

31. A culture device as described in claim 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 expanding and contracting the three-dimensional tissue by the vibrations generated by the vibrating body.

32. The culture device according to claim 26, wherein the mechanical stimulus applied by the application unit includes at least a compressive stimulus.

33. A culture device according to claim 26, wherein the application section includes: a base section having a recess into which the three-dimensional tissue body fits; and a pressing section that presses the three-dimensional tissue body fitted into the recess.

34. A culture apparatus as described in claim 26, wherein the application unit includes: a tension unit that pulls the three-dimensional tissue; and a pressing unit that presses the three-dimensional tissue to which a stretching stimulus has been applied by the tension unit in a direction different from the pulling direction of the tension unit.

35. The culture apparatus according to claim 26, further comprising a circulation section for circulating the culture medium in the culture vessel.

36. The culture apparatus according to claim 26, further comprising a gas regulator for regulating gas inside the culture vessel.

37. The culture apparatus according to claim 26, further comprising a temperature control unit for controlling the temperature inside the culture vessel.

38. A culture apparatus as described in claim 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.

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